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Japan Australia Meeting on Cell Death
21st – 23rd October, 2015
WEHI, Melbourne
jamoncelldeath.wordpress.com
SCIENTIFIC PROGRAM & ABSTRACT BOOK
Sponsors & Supporters
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PROGRAM OVERVIEW
Wednesday, 21 October 2015
1:00pm Registration Opens 3:00 pm Welcome 3:30 pm Selected Short Poster Talks
Chair: Grant Dewson 4:10 pm Break 4:20 pm Plenary Session 1: Shigekazu Nagata
Chair: Andreas Strasser 5:10 pm Poster session and drinks
Thursday, 22 October 2015
9:00 am Session 1: Apoptosis / Inflammation / Immunity
Chair: Joe Trapani 10:35 am Morning Tea 11:00 am Session 2: Apoptosis
Chair: Ruth Kluck 12:30 pm Lunch 1:20 pm Session 3: Necroptosis
Chairs: James Murphy & Isabelle Lucet 3:00 pm Selected Short Poster Talks
Chair: David Vaux 3:50pm Afternoon Tea 4:20 pm Session 4: Autophagy
Chairs: Kelli MacDonald & Justine Mintern 5:45 pm Plenary Session 2: Sharad Kumar
Chair: David Vaux 6:25 pm Poster session, drinks, canapés
Friday, 23 October 2015
9:15 am Session 5: Pyroptosis & Inflammation Chairs: Ana Traven & Thomas Naderer
10:50 am Morning Tea 11:15 am Session 6: Infection & Cell Death
Chair: Chris Andoniou 12:30 pm Lunch 1:15 pm Session 7: Cell Death and the Clinic I
Chair: Guillaume Lessene 2:40 pm Afternoon Tea 3:05 pm Session 8: Cell Death and the Clinic II
Chair: Paul Ekert 4:10 pm Plenary Session 3: Clare Scott
Chair: Paul Ekert 5:00 pm Wrap Up & Prizes
Conference Organising Committee
Assoc. Prof. John Silke, The Walter & Eliza Hall Institute
Dr Grant Dewson, The Walter & Eliza Hall Institute Prof. Hiroyasu Nakano, Toho University School of Medicine
Dr Peter Czabotar, The Walter & Eliza Hall Institute Dr Misty Jenkins, Peter MacCallum Cancer Centre
Dr Kate Schroder, University of Queensland Dr Justine Mintern, University of Melbourne
Dr James Vince, The Walter & Eliza Hall Institute Prof. Masato Tanaka, Tokyo University of Pharmacy & Life Sciences
Prof. Sho Yamasaki, Medical Institute of Bioregulation, Kyushu University Prof. Catherine Day, University of Otago, New Zealand
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DETAILED PROGRAM OVERVIEW Wednesday, 21 October 2015
1:00 pm Registration 3:00 pm Welcome to Country (Aunty Joy)
Welcome to WEHI (Prof. Doug Hilton) Housekeeping & Welcome (Assoc. Prof. John Silke)
3:30 pm Selected Short Poster Talks Chair: Grant Dewson
3:30 pm 1 Poster talk 3:33 pm 2 Poster talk 3:36 pm 3 Poster talk 3:39 pm 4 Poster talk 3:42 pm 5 Poster talk 3:45 pm 6 Poster talk 3:48 pm 7 Poster talk 3:51 pm 8 Poster talk 3:54 pm 9 Poster talk 3:57 pm 10 Poster talk 4:00 pm 11 Poster talk
4:05 am Short Break
4:15 pm 12 Plenary Session 1: Shigekazu Nagata Exposure of phosphatidylserine during apoptosis and engulfment of dead cells
Chair: Andreas Strasser
5:00 pm Poster session and drinks Stephen Ma on Piano
7:30 pm End Day 1
Thursday, 22 October 2015 – Morning
9:00 am Session 1: Apoptosis, Inflammation & Immunity Chair: Joe Trapani
9:05 am 13 Ben Kile – Apoptotic caspases suppress DAMP signaling in vivo
9:25 am 14 Masato Tanaka – The role of CD169 macrophages in dead cell clearance and inflammatory regulation
9:45 am 15 Sho Yamasaki – Recognition of damaged self through MINCLE
10:05 am 16 Ivan Poon – Elucidating the molecular mechanism of apoptotic cell disassembly
10:25 am 17 Tatsuya Saitoh - Mitochondrial damage elicits a viral RNA-degrading innate immune response
10:35 am Morning Tea
11:00 am Session 2: Apoptosis Chair: Ruth Kluck
11:05 am 18 Andreas Strasser – How does tumour suppressor p53 protect us against cancer development?
11:25 am 19 Mark Van Delft – Discovery and characterization of small molecule inhibitors of Bak-mediated apoptosis
11:40 am 20 Erinna Lee - Repurposing anti-cancer drugs for the treatment of parasitic infections
12:00 pm 21 Sweta Iyer – A new activation site in Bak switches Bak to a death-inducing oligomer
12:10 pm 22 Hamsa Puthalakath – Identification of small molecular inhibitors of BIM expression for treating cardiomyopathy
12:30 pm Lunch
Thursday, 22 October 2015 - Afternoon
1:20 pm Session 3: Necroptosis Chairs: James Murphy & Isabelle Lucet
1:25 pm 23 Kim Newton – Regulation of cell death by the kinase RIPK1
1:45 pm 24 Hiroyaso Nakano – Targeted integration of CFLIPs on the X chromosome in mice results in identification of genes that promote compensatory proliferation
2:05 pm 25 Mikiko Sodeoka – Chemical Approach To Oxidative Stress-Induced Necrotic Cell Death
2:25 pm 26 Liz Hartland – A bacterial cysteine protease effector cleaves RHIM proteins to block necroptosis and inflammation
2:45 pm 27 Maria Tanzer – MLKL activation is tuned by phosphorylation outside its pseudokinase activation loop
Short Preparation break
3:10 pm Selected Short Poster Talks Chair: David Vaux
3:15 pm 28 Poster talk 3:18 pm 29 Poster talk 3:21 pm 30 Poster talk 3:24 pm 31 Poster talk 3:27 pm 32 Poster talk 3:30 pm 33 Poster talk 3:33 pm 34 Poster talk 3:36 pm 35 Poster talk 3:39 pm 36 Poster talk 3:39 pm 37 Poster talk 3:42 pm 38 Poster talk
3:50 pm Afternoon Tea
4:15 pm Session 4: Autophagy Chairs: Kelli Macdonald & Justine Mintern
4:20 pm 39 Shigeomi Shimizu – Biological roles of autophagic cell death
4:40 pm 40 Jim Harris – Inflammaphagy: Regulation of inflammatory mediators by autophagy
5:00 pm 41 Lisa Lindqvist – Bcl-2 and related pro-survival proteins require BAK and BAX to affect autophagy
5:20 pm 42 Michael Lazarou – Culling bad mitochondria: the molecular mechanisms of PINK1/Parkin mitophagy
5:40 pm 43 Plenary Session 2: Sharad Kumar Modes And Mechanisms Of Developmental Cell Death: What Have We Learned From ‘The Fly’? Chair: David Vaux
6:20 pm Poster session, drinks & canapés Beer by Dainton Brewery Music by Moreland City Soul Revue
8:20 pm End Day 2
Friday, 23 October 2015 - Morning
9:15 am Session 5: Pyroptosis & Inflammation Chairs: Ana Traven & Thomas Naderer
9:20 am 44 Takashi Suda - Novel Anti-Inflammatory Function Of Vitamin B6 By Inhibition Of The Nlrp3 Pathway
9:40 am 45 Kate Schroder - Neutrophil inflammasomes selectively drive interleukin-1β production but do not elicit pyroptotic or apoptotic cell death
10:00 am 46 Yoshifumi Yamaguchi - Live-imaging analysis of apoptosis and pyroptosis at a single cell resolution
10:20 am 47 Kate Stacey - Modes of cell death in response to invading DNA: pyroptosis, apoptosis, and “Nazaroptosis”
10:40 am 48 Rebecca Feltham - Mindbomb-2 is a novel RIPK1 interacting E3 Ub-ligase that protects cells from the cytotoxic effects of TNF
11:50 am Morning Tea
11:15 am Session 6: Infection and Cell Death Chair: Chris Andoniou
11:20 am 49 Marc Pellegrini - Dying to survive 11:40 pm 50 Gemma Kelly - Investigating How The Epstein-Barr
Virus-Encoded vBCL-2 Homologue Bhrf1 Functions To Protect Lymphoma Cells From Apoptosis
12:00 pm 51 Marc Kvansakul - Structural Insight Into Vaccinia Virus Mediated Inhibition Of Apoptosis
12:20 pm 52 Marcel Doerflinger - Mechanism of Bim-mediated apoptosis during sepsis-induced lymphopenia
12:30 pm Lunch
Meetings don't end like light from dying stars they
linger past the expiry date
Friday, 23 October 2015 - Afternoon
1:15 pm Session 7: Cell Death and the Clinic I Chair: Guillaume Lessene
1:20 pm 53 Koji Yasutomo - Genetic dissection of familial inflammatory disorders
1:40 pm 54 MaryAnn Anderson - The emerging role of BH3 mimetics in hematological malignancy
2:00 pm 55 Minoru Tanaka - Study on the molecular mechanisms linking between hepatic cell death and fibrosis
2:20 pm 56 Ricky Johnstone - Targeting the epigenome to induce cancer cell death and differentiation
2:40 pm Afternoon Tea
3:05 pm Session 8: Cell Death and the Clinic II Chair: Paul Ekert
3:10 pm 57 Andrew Wilks - Lost In Translation: Converting Cutting Edge Research Into The Therapies Of Tomorrow
3:30 pm 58 Misty Jenkins - Increased cytotoxic lymphocyte synapse dwell time causes cytokine storm
3:50 pm 59 Chun Fong - Evading The Storm: Resistance To Targeted Epigenetic Therapies And The Leukaemia Stem Cell
4:10 pm 60 Plenary Session 3: Clare Scott Chair: Paul Ekert
5:00 pm Wrap Up & Prizes
Invited Speaker Abstracts
Plenary Session
12. EXPOSURE OF PHOSPHATIDYLSERINE DURING APOPTOSIS AND ENGULFMENT OF DEAD CELLS
Shigekazu Nagata
Laboratory of Biochemistry and Immunology, WPI Immunology Frontier Research Center, Osaka 565-0871, Japan
When cells undergo apoptosis, they expose phosphatidylserine (PtdSer) on their surface. How PtdSer is exposed to the cell surface had been elusive. We recently identified two membrane proteins (TMEM16F and Xkr8) that are involved in scrambling of phospholipids in plasma membrane. TMEM16F carries 10 transmembrane regions, and requires Ca2+ to mediate phospholipid scrambling. It plays a role in the PtdSer-exposure in activated platelets for blood clotting. Xkr8 carries 6 transmembrane regions, and caspase-cleavage in its C-terminus confers it the scramblase activity. In addition to the activation of scramblase, a flippase that translocates PtdSer from outer to inner leaflets is inactivated during apoptosis. We found that ATP11C, a P4-type ATPase that works as a flippase, is cleaved by caspase during apoptosis. Live cells lacking the flippase constitutively expose PtdSer, and are engulfed by macrophages, indicating that PtdSer on the cell surface is necessary and sufficient to be recognized by macrophages for engulfment. MFG-E8, Tim-4, Gas6, and Protein S specifically bind PtdSer with high affinity. We found that mouse resident peritoneal macrophages require both Tim4 and Protein S for engulfment. Tim4 is involved in tethering of apoptotic cells, while Protein S promotes the engulfment of apoptotic cells by binding to MerTK, a tyrosine kinase receptor. Here, I discuss how PdtSer is exposed during apoptotic cell death, and how dead cells are engulfed by macrophages.
Session 1: Apoptosis, Inflammation & Immunity 13. APOPTOTIC CASPASES SUPPRESS DAMP SIGNALLING IN VIVO
Kate McArthur, Michael J White, Donald Metcalf, John Cambier, Sammy Bedoui, Matthew Ritchie, Marco Herold, David CS Huang, Guillaume Lessene and Benjamin T Kile*
ACRF Chemical Biology Division, The Walter & Eliza Hall Institute of Medical Research, Parkvil le, Austral ia
Activated caspases are a hallmark of apoptosis induced by the intrinsic (or “mitochondrial”) pathway, but they are ultimately dispensable for cell death, and for the apoptotic clearance of cells in vivo. In the light of emerging evidence that caspases can inactivate damage-associated molecular pattern molecules (DAMPs), this has led to the suggestion that caspases are primarily activated not to kill, but to prevent dying cells from triggering a host immune response. Here we show that activation of Bak and Bax, the essential mediators of the intrinsic apoptosis pathway, induces mitochondrial (mt) membrane permeabilisation that results in the efflux of mtDNA into the cytosol. In the absence of the apoptotic caspase cascade, the apoptotic cell behaves as if virally infected. Cytosolic mtDNA activates the DNA sensor cGAS, which then initiates STING-mediated type I interferon (IFN) production. In a wild-type cell, concurrent activation of the caspase cascade (key members of which include the initiator caspase, Caspase-9, and the effector caspases, Caspase-3 and Caspase-7) by mitochondrial cytochrome c prevents IFN production. Disabling the caspase cascade by pharmacological inhibition or genetic deletion of Caspase-9, Apaf-1, or Caspase-3/7 triggers secretion of IFN-β by apoptotic cells. This is true of mouse and human cells, and cells of both hematopoietic and non-hematopoietic origin. In vivo, loss of Caspase-9 precipitates an elevation in IFN-β levels and consequent hematopoietic stem cell dysfunction, which is corrected in Bak/Bax/Caspase-9-deficient mice. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.
14. THE ROLE OF CD169 MACROPHAGES IN DEAD CELL CLEARANCE AND INFLAMMATORY REGULATION
Masato Tanaka
Laboratory of Immune Regulation, School of Life Science, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
When cell death occurs in vivo, cell corpses are not left untreated, but are recognized and engulfed by phagocytes, such as macrophages and dendritic cells. Previously, we reported that CD169+ macrophages in the marginal zone of the spleen capture blood-borne apoptotic cells, and induce dead cell antigen-specific tolerance. We also demonstrated that CD169+ macrophages in the lymph node sinus phagocytose dead tumor cells that flow into draining lymph node via lymphatic flow and crosspresent tumor cell-associated antigen to CD8 T cells. These results indicate that CD169+ macrophages localizing at the border region of lymphoid organ, monitor the entry of dead cells via blood stream or lymphatic flow, and suppress or activate dead cell antigen-specific CD8 T cells.
In order to explore the distribution of CD169+ cells outside secondary lymphoid organs, we generated mice that harbor the Cre recombinase gene in the CD169 loci (CD169-Cre mice), and crossed those mice with ROSA26-yellow fluorescent protein reporter mice to analyze recombinase activity. Using the mice, we detected gene recombination not only in macrophages located in lymphoid organs, but also in some tissue resident macrophages in several organs such as intestine and kidney. Among them, we focused on CD169+ intestine-resident macrophages, and examined the roles of these macrophages in the experimental colitis. We found that CD169+ macrophages reside not at the villus tip, but at the bottom-end of the lamina propria microenvironment. Following mucosal injury, the CD169+ macrophages recruit inflammatory monocytes by secreting CCL8. Selective depletion of CD169+ macrophages or administration of neutralizing anti-CCL8 antibody ameliorates the symptoms of experimentally induced colitis in mouse. These findings suggest that CD169+ macrophage-derived CCL8 serves as an emergency alert for the collapse of barrier defense, and is a promising target for the suppression of deteriorating mucosal injury.
15. RECOGNITION OF DAMAGED SELF THROUGH MINCLE
Sho Yamasaki* Medical Institute of Bioregulation, Kyushu University *[email protected]
C-type lectin receptors (CLRs) comprise a large family of proteins that share a common structural motif and are involved in various immune responses. Among them, we found that Mincle (macrophage inducible C-type lectin) is an activating receptor for “damaged self” as well as “non-self pathogens”. From those pathogens and dead cells, characteristic glycolipids were identified as Mincle ligands, all of which possessed adjuvant activities. These findings shed light on CLRs as emerging immune receptor family for wide spectrum of “danger” derived from self and non-self. In this symposium, the recent progress and perspective on the physiological/pathological function of Mincle and related CLRs will be discussed.
16. ELUCIDATING THE MOLECULAR MECHANISM OF APOPTOTIC CELL DISASSEMBLY
Georgia K Atkin-Smith, Rochelle Tixeira, Lanzhou Jiang, Stephanie Paone, Sarah Caruso, Thomas Spink, Jenny DY Chow, Ivan K H Poon*
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science
Background: Apoptosis occurs in essentially all tissues as part of development, homeostasis, and pathogenic processes including infection and cardiovascular disorders. Apoptotic cells often disassemble into smaller membrane-bound particles called apoptotic bodies (a process known as apoptotic cell disassembly). Under normal physiological conditions, the generation of apoptotic bodies during apoptosis can facilitate efficient removal of apoptotic cells by phagocytes, to prevent intracellular factors leaking from dying cells and promoting unwanted inflammation. However, under certain pathological conditions, cellular materials such as cytokines, cell surface molecules and microRNA can be packaged into apoptotic bodies as a mechanism to regulate immunity and tissue repair. Since billions of cells undergo apoptosis daily, the importance of apoptotic cell disassembly and clearance for health and disease is fundamental, yet the mechanisms involved in the formation of apoptotic bodies are poorly understood. These mechanisms also represent attractive targets for the development of novel therapeutics.
Aim: To determine the molecular mechanism of apoptotic cell disassembly.
Results: Here, we describe two new mechanisms of cell disassembly by apoptotic T lymphocytes and monocytes via the formation of a novel membrane protrusion called apoptopodia. Mechanistically, we have identified ROCK1 kinase and pannexin 1 membrane channels as key regulators of apoptotic cell disassembly. Additionally, have identified a novel selection of drugs that can modulate apoptotic body formation.
Conclusion: Understanding the mechanistic basis of this process will generate fundamental knowledge of the downstream consequence of cell death and has significant implication in health and diseases.
ANOIKIS The homeless cell is
Detached from the matrix Dies of loneliness
Session 2: Apoptosis 18. HOW DOES TUMOUR SUPPRESSOR P53 PROTECT US AGAINST CANCER DEVELOPMENT? Ana Janic, Liz Valente, Brandon Aubrey, Haoyu Yang, Stephanie Grabow, Liz Milla, Sam Wilcox, Liam O’Connor, Scott Lowe, Gemma Kelly, Marco Herold, Andreas Strasser* Molecular Genetics of Cancer Division, The Walter & Eliza Hall Institute of Medical Research, Parkvil le, Austral ia
The tumour suppressor p53 is mutated in ~50% of human cancer and also functions as a major regulator of cellular responses to genotoxic cancer therapy. p53 is a transcription factor that can activate several cellular responses, including cell death, cell cycle arrest/senescence and DNA repair through induction of target genes. Surprisingly, no spontaneous tumours arose in mice lacking Puma, Noxa and p21, the essential mediators of p53-induced apoptosis and cell cycle arrest/senescence, respectively. To define the mechanisms of p53-mediated tumour suppression we screened an shRNA library to identify p53 targets that when knocked-down in haematopoietic stem/progenitor cells (HSPCs) deficient for p53-driven apoptosis, cell cycle arrest/senescence (Puma-/-p21-/-) promote lymphoma development. This library was also screened for shRNAs that could accelerate MYC-driven lymphomagenesis even when pro-apoptotic PUMA was absent (Eµ-Myc;Puma-/- HSPCs). These screens identified p53 target genes implicated in the control of DNA repair (Mlh1) and cell proliferation (Cop1). Interestingly, knockdown of these genes only promoted tumour development when p53-mediated apoptosis, cell cycle arrest/senescence were also impaired (i.e. Puma-/-p21-/- background). This reveals that several p53-activated effector processes must be compromised to facilitate tumorigenesis. Thus, p53 is such a potent tumour suppressor precisely because it coordinates so many cell growth inhibitory responses.
19. DISCOVERY AND CHARACTERIZATION OF SMALL MOLECULE INHIBITORS OF BAK-MEDIATED APOPTOSIS
Mark F van Delft*1,2, Stephane Chappaz1,2, Kate McArthur1,2, Yelena Khakham1,2, Kym N Lowes1,2, Kurt Lackovic1,2, Peter E Czabotar1,2, Grant Dewson1,2, Benjamin T Kile1,2, David C Huang1,2, and Guillaume Lessene1,2,
1The Walter and Eliza Hall Institute, Melbourne, Austral ia 2The University of Melbourne, Medical Biology, Melbourne, 3The University of Melbourne, Pharmacology and Therapeutics
Activating apoptosis with BH3-mimetics such as ABT-263 or ABT-199 is now becoming a clinically validated approach to treat cancer, but the converse tactic of using small molecules to block apoptosis has been much less explored. Caspase inhibitors have not enjoyed much clinical success and it is now believed that they act too late in the apoptotic cascade to rescue cells from death. Thus, the therapeutic impact of small molecules that can truly maintain cell survival in the presence of apoptotic stress signals remains unknown. There are, however, a number of indications, including ischemia-reperfusion injuries, where acute inhibition of cell death could be beneficial. Using a phenotypic screen in engineered mouse embryonic fibroblasts, we identified a series of small molecule inhibitors of apoptosis. Through extensive characterization, we have demonstrated that these first-in-class compounds specifically inhibit Bak-mediated apoptosis by blocking an early step of Bak activation and thereby preventing its subsequent conformational change and oligomerisation. Contrary to caspase inhibitors, apoptosis inhibition by these small molecules permits the clonogenic survival and sustained growth of cells rescued from apoptotic stress. Moreover, we have shown that the inhibitors can block apoptosis in a range of primary cell types. Altogether, our work demonstrates for the first time that blocking apoptosis upstream of the mitochondria is feasible and presents a clear advantage over inhibiting caspase activity. These entirely novel compounds will enable proof-of-concept experiments in vivo and help to evaluate the therapeutic potential of inhibiting apoptosis in various disease models.
20. REPURPOSING ANTI-CANCER DRUGS FOR THE TREATMENT OF PARASITIC INFECTIONS
Erinna F. Lee1,2,3*, Neil D. Young4, Christoph Grevelding5, Brad E. Sleebs6, G. Lessene6, Peter M. Colman6, Robin B. Gasser4, W. Douglas Fairlie1,2,3
1Olivia Newton-John Cancer Research Institute, Austral ia 2School of Cancer Medicine, La Trobe University, Melbourne 3La Trobe Institute for Molecular Science, La Trobe University 4Department of Veterinary Science, University of Melbourne 5Institut für Parasitologie, Justus-Liebig-Universität, Germany 6The Walter and Eliza Hall Institute, Parkvil le, Austral ia
Parasitic worms (helminths) affect more than a third of the world’s human population, as well as having significant impact on animals. The limited number of effective treatments against worms in humans has provided significant impetus for the development of new anti-helminthic drugs in the absence of effective vaccines. Here, we describe the identification and characterisation of a Bcl-2-regulated apoptosis pathway in schistosomes, the causative agent of schistosomiasis, which is a major global health problem. Genomic, biochemical and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by pro-survival Bcl-2-like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilisation. Our preliminary studies have also identified a number of different classes of “BH3-mimetic” drugs that bind to the schistosome Bcl-2 pro-survival protein and high-resolution structural data demonstrates how these compounds engage its target. Moreover, we show that BH3-mimetic compounds can profoundly influence schistosome biology and may have uses beyond cancer treatment.
21. A NEW ACTIVATION SITE IN BAK SWITCHES BAK TO A DEATH-INDUCING OLIGOMER
Sweta Iyer1*, Khatira Anwari1#, Amber E Alsop1, Wai Shan Yuen2, David C S Huang1, John Carroll2, Nicholas A Smith3, Brian J Smith3, Grant Dewson1 and Ruth M Kluck1
1The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Austral ia 2Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University 3La Trobe Institute for Molecular Sciences, La Trobe University, Victoria 3086, Austral ia
Bak, a pro-apoptotic member of the Bcl-2 family, mediates the mitochondrial pathway of apoptosis. Following an apoptotic stimulus, the binding of BH3-only relatives at a hydrophobic surface groove (α2-α5) triggers Bak activation (i.e. conversion into a pore-forming protein). Activation involves a series of conformational changes in Bak resulting in the formation of symmetric homodimers, which then associate to form the apoptotic pore in the mitochondrial outer membrane.
In the present study, we identified a second activation site in Bak, the α1-α2 loop region. As shown by cysteine linkage and limited proteolysis, activation at this new site triggered global conformational changes in Bak similar to those triggered by BH3-only proteins binding at the α2-α5 groove. However, the first step in loop-mediated Bak activation involved displacement of the α1 helix, as shown by cysteine tethering and in silico approaches.
We are currently investigating whether endogenous proteins can target this new site, and whether this site might provide a new target for developing therapies.
22. IDENTIFICATION OF SMALL MOLECULE INHIBITORS OF BIM EXPRESSION FOR TREATING CARDIOMYOPATHY
George Mbogo, Benjamin Richards, Belinda Abbott, Brian Smith and Hamsa Puthalakath*
La Trobe Institute for Molecular Science, Kingsbury Drive, Bundoora 3086, Austral ia
Background : Excessive stimulation of the β-ARs, 1,2 results in cardiomyocyte apoptosis leading to heart failure 3. Previous work in our lab had demonstrated the crucial role of the pro-apoptotic protein Bim in mediating this cell death process 4. We also have deciphered the molecular pathway involved in the transcriptional regulation of Bim during β-AR signalling enabling us to identify molecular targets for treating this disease.
Aims : To conduct a high throughput screening of a chemical library and identify drug hits that specifically target the apoptotic arm of β-AR signalling.
Results : Library screening resulted in the identification of 38 potential drug hits. Further characterization of these hits based on their ability to block Bim induction without affecting PKA activity (i.e. maintaining the functional compensation) yielded 4 drug hits. These compounds work within the 5-10 µM range and are cell permeable. Analogue synthesis and evaluation is underway to understand the structure-activity relationship (SAR) to optimise these drug hits to work within nano molar range. Studies on pharmacokinetics using rat liver microsomes also are being undertaken.
Conclusion : Though β-Blockers have been extensively used to treat heart failure for the last 60 years, heart failure remains poorly controlled with a 5-year survival rate of only 50%. This is due to the fact that β-Blockers block the entire signal pathway involved in the heart muscle contraction. Our drug hits offer potential superior treatment i.e. one that maintains the β-AR-mediated functional compensation and at the same time is capable of blocking the apoptotic arm of the β-AR pathway.
Session 3: Necroptosis
23. REGULATION OF CELL DEATH BY THE KINASE RIPK1
Kim Newton, Kate Wickliffe, Allie Maltzman, Debra L. Dugger, Joshua Webster, and Vishva M. Dixit
Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080
Kinase RIPK1 suppresses apoptosis and necroptosis in vivo independent of its enzymatic activity, whereas its kinase activity is required for death in response to certain stimuli. We have compared RIPK1-deficient mice with mice expressing either catalytically inactive RIPK1 or RIPK1 with a mutated RHIM (RIP homology interaction motif) to dissect how RIPK1 executes its opposing functions. Analyses using antibodies that recognize the autophosphorylation sites on mouse RIPK1 or RIPK3 have provided unexpected insights into RIPK1 signaling.
Necroptosis A death by fire
Or just indecision by a caspase?
24. TARGETED INTEGRATION OF cFLIPS ON THE X CHROMOSOME IN MICE RESULTS IN IDENTIFICATION OF GENES THAT PROMOTE COMPENSATORY PROLIFERATION
Hiroyasu Nakano
Department of Biochemistry, Toho University School of Medicine, 5-21-16 , Omori-Nishi, Ota-ku, Tokyo 143-8540, Japan
Compensatory proliferation is a process in which dying cells produce growth factors to promote proliferation of neighboring cells. However, the mechanisms are not fully understood. Here we generated knock-in (KI) mice harboring human short form of cellular FLICE-inhibitory protein (cFLIPs) gene that promotes necroptosis in vitro on the X chromosome. While all male CFLIPs KI mice died in utero, most female CFLIPs KI mice were born, grew without apparent abnormality, and fertile. Consistent with X-chromosome inactivation, intestinal epithelial cell (IEC)s of heterozygous female CFLIPs KI mice expressed CFLIPs and died in a mosaic pattern in utero. We found that tissue repair genes including Regenerating islet-derived gene (Reg)3b and Reg3g were significantly elevated in IECs of CFLIPs KI mice in a STAT3-dependent manner. Deletion of Receptor-interacting protein kinase (Ripk)3 suppressed cell death of IECs along with downregulation of Reg3β and Reg3γ. Together, RIPK3-dependent cell death might promote compensatory proliferation through upregulation of Reg3β and Reg3γ.
25. CHEMICAL APPROACH TO OXIDATIVE STRESS-INDUCED NECROTIC CELL DEATH
Mikiko Sodeoka* and Kosuke Dodo
RIKEN
Recently non-apoptotic cell death attracts increasing attention. We have been working on the development of small molecules, which can selectively inhibit or activate a specific type of cell death aiming to elucidate its molecular mechanism using these molecules as chemical probes. We succeeded to develop selective inhibitors of necrotic cell death induced by oxidative stress, IM-54 and its derivatives [1-3]. IM-54 did not inhibit apoptotic cell death induced by oxidative stress, anticancer drugs such as etoposide, or a physiological death ligand. IM-54 was also ineffective to necroptosis. IM derivatives were found to be effective for rat in vivo ischemia-reperfusion injury models suggesting that the IM-suppressive necrosis would play critical role in ischemia-reperfusion injury. Mitochondrial localization of fluorescent-labelled IM derivative was observed. Furthermore, their binding proteins were identified by using IM-immobilized affinity gels. Functions of their binding proteins were also analysed [4]. [1] M. Katoh, K. Dodo, M. Fujita, M. Sodeoka, Bioorg. Med. Chem. Lett. 15, 3109 (2005). [2] K. Dodo, M. Katoh, T. Shimizu, M. Takahashi, M. Sodeoka, Bioorg. Med. Chem. Lett. 15, 3114 (2005). [3] M. Sodeoka, K. Dodo, Chemical Record, 10, 308 (2010). [4] M. Okazaki, K. Kurabayashi, M. Asanuma, Y. Saito, K. Dodo, M. Sodeoka, Biochim. Biophys. Acta, Biomembranes, in press.
Cytokine - a dangerous lover: one kiss warns you,
salvages you, ki l ls you.
26. A BACTERIAL CYSTEINE PROTEASE EFFECTOR CLEAVES RHIM PROTEINS TO BLOCK NECROPTOSIS AND INFLAMMATION
Elizabeth L. Hartland*
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Austral ia
Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as RIPK1, RIPK3, TRIF and ZBP1/DAI that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling. RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis. Here we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM containing proteins, RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This required a previously unrecognised tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaved within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL led to rapid cleavage of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of TNF, LPS or poly(I:C)-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibited TNF-induced phosphorylation and plasma membrane localization of MLKL in an EspL dependent manner. In vivo, Ripk3-/- but not Mlkl-/- mice exhibited increased pathology upon infection with the EPEC-like mouse pathogen Citrobacter rodentium compared to wild type C57BL/6 mice. The activity of EspL defines a new family of T3SS cysteine protease effectors found in a range of bacteria and reveals that gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.
27. MLKL ACTIVATION IS TUNED BY PHOSPHORYLATION OUTSIDE ITS PSEUDOKINASE ACTIVATION LOOP
Maria Tanzer*, Anne Tripaydonis, Joanne Hildebrand, John Silke, James Murphy
The Walter and Eliza Hall Institute *[email protected]
The pseudokinase, MLKL (Mixed Lineage Kinase Domain Like), is the most terminal obligatory component of the necroptosis cell death pathway known. Phosphorylation of the activation loop in the MLKL pseudokinase domain by the protein kinase, receptor interacting protein kinase-3 (RIPK3) is known to be the key step in MLKL activation. This phosphorylation event is believed to trigger a molecular switch, leading to exposure of MLKL's N-terminal four helix bundle (4HB) domain, its oligomerisation, membrane translocation and ultimately cell death. To examine this process in detail we firstly tested whether other phosphorylation events modulate MLKL activation. Therefore we reconstituted Mlkl-/- Ripk3-/- MDFS (mouse dermal fibroblasts) with full length MLKL harbouring mutations at one of three phospho-sites identified by Mass spectrometry. We found that phosphorylation independent of its activation loop phosphorylation can fine tune the ability of MLKL to induce necroptosis. Furthermore we investigated whether the killing function of MLKL is evolutionarily conserved amongst various MLKL orthologs. Surprisingly unlike their mouse, horse and frog counterparts, human, chicken and stickelback 4HB domain were unable to induce cell death in murine fibroblasts. However, recombinant protein of mouse, frog, human and chicken permeablized liposomes. This indicates that although the membrane permeabilization function of the 4HB domain is evolutionarily conserved, execution of necroptosis relies on additional factors that are poorly conserved. MLKL,
You pretty litt le ki l ler, Are you the end or just its beginning?
Session 4: Autophagy
39. BIOLOGICAL ROLES OF AUTOPHAGIC CELL DEATH
Shigeomi Shimizu* and Satoko Arakawad
Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University
Background: Programmed cell death (PCD) is a crucial process required for the normal development and physiology of metazoans. The three major mechanisms that induce PCD are called type I (apoptosis), type II (autophagic cell death), and type III (necrotic cell death). Dysfunctional PCD leads to diseases such as cancer and neurodegeneration. Apoptosis is the most common form of PCD and is regulated by the members of the BCL2 family proteins, among which BAX and BAK act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagic cell death in response to various types of cellular stressors.
Aims: We extended our study to determine the physiological role of autophagic cell death and generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice.
Results: Embryonic fibroblasts and thymocytes from TKO mice underwent far less frequent autophagy, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence for the occurrence of ATG5-dependent death of DKO cells. Compared with wild-type embryos, loss of the interdigital web was significantly delayed in DKO embryos and further delayed in TKO embryos.
Conclusion: These data suggest that ATG5-dependent cell death contributes to embryonic development of DKO mice, implying that autophagic cell death compensates for deficient apoptosis.
40. INFLAMMAPHAGY: REGULATION OF INFLAMMATORY MEDIATORS BY AUTOPHAGY
James Harris
Lupus Research Group, Centre for Inflammatory Diseases, School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Austral ia Autophagy is a catabolic mechanism for the delivery of cellular constituents, including organelles, to lysosomes for degradation. During times of nutrient deprivation, autophagy can facilitate the recycling of amino acids, thus promoting cell survival. More recently, autophagy has been show to play a number of important roles in the survival and function of immune cells. In particular, autophagy intersects with antigen presenting pathways, aids the intracellular killing of some pathogenic microorganisms (xenophagy) and plays a role in thymic selection. In addition, autophagy regulates the secretion of specific pro-inflammatory cytokines, particularly IL-1 family cytokines. This is achieved through the removal of stimulatory signals, such as reactive oxygen species and mitochondrial DNA, as well as through the regulation of inflammasome components. We have also now demonstrated that loss of autophagy leads to hyper-secretion of macrophage migration inhibitory factor (MIF), a pluripotent pro-inflammatory molecule. MIF plays a pathogenic role in a number of inflammatory diseases, including rheumatoid arthritis, and has been linked to tumour progression in a number of different cancers. Loss of autophagy in macrophages and dendritic cells has previously been shown to induce reactive oxygen species (ROS)-dependent secretion of IL-1 family cytokines. Here, we demonstrate that loss of autophagy also results in increased MIF secretion by human and mouse monocytes and macrophages. Induction of autophagy with mTOR inhibitors had no effect on MIF secretion or on intracellular levels of the cytokine, but amino acid starvation increased secretion in a ROS-dependent manner. This starvation-induced MIF secretion was apparently independent of autophagy induction, but may be the result of a net loss of autophagy due to increased turnover of autophagosomes, coupled with a loss of de novo synthesis of autophagy proteins. These data further demonstrate that autophagic regulation of ROS plays a pivotal role in the regulation of inflammatory cytokine secretion in macrophages, with potential implications for the pathogenesis of inflammatory diseases.
41. BCL-2 AND RELATED PRO-SURVIVAL PROTEINS REQUIRE BAK AND BAX TO AFFECT AUTOPHAGY
Lisa M. Lindqvist,* Boris Reljic, Erinna F. Lee, W. Douglas Fairlie, Melanie Heinlein, David C. S. Huang, and David L. Vaux
Cell Signall ing & Cell Death Division, The Walter & Eliza Hall Institute of Medical Research, Parkvil le, Austral ia
Background: Autophagy is a catabolic process that envelopes, degrades, and recycles cytoplasmic material especially during times of stress. For the last 10 years there has been an enduring dogma that the pro-survival Bcl-2 family members inhibit autophagy by directly binding to the autophagy protein Beclin 1.
Aims: However, because Bcl-2, Bcl-xL, and Mcl-1 also inhibit the pro-apoptotic activity of Bax and Bak, and many inducers of autophagy also cause cell death, we wondered whether the current model might be spurious. To distinguish whether Bcl-2, Bcl-xL, or Mcl-1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking Bax and Bak.
Results: In cells with Bax and Bak, inhibiting the pro-survival Bcl-2 family members induced both autophagy and cell death, but when cells were unable to undergo mitochondria-mediated apoptosis, neither inhibiting nor over-expressing Bcl-2, Bcl-xL or Mcl-1 caused any detectable effect on LC3B lipidation, LC3B turnover, or autophagosome formation (1).
Conclusion: These results challenge the notion that Bcl-2, Bcl-xL or Mcl-1 function by interacting with Beclin 1 to regulate autophagy, but instead show that they affect autophagy indirectly.
(1) Lindqvist LM, et al. (2014) Proc Natl Acad Sci U S A. 111:8512-7.
42. CULLING BAD MITOCHONDRIA: THE MOLECULAR MECHANISMS OF PINK1/PARKIN MITOPHAGY
Michael Lazarou*, Danielle A. Sliter, Lesley A. Kane, Shireen A. Sarraf, Chunxin Wang, Jonathon L. Burman, Dionisia P. Sideris, Adam I. Fogel and Richard J. Youle
Monash University
*Michael. [email protected]
Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, PINK1 phosphorylates ubiquitin to activate Parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins to activate mitophagy. Using genome editing to knock out five autophagy receptors, we find that two previously linked to xenophagy, NDP52 and Optineurin, are the primary receptors for PINK1/Parkin-mediated mitophagy.
The ubiquitin kinase PINK1 recruits NDP52 and Optineurin, but not p62, to mitochondria to directly activate mitophagy independent of Parkin. Once recruited to mitochondria, NDP52 and Optineurin recruit ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria revealing a function for these autophagy receptors upstream of LC3. This supports a new model that PINK1 generated phospho-ubiquitin serves as the autophagy signal on mitochondria and that Parkin amplifies it. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathway.
Apoptopodia Apoptotic bodies' nostalgia
Or apoptotic cells' desperate love
Autophagy and Recycling sound nicer than
Eat yourself and die.
Plenary Session 43. MODES AND MECHANISMS OF DEVELOPMENTAL CELL DEATH: WHAT WE HAVE LEARNT FROM ‘THE FLY’
Sharad Kumar
Centre for Cancer Biology, University of South Austral ia, Adelaide
For over a century the humble vinegar fly, Drosophila melanogaster, has been used as a model organism for genetic studies. It is an ideal organism for the study of animal development, complex biological pathways and genetic diseases. Alongside our mammalian studies, we have used the fly for the discovery of key components of the cell death machinery and to understand how the machine is activated and regulated to precisely delete obsolete tissues during development. The use of Drosophila has also allowed us to discover an apparent non-apoptotic form of cell death. In my talk I will attempt to summarize our past and current studies using Drosophila as a model to uncover the mysteries of developmental cell death.
Session 5: Pyroptosis & Inflammation
44. NOVEL ANTI-INFLAMMATORY FUNCTION OF VITAMIN B6 BY INHIBITION OF THE NLRP3 PATHWAY
Peipei Zhang1, Takeshi Kinoshita1, Hiroko Kushiyama1, Sofya Suidasari2, Norihisa Kato2, Takashi Suda1*
1Cancer Research Institute, Kanazawa University 2Graduate School of Biosphere Science, Hiroshima University
Background and Aims: Vitamin B6 represents six water soluble vitamers; pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), and their phosphorylated forms. Several lines of evidence have suggested that vitamin B6 has potential therapeutic activity in a variety of inflammatory diseases, and patients with inflammation had significantly lower blood levels of pyridoxal 5'-phosphate (PLP) compared with the control subjects. In addition, one of us (NK) previously observed that vitamin B6 inhibited LPS induced NF-κB activation. To further explore anti-inflammatory function of vitamin B6, we investigated whether vitamin B6 exerts its anti-inflammatory activity via inhibition of NLRP3 inflammasome activation. Results: Incubation of LPS-primed macrophages with PL and PLP but not with PM and PN inhibited secretion of IL-1β and IL-18, and delayed pyroptotic cell death induced by NLRP3 agonists (ATP, nigericin, R837, and monosodium urate crystals). Consistently, PL and PLP blocked proteolytic maturation of pro-caspase-1 and pro-IL-1β. In contrast, none of the B6 vitamers inhibited IL-6 secretion under these conditions. PL and PLP supressed IL-1β production induced by Staphylococcus aureus infection (NLRP3-dependent), but not that induced by Salmonella typhimurium (NLRC4-dependent) and Listeria monocytogenes (AIM2-dependent). Importantly, in a mouse model of LPS-induced IL-1β production, PL and PLP reduced IL-1β levels in both serum and peritoneal lavage. Moreover, PL and PLP protected mice from lethal endotoxic shock induced by LPS injection. Conclusion: Collectively, these findings reveal a novel anti-inflammatory function of vitamin B6 through inhibition of the NLRP3 pathway, and suggest a potential use of vitamin B6 in preventing NLRP3-driven inflammatory diseases.
4 am in Hyderabad I make haiku about a dead cell
What a miserable l ife
45. NEUTROPHIL INFLAMMASOMES SELECTIVELY DRIVE INTERLEUKIN-1ß PRODUCTION BUT DO NOT ELICIT PYROPTOTIC OR APOPTOTIC CELL DEATH
Kate Schroder*
Macrophage and dendritic cell inflammasomes drive potent innate immune responses against intracellular pathogens, by eliciting rapid caspase-1-dependent pro-inflammatory cytokine production (e.g. interleukins (IL)-1β and -18) and pyroptotic cell death, as well as caspase-8-directed apoptotic cell death. The contribution of other cell types to inflammasome-mediated host defense had not been examined in detail. Here we demonstrate that neutrophils, typically viewed as cellular targets of IL-1β, themselves activate the NLRC4 inflammasome during acute Salmonella infection, and are a major cell compartment for IL-1β production during acute peritoneal challenge in vivo. Importantly, unlike macrophages, neutrophils do not undergo pyroptosis upon in vitro or in vivo NLRC4, NLRP3 or AIM2 inflammasome activation. Furthermore, neutrophils also resisted inflammasome/caspase-8-directed apoptotic cell death. The ability of neutrophils to resist inflammasome-mediated death is unique amongst inflammasome-signaling cells so far described. Their continued viability allows neutrophils to sustain IL-1β production at a site of infection, and exert their crucial inflammasome-independent antimicrobial effector functions to clear infection. This work reveals neutrophils as a surprising new cellular player in inflammasome-mediated host defence during in vivo bacterial infection, and highlights how myeloid cell identity can have a major impact on innate immune signalling pathways.
46. LIVE-IMAGING ANALYSIS OF APOPTOSIS AND PYROPTOSIS AT A SINGLE CELL RESOLUTION
Yoshifumi Yamaguchi 1,2*, Ting Liu1 Yoshitaka Shirasaki3,4, Osamu Ohara3,5, Naomi Shinotsuka1 and Masayuki Miura1,6
1Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo 2PRESTO, Japan Science and Technology Agency 3Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences ( IMS-RCAI) 4Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo 5Department of Human Genome Research, Kazusa DNA Research Institute 6CREST, Japan Agency for Medical Research and Development
Background: Caspases are evolutionarily conserved cysteine proteases that regulate not only apoptotic cell death but also inflammatory cell death termed pyroptosis in response to various kinds of stimuli. Dynamics of the two types of cell death in vivo remained to be elucidated.
Results: We have developed genetically encoded FRET sensors, SCAT3 and SCAT1, for detecting caspase-3 and caspase-1 activation, respectively. SCAT3 allowed us to observe dynamics and unexpected behaviour of apoptotic dying cells in developing embryos. SCAT1 enables to capture cells undergoing pyroptosis with caspase-1 activation. We found, as in the case of caspase-3 activation during apoptosis, all-or-none activation of caspase-1 occurred in pyroptotic cells at the single-cell level, with similar activation kinetics irrespective of the type of inflammasome or the intensity of the stimulus. Release of IL-1ß from dying macrophages that exhibited caspase-1 activation was also confirmed by live-imaging analysis.
Conclusion: Apoptosis and pyroptosis can affect their surroundings, thereby facilitating developmental processes and inflammatory responses. Our results demonstrate that live-imaging analysis of cell death offers novel opportunities for understanding the significance of cell death in multicellular organisms.
Just wondering now What time the Jam On will end
We need to book flights
47. MODES OF CELL DEATH IN RESPONSE TO INVADING DNA: PYROPTOSIS, APOPTOSIS, AND “NAZAROPTOSIS”
Vitaliya Sagulenko, Parimala Vajjhala, Nazarii Vitak, Katryn J. Stacey*
The University of Queensland, School of Chemistry and Molecular Biosciences
Background: Extra-chromosomal DNA presents a potentially dangerous situation to the cell; it could derive from infectious organisms, transposons, or damaged self DNA. Cell death is an appropriate defensive response to any of these situations. The AIM2 (absent in melanoma 2) inflammasome is activated by cytosolic double stranded DNA, and leads to caspase-1-dependent lytic cell death termed pyroptosis. However, we have also demonstrated simultaneous AIM2-dependent activation of caspase-8, caspase-3 and apoptosis. Such responses are only well characterised in mammalian macrophages. Aims: To elucidate AIM2-dependent and –independent pathways of response to cytosolic DNA in a range of species and cell types. Results: (1) Caspase-8 may be activated by the inflammasome as a back-up pathway for pathogen-mediated inhibition of caspase-1. We have demonstrated that heterotypic death domain interactions mediate initiation of procaspase-8 filaments by the inflammasome adapter molecule, ASC. (2) In a further redundancy of cell death pathways, we have shown that caspase-3 can be cleaved downstream of both caspase-8 and caspase-1, leading to apoptosis. (3) Examination of fly and chicken responses to cytosolic DNA demonstrates AIM2-independent lytic cell death initiated within 10 minutes of introduction of DNA, termed “Nazaroptosis” here. This mode of cell death is also seen in mammalian non-macrophages, and unlike AIM2 responses does not require DNA to be in double stranded form. Conclusion: The evolution of several different cell death pathways elicited by extra-chromosomal DNA, as well as the redundancy of downstream pathways initiated by AIM2, stresses the importance of death as a response to invading DNA.
Session 6: Infection and Cell Death 49. DYING TO SURVIVE
Greg Ebert, Simon Preston, Cody Allison, James Cooney, Michael Stutz, Jesse Toe, Samar Ojaimi and Marc Pellegrini*
Infection & Immunity Division, The Walter & Eliza Hall Institute of Medical Research
We have developed several mouse models of hepatitis B virus (HBV), HIV-1, Mycobacterium tuberculosis and other infections to examine the role of host cell signalling and cell death pathways on host-pathogen interactions. Using these models we identified cellular inhibitors of apopotosis proteins (cIAPs) as major regulators of pathogen persistence. With the use of gene targeted mice and small molecule drugs we showed that inhibition of cIAP function promotes elimination of infected cells without causing collateral tissue damage. In the case of HBV infection, these interventions resulted in clearance of infection.
Cell death Cell done to death
And postdoc to despair
50. INVESTIGATING HOW THE EPSTEIN-BARR VIRUS-ENCODED VBCL-2 HOMOLOGUE BHRF1 FUNCTIONS TO PROTECT LYMPHOMA CELLS FROM APOPTOSIS
L Fitzsimmons1, R Cartlidge1, L Galbraith2,3, R Tierney1, C Shannon-Lowe1, M Takiguchi2,3, A Bell1, A Rickinson1, D Huang2,3, A Strasser2,3, M Kvansakul4, M Rowe1 and G Kelly2,3*
1 School of Cancer Sciences, The University of Birmingham, UK 2 The Walter and Eliza Hall Institute, Royal Parade, Parkvil le 3 Department of Medical Biology, The University of Melbourne. 4 Department of Biochemistry, La Trobe University, Melbourne.
Viruses have evolved multiple strategies to prevent host cell death in order to ensure efficient virus production. Some viruses have evolved to encode homologues of the cellular anti-apoptotic BCL-2 family of proteins that inhibit the intrinsic apoptotic pathway, termed viral (v)BCL-2 proteins. The oncogenic Epstein-Barr Virus (EBV) is one such virus and encodes two vBCL-2s, called BHRF1 and BALF1. Until recently it was thought that vBCL-2 proteins were expressed exclusively during viral replication but we now know that BHRF1 can be detected in ~15% of endemic Burkitt lymphomas (BL) and that BHRF1 and BALF1 are required for B cell transformation. This raises the possibility that BHRF1 could be a therapeutic target for the treatment of aggressive BL. We aim to: 1) investigate the role of BHRF1 in lymphoma development, growth and sensitivity to anti-cancer drugs and 2) identity the critical interactions of BHRF1 with cellular BCL-2 family proteins required for inhibition of apoptosis. Interestingly, we have found that BHRF1 expression in early pre-malignant cells can accelerate MYC-driven lymphoma development. Furthermore, we have used a site-directed mutagenesis approach to create a panel of BHRF1 mutant proteins that have reduced binding affinity for select cellular BCL-2 family proteins. By testing the ability of these BHRF1 mutants to protect lymphoma cells from death stimuli we have identified key residues within the protein that are critical for its function. These findings advance our understanding of the requirements for apoptosis inhibition by oncogenic viruses during tumorigenesis and also inform the design of BH3-mimetic drugs that could target BHRF1 for the treatment of aggressive BL.
51. STRUCTURAL INSIGHT INTO VACCINIA VIRUS MEDIATED INHIBITION OF APOPTOSIS
Marc Kvansakul*
La Trobe University
Apoptosis is a tightly regulated process that plays a crucial role in the removal of virally infected cells; a process controlled by both pro- and anti-apoptotic members of the Bcl-2 family. The pro-apoptotic proteins Bak and Bax are regulated by anti-apoptotic Bcl-2 proteins and are also activated by a subset of proteins known as BH3-only proteins, which contain dual functions by directly activating Bak and Bax, or by sequestering and neutralizing anti-apoptotic members. Numerous viruses express proteins that prevent premature host cell apoptosis. Vaccinia virus encodes F1L, an anti-apoptotic protein essential for survival of infected cells that bears no discernible sequence homology to mammalian cell death inhibitors. Despite the limited sequence similarities, F1L has been shown to adopt a novel dimeric Bcl-2-like fold, which enables hetero-oligomeric binding to both Bak and the pro-apoptotic BH3-only protein Bim that ultimately prevents Bak and Bax homo-oligomerization. However, no structural data on the mode of engagement between F1L and its Bcl-2 counterparts is presently available. Here we solved the crystal structures of F1L in complex with two ligands, Bim and Bak. Our structures indicate that F1L can engage two BH3 ligands simultaneously via the canonical Bcl-2 ligand binding grooves. Furthermore, with the use of structure guided mutagenesis, we generated point mutants within the binding pocket of F1L in order to elucidate residues responsible for both Bim and Bak binding and prevention of apoptosis. We propose that the sequestration of Bim by F1L is primarily responsible for preventing apoptosis during vaccinia virus infection.
Session 7: Cell Death and the Clinic I 53. GENETIC DISSECTION OF FAMILIAL INFLAMMATORY DISORDERS
Koji Yasutomo
Department of Immunology, Graduate School of Medicine, Tokushima University
Inflammatory disorders are caused by interplay between genetic and environmental factors. The genetic studies of familial diseases caused by single gene mutation could provide crucial information about molecular basis of human disorders. In that sense, we have tried to identify the causative genes for familial autoinflammtory disorders. We have identified NLRC4 as a causative gene for familial cold-induced autoinflammtory disorders. The mutation in NLRC4 facilitated the oligomer formation of NLRC4, which increased IL-1ß production and pyroptosis. The transgenic mice in which mutant Nlrc4 is expressed under invariant chain promoter developed severe inflammatory responses. The inflammatory response were suppressed by blockade of IL-1ß. However the blockade of IL-1ß could not completely suppress the inflammation, suggesting the involvement of other mechanisms for exerting inflammation. We also identified a causative gene for familial lung fibrosis. I would like to discuss the molecular mechanism of lung fibrosis by focusing on cell death in this symposium.
54. THE EMERGING ROLE OF BH3 MIMETICS IN HEMATOLOGICAL MALIGNANCY
MaryAnn Anderson
Cancer & Haematology Division, The Walter & Eliza Hall Institute of Medical Research
The first extensively validated BH3 mimetic ABT-737 demonstrated promising preclinical efficacy in a range of B cell malignancies, including chronic lymphocytic leukemia (CLL), by targeting BCL2, BCLxL and BCLw. This agent was not suitable for clinical development but its orally available analogue navitoclax (ABT-263) demonstrated 35% overall response rates (ORR) in relapsed and refractory (r/r) CLL. The true potential of this agent was not realized as dose escalation was limited by on target BCLxL mediated thrombocytopenia. Since 2011 we have been conducting various clinical trials with a BCL2 selective agent, venetoclax (ABT-199). As a single agent in r/r CLL venetoclax results in an ORR of 77% including a 23% complete response rate even amongst patients with high risk deletion 17p and fludarabine refractory disease. As of April 2014 the median progression free survival was 18 months. With appropriate safety precautions the drug can be administered safely despite initial concerns related to early tumor lysis syndrome.
Apoptotic pore Omnipresent, yet absent
Structure elus ive
My love of cell death Drove me to DRONC and DEBCL
And now I am as confused as death by autophagy
55. STUDY ON THE MOLECULAR MECHANISMS LINKING BETWEEN HEPATIC CELL DEATH AND FIBROSIS
Tomoki Yagai, Atsushi Miyajima and Minoru Tanaka*
National Center for Global Health and Medicine
Background and Aims: In the liver, chronic injury accompanied by continuous hepatic cell death will often lead to liver fibrosis. Patients with advanced liver fibrosis have poor prognosis because of impaired liver functions and risk for carcinogenesis. Sustained activation of hepatic stellate cell (HSC) and liver sinusoidal endothelial cell (LSEC), which constitute blood capillary, is known to cause liver fibrosis during chronic injury. Although many molecules are currently supposed to be involved in liver fibrogenesis, effective therapy for liver fibrosis has not been developed except liver transplantation. Our aim is to clarify the molecular mechanisms linking between hepatic cell death and fibrosis from the point of view of cell-cell communication.
Results: By the cDNA microarray analysis between normal and chronically injured mouse livers, we found that Semaphorin 3E (Sema3e) was upregulated in injured liver. Interestingly, Sema3e expression was induced in dying hepatocytes. In the primary culture of LSEC, Sema3e induced the contraction of LSEC morphology in vitro. Furthermore, consecutive expression of exogenous Sema3e cDNA in liver by the Hydrodynamic Tail Vein injection method caused the contraction of LSEC with disorganized blood capillary, resulting in the activation of HSC in vivo. In contrast, Sema3e-knockout mice showed the amelioration of liver fibrosis compared to wild-type mice in a chronic liver injury model.
Conclusion: Our results demonstrate that Sema3e secreted from damaged hepatocytes primarily affects LSEC in a paracrine manner, which leads to liver fibrosis by activating HSC, suggesting that Sema3e is a novel therapeutic target for liver fibrosis. Reference: Yagai T, Miyajima A and Tanaka M. Am J Pathol.184: 2250-9 (2014).
56. TARGETING THE EPIGENOME TO INDUCE CANCER CELL DEATH AND DIFFERENTIATION
Ricky W Johnstone
Peter MacCallum Cancer Centre
Altered expression, function or localisation of epigenetic enzymes and/or their partner proteins can play a crucial role in cancer onset and progression. Histone writers and erasers that regulate histone acetylation or methylation, or histone readers that recognise specific histone marks, play important roles in tightly regulating gene expression through the remodeling of chromatin and these proteins are promising targets for therapeutic interventions intended to reverse aberrant epigenetic states associated with cancer. I will outline our current understanding of altered epigenetic regulation in cancer onset and progression, the development of small molecule inhibitors of epigenetic enzymes and/or key partner proteins and the molecular, biological and clinical consequences of inhibiting these proteins.
It 's windy but bright She adds it …we're al l warm now
It's dark. I 'm floating
Session 8: Cell Death and the Clinic II 57. LOST IN TRANSLATION: CONVERTING CUTTING EDGE RESEARCH INTO THE THERAPIES OF TOMORROW
Andrew F Wilks1,2,3*, Guillaume Lessene3, James Murphy3, Christopher Burns3 Jean-Marc Garnier3, Pooja Sharma3, Peter Czabotar3, Joanne Hildebrand3, Maria Tanzer3, John Silke3, Isabelle Lucet3, Pat Sharp3, John Feutrill2, Anthony Cuzzupe2
1. Catalyst Tx, 2. SYNthesis Research, and 3. The Walter and Eliza Hall Institute
If we, as basic scientists, are not translating (or at least helping to facilitate the translation of) our research for the greater good, we are doing ourselves, and those who fund our research, a disservice. Whilst the preceding statement may (intentionally) seem rather provocative, it is not hard to justify. Nonetheless, it remains exceedingly challenging to fulfil that obligation; funding sources for early stage discovery research are hard to find, and the dispiritingly high attrition rate of successful translation of basic research means that only the brave seek to venture there. Catalyst Therapeutics was established as a joint venture between the Walter and Eliza Hall Institute and SYNthesis Research, in order to try to bridge the translation gap for promising, but therapeutically unproven, research discoveries emerging from the WEHI research labs. This presentation will focus on the challenges (and opportunities) of undertaking early-stage translational drug development at the cutting edge of fundamental research. We will focus on our Necroptosis Programme, as an example of a promising yet challenging opportunity that Catalyst Tx took on, and discuss the prospect of a new class of therapeutics emerging that targets a hitherto unproven opportunity for the treatment of inflammatory diseases.
58. INCREASED CYTOTOXIC LYMPHOCYTE SYNAPSE DWELL TIME CAUSES CYTOKINE STORM
Misty R Jenkins1,2*, Jesse Rudd-Schmidt1,2, Laura Kilpatrick1, Jamie A Lopez1, Stuart I Mannering4,5, Ilia Voskoboinik1,2, Joseph A Trapani1,2
1Peter MacCallum Cancer Centre, East Melbourne 2 The Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkvil le 3 Department of Genetics, The University of Melbourne, Parkvil le 4 Immunology and Diabetes Unit, St Vincent’s Institute of Medical Research, Fitzroy 5 Department of Medicine, University of Melbourne, St Vincent’s Hospital, Fitzroy
Failure of cytotoxic T lymphocytes (CTL) or natural killer cells (NK) to kill target cells by perforin/granzyme-induced apoptosis causes severe immune dysregulation. Infants deficient in perforin suffer a fatal ‘cytokine storm’ resulting from macrophage over-activation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTL/NK cells and that interferon-g directly invokes the activation and secondary over-production of pro-inflammatory IL-6 from naïve macrophages. Further, using live cell microscopy to visualise hundreds of synapses formed between WT, perforin-null or granzyme A/B-null CTL/NK and their targets in real time, we show that hyper-secretion of cytokine and chemokines is linked to failed disengagement of perforin- or granzyme-deficient lymphocytes from their targets, with mean synapse time increased five-fold. The signal for detachment arose from the dying target cell and was caspase-dependent, as delaying target cell death with various forms of caspase-blockade also prevented their disengagement from fully competent CTL/NK cells and caused cytokine hyper-secretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells. In recent data, we will provide evidence which will shed light on the mechanisms of synapse detachment.
59. EVADING THE STORM: RESISTANCE TO TARGETED EPIGENETIC THERAPIES AND THE LEUKAEMIA STEM CELL
Chun Yew Fong1,2, Mark A. Dawson1,2,3
1Cancer Epigenetics Laboratory, Peter MacCallum Cancer Centre, , 2Sir Peter
MacCallum Department of Oncology, 3Department of Haematology, Peter
MacCallum Cancer Centre
Bromodomain and Extra Terminal protein (BET) inhibitors are targeted therapies that deliver a new therapeutic paradigm by directly targeting epigenetic readers. Early clinical trials have shown significant promise especially in acute myeloid leukaemia (AML); therefore the evaluation of resistance mechanisms, an inevitable consequence of cancer therapies, is of utmost importance to optimise the efficacy of these drugs. Using primary murine stem and progenitor cells immortalised with MLL-AF9, we have generated 20 cell lines derived from single cell clones demonstrating stable resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through altered drug efflux or metabolism but emerges from leukaemia stem cells (LSC). Resistant clones display a leukaemic granulocyte-macrophage progenitor (L-GMP) transcriptome and immunophenotype (Lin-, Sca-, cKit+, CD34+, FcgRII/RIII+), functionally exhibit increased clonogenic capacity in vitro and markedly shorter leukaemia latency in vivo. Chromatin bound BRD4 is globally reduced in resistant cells, however expression of key oncogenes such as MYC remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors is in part a consequence of increased Wnt/B-catenin signaling. Negative regulation of this pathway results in differentiation of resistant cells into mature leukaemic blasts, inhibition of MYC expression and restoration of sensitivity to I-BET in vitro and in vivo. Finally, we show that the sensitivity of primary human AML cells to I-BET correlates with the baseline expression of Wnt/ß-catenin target genes. Together these findings highlight the potential therapeutic limitations of BET inhibitors and identifies strategies that may overcome resistance and enhance the clinical utility of these unique targeted therapies. Furthermore, our ability to sustain a highly enriched population of LSC in culture indefinitely provides a unique resource to interrogate and molecularly characterise LSCs thereby enabling screening of a range of therapies in vitro and in vivo against this previously elusive population and provides novel insights into the biology of AML.
Plenary Session 60. KILLING CANCER CELLS – DOES UNDERSTANDING THE ENEMY HELP?
Clare Scott
Stem Cells & Cancer Division, The Walter & Eliza Hall Institute of Medical Research
Effective cancer therapy requires efficient cancer cell death, such that relapse of drug-resistant cells does not occur. The aim should be long-term cancer control or cure, including by harnessing the immune system. Unfortunately, all too often, cancer cells evolve to evade whatever pressure they are placed under. By understanding the drivers and susceptibilities of each individual cancer, we may deliver more efficient therapy, essential for maximal cell killing.
Treatment for epithelial ovarian cancer revolves around a “one size fits all” approach with the same chemotherapeutics today as were used more than a decade ago. Yet the identification of distinct molecular subsets of ovarian cancer and clonal evolution occurring with successive rounds of therapy suggests that matching treatment to the cancer molecular phenotype may be important and more effective. In vivo pre-clinical models, such as well-annotated patient-derived xenografts which enable us to drive drug resistance, facilitate exploration of novel targeted therapeutic approaches with direct relevance for patients in the clinic.
Poster Abstracts
A
DISSOCIATION OF BAK Α1 FROM THE CORE AND LATCH DOMAINS IS REQUIRED FOR APOPTOSIS
Amber E Alsop1,2,*, Stephanie C Fennell1, Ray C Bartolo1, Iris KLTan1, Grant Dewson1,2 and Ruth M Kluck1,2
1 The Walter and Eliza Hall Institute of Medical Research, Parkvil le, Victoria, Austral ia 2 Department of Medical Biology, University of Melbourne, Parkvil le, Victoria, Austral ia
During apoptosis Bak permeabilizes mitochondria after undergoing major conformational changes, including BH3 domain exposure and poorly-defined N-terminal changes. To characterize those changes, and their timing, eleven antibodies were epitope mapped using peptide arrays and mutagenesis. Multiple epitopes map to regions of the N-terminus that are buried in inactive Bak, including the BH4 domain in the center of α1. Mutations in the BH4 domain destabilized Bak, demonstrating its role in maintaining Bak in a correctly-folded inert state. After Bak activation by Bid, all epitopes in the α1 helix are exposed indicating dissociation of α1 from α2 in the core and from α6-α8 in the latch. Disulfide tethering of α1 to α2 or α6 blocks cytochrome c release, suggesting that α1 dissociation is required for further conformational changes during apoptosis. The sequence of conformational events was determined using FACS and proteolysis assays performed when α1 or the core and latch were tethered. These showed that Bid triggers α2 movement, followed by α1 dissociation. Furthermore, Blue Native PAGE demonstrated that dissociation of α1 from α2 is essential for BH3:groove dimer formation. Note, however, that α1 dissociation from the α6-α8 latch is also necessary for α2 to reach the position required for dimerization. Thus, α1 dissociation is a key step in unfolding Bak into three major components, the N-terminus, the core (α2-α5), and the latch (α6-α8).
MLKL PROMOTES EMBRYONIC LETHALITY CAUSED BY LOSS OF CASPASE-8 OR FADD AND THEIR COMBINED ABSENCE CAUSES FATAL LYMPHADENOPATHY AND AUTOIMMUNE DISEASE
Silvia Alvarez-Diaz1, 2,*, Christopher P Dillon3, Najoua Lalaoui1, 2, Maria C Tanzer1, 2, Diego A Rodriguez3, Ann Lin1, Marion Lebois1, Emma C Josefsson1, 2, Lorraine A O’Reilly1, 2, John Silke1, 2, Warren S Alexander1,
2, Douglas R Green3, Andreas Strasser1, 2
1The Walter and Eliza Hall Institute of Medical Research, Parkvil le, Victoria 3052, Austral ia 2Department of Medical Biology, University of Melbourne, Parkvil le, Victoria 3050, Austral ia 3Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
Necroptosis has emerged as a programmed cell death process. RIPK1, RIPK3 and MLKL have been identified as key regulators of this signaling pathway although a role for MLKL within the whole animal has not yet been established. Here, we show that loss of MLKL rescues the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8-/-Mlkl-/- and Fadd-/-
Mlkl-/- double mutant mice are viable and fertile but they rapidly develop severe lymphadenopathy, systemic autoimmune disease and thrombocytopenia. These morbidities occur more rapidly and with increased severity in Casp8-/-Mlkl-/- and Fadd-/-Mlkl-/- mice compared to Casp8-/-Ripk3-/- or Fadd-/-Ripk3-/-mice. These results demonstrate that MLKL is essential for necroptosis within the whole animal, most likely functioning in the effector phase of this process. Furthermore, both RIPK3 and MLKL appear to exert functions independently of necroptosis that inhibit or promote lymphadenopathy and autoimmune disease, respectively, when Caspase-8 or FADD is absent.
STRUCTURAL AND FUNCTIONAL STUDIES OF VIRAL PROTEINS E1B19K AND FPV039
Mohd Ishtiaq Anasir*, Mike Ryan, and Marc Kvansakul
La Trobe University
Background: Apoptosis is one of the host defense mechanisms to fight viral infections. In order to survive host defence mechanism, many viruses encode anti-apoptotic proteins that can prevent the clearing of infected cells through apoptosis. We are interested in E1B19K and FPV039, which are anti-apoptotic proteins, expressed by adenovirus and fowlpox virus respectively.
Aims: Our aim is to express and purify these proteins to high yield and purity for structural studies using X-ray Crystallography and Small Angle X-Ray Scattering. Besides that, we are also interested to identify the interactions of these proteins with pro-apoptotic proteins such as Bak and Bax using Isothermal Titration Calorimetry technique.
Results: We are able to purify FPV039 to high yield and purity using two-step purification system consisting of GST-affinity chromatography and size exclusion chromatography. We also identified pro-apoptotic proteins that bind to FPV039. FPV039 binds to pro-apoptotic Bak and all the pro-apoptotic BH3-only proteins. This suggests that FPV039 able to directly inhibit Bak-mediated apoptosis and indirectly inhibit Bak and Bax-mediated apoptosis by inhibiting BH3-only proteins, which are the activators of Bak and Bax.
Conclusion: In conclusion, we are able to purify FPV039 to high yield and purity and identify binding partners of FPV039.
INHIBITOR OF APOPTOSIS PROTEINS (IAPS) LIMIT INFLAMMATION IN THE SKIN
Holly Anderton, Najoua Lalaoui, James Rickard, John Silke
Cell Signall ing & Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Parkvil le, Austral ia
Inhibitor of APoptosis proteins (IAPs) are known to play a crucial role in innate immunity through regulation of apoptosis, necroptosis and survival from the TNF super family but also through Toll Like Receptors (TLR). It has been shown that loss of IAPs, whether genetic or induced by IAP antagonist (smac-mimetic) drugs, sensitises cells to cell death through TNFR1 signalling. Given their crucial role in regulating inflammation through TNF and TLR signalling, we were interested in understanding the role of cellular IAPs (cIAPs) in skin development and homeostasis. We found that genetic deletion of cIAP1 in the skin and ubiquitous cIAP2 deletion is lethal due to skin inflammation by post partum day 9. To investigate the molecular mechanisms involved in the lethality induced by loss of cIAPs, we injected smac-mimetic subcutaneously into mice knock-out for proteins that regulate apoptosis, necroptosis and TLR signalling. We found that loss of some proteins involved in those pathways ameliorated the inflammation caused by injection of smac-mimetic. We focused on the effect of RIPK1 on the inflammation induced by loss of cIAPs. Remarkably, genetic deletion of just one allele of RIPK1 extends the lifespan of the cIAP1/2 skin knock-out mice significantly. Our work demonstrates the complex role of IAPs in regulating skin inflammation through multiple signalling pathways.
The meaning of death Life in the balance, then you
Try the salmon mousse
IMPACT OF OVEREXPRESSION OF PRO-SURVIVAL PROTEINS ON THE DEVELOPMENT AND TREATMENT OF MLL-AF9 ACUTE MYELOID LEUKAEMIA
Natasha Anstee*, Rebecca Bilardi, Stefan Glaser, Cassandra Vandenberg, Suzanne Cory
1The Walter and Eliza Hall Institute of Medical Research, Parkvil le, Austral ia *[email protected]
Despite considerable progress in delineating the molecular lesions causing acute myeloid leukaemia (AML), better treatment is urgently needed for this devastating disease. Chromosome translocations involving the mixed lineage leukaemia (MLL) gene account for 5-6% of paediatric and adult AMLs, the most common being the MLL-AF9 fusion.
Deregulation of apoptosis is a critical step in tumour development and mediates resistance to therapy. The Bcl-2 family of proteins regulates one of the key pathways of apoptosis. High expression of the pro-survival members Bcl-2 and Mcl-1 in AML correlates with poor prognosis and drug resistance.
We are using a preclinical mouse model to study the impact of these pro-survival proteins on the development and treatment of MLL-AF9 induced AML. Haemopoietic stem cells from vavP-BCL-2 or vavP-mcl-1 transgenic mice were infected with an MLL-AF9 retrovirus and transplanted into lethally-irradiated mice. Overexpression of Mcl-1 or BCL-2 increased the leukaemic burden in the spleen and blood of sick mice although it did not accelerate morbidity. Furthermore, AMLs overexpressing Mcl-1 or BCL-2 tended to have a high proportion of mature cells and a reduced capacity for transplantation compared to ‘wild type’ MLL-AF9 leukaemias.
Our cohort of murine MLL-AF9-induced AMLs represents a valuable resource for pre-clinical testing of new therapeutic regimens, including for AMLs that overexpress Mcl-1 or BCL-2. In vitro treatment experiments have shown, as expected, that overexpression of Mcl-1 and Bcl-2 increases resistance to standard therapeutics. However, promising results have been identified with several recently developed drugs, the most promising of which will now be tested in vivo.
BEADED-APOPTOPODIA, A NOVEL REGULATOR OF APOPTOTIC CELL DISASSEMBLY
Georgia K. Atkin-Smith*, Rochelle Tixeira, Stephanie Paone, Suresh Mathivanan, Christine Collins, Michael Liem, Katharine J. Goodall, Kodi S. Ravichandran, Mark D. Hulett & Ivan K.H Poon
La Trobe Institute for Molecular Sciences *[email protected]
Efficient disassembly and clearance of dying cells is important to avoid unwanted accumulation of cellular debris, and thus the release of harmful intracellular contents. Apoptotic cell disassembly is characterised by the formation of smaller fragments (a form of extracellular vesicle called apoptotic bodies) which can facilitate rapid removal of apoptotic debris and intercellular communication. However, the mechanism underpinning this process is unclear and therefore we aimed to further elucidate the molecular mechanism of apoptotic cell disassembly and apoptotic body formation. To achieve this we conducted a series of cell biological and proteomic analysis.
While observing monocytes undergoing apoptosis by time-lapse microscopy, we discovered a new type of membrane protrusion that resembles a ‘beads-on-a-string’ structure, termed ‘beaded-apoptopodia’. Interestingly, the ‘beads’ are frequently sheared off the ‘string’ to form apoptotic bodies and did not contain nuclear contents. Furthermore, in an unbiased drug screen, we identified the ability of sertraline (a commonly used antidepressant) and monensin (a common inhibitor of vesicle trafficking) to block the formation of beaded-apoptopodia, therefore giving insights into the molecular mechanism responsible.
The generation of these ‘beaded-apoptopodia’ demonstrates for the first time a novel mechanism for both the formation and release of apoptotic bodies. Additionally, the generation of apoptotic bodies via this mechanism can facilitate a selective sorting process of cellular contents and may be regulated by a combination of cellular process.
Overall, these data uncover a new, regulated mechanism of apoptotic body formation and compounds that can modulate this process.
INVESTIGATING THE ROLE OF MUTANT P53 IN THE DEVELOPMENT, GROWTH AND CHEMOSENSITIVITY OF LYMPHOMA
Brandon Aubrey1,2 Lin Tai1, Marco Herold 1,2, Andreas Strasser,1,2 Gemma Kelly1,2
1Molecular Genetics of Cancer Division, Walter and Eliza Hall Institute of Medical Research, Parkvil le, 2Department of Medical Biology, University of Melbourne, Parkvil le
Deregulated c-MYC expression and p53 mutations are among the most common genetic abnormalities detected in human cancer. Mutant p53 confers a poor prognosis through both loss of wild-type p53 activity as well as various proposed gain-of-function properties. We have previously shown that MYC-driven lymphomas are exquisitely dependent on the anti-apoptotic BCL-2 protein MCL-1 for their sustained survival and growth. This dependency is reduced, but not completely ablated, by p53 mutation. We hypothesize that mutant p53 confers upon lymphoma cells a survival advantage through still poorly understood gain-of-function activities. We have investigated the effects of five different mutant mouse p53 proteins (V170M, I192S, G280, R246Q, R270H; corresponding to mutations frequently found in human cancers) on lymphoma growth and development. The mutant p53 proteins exhibit distinct and context-specific effects during tumorigenesis. The R246Q mutant p53 protein was most potent in accelerating lymphoma development in the context of MYC over-expression. Strikingly, over-expression of mutant p53 in Eμ-Myc lymphoma-derived cell lines containing wild-type p53 impaired induction of apoptosis by Nutlin-3a, an inhibitor of the E3 ligase Mdm-2, the critical negative regulator of p53, although the p53 apoptosis effectors, Puma and Noxa, were still potently induced. Whole transcriptome RNA sequencing was performed to evaluate the extent of dominant negative effect exerted by the mutant p53 protein during wild-type p53 activation and to identify novel mutant p53 transcriptional targets. In summary, our findings suggest a gain-of-function oncogenic role of mutant p53 in haematopoietic cells that provides a particularly potent selective advantage in the context of MYC over-expression. Different mutant p53 proteins exhibit distinct functional properties, suggesting that different p53 mutations are likely to be associated with distinct risks in human neoplastic disease.
B
NLRP3 INFLAMMASOME ACTIVATION DOWNSTREAM OF CYTOPLASMIC LPS RECOGNITION BY BOTH CASPASE-4 AND CASPASE-5
Paul J Baker1,8,*, Dave Boucher4,8, Damien Bierschenk4, Christina Tebartz5, Paul G Whitney5, Damian B D’Silva1, Maria C Tanzer1, Mercedes Monteleone4, Avril AB Robertson4, Matthew A Cooper4, Silvia Alvarez-Diaz1, Marco J Herold1, Sammy Bedoui1, Kate Schroder4,9, Seth L Masters1,9
1The Walter and Eliza Hall Institute of Medical Research 4Cell Biology and Molecular Medicine division, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072 5The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Austral ia 8,9These authors contributed equally. *[email protected]
Caspase-4 and caspase-5 are human-specific inflammatory proteases that detect cytoplasmic lipopolysaccharide (LPS), and are thus comparable to the now well-described, murine-specific caspase-11. When activated, these trigger pyroptotic cell death and caspase-1-dependent IL-1β production. We have shown that a specific NLRP3 inhibitor, MCC950, prevents caspase-4/5-dependent IL-1β production elicited by intracellular LPS. Given that caspase-4 and caspase-5 can both detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, using CRISPR-based technology, we generated human monocytic cell lines in which caspase-4 and caspase-5 were genetically deleted either individually or together. We found that the deletion of caspase-4, but not caspase-5 suppressed cell death and IL-1β production upon transfection of LPS into the monocyte cytoplasm, whereas deletion of either caspase inhibited both of these phenotypes following infection of cells with Salmonella Typhimurium. Furthermore, double deletion of caspase-4 and -5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL-1β maturation, downstream of cytoplasmic LPS detection by caspase-4/5. We also show that both caspase-4 and caspase-5 are functionally important for human monocytes to respond to an invasive, gram-negative bacterial pathogen.
THE MOLECULAR BASIS OF IMPROVED HAEMATOPOIESIS IN PATIENTS WITH MYELODYPLASTIC SYNDROME TREATED WITH IRON CHELATION THERAPY
Ashish Banerjee, Vanessa Orlowsky, Shahla Vilcassim, Tanu Shree Arora, Nicole Mifsud, Abhilasha Tiwari, Graham Jenkin, Paul Ekert, Norbert Gattermann, Stephen Opat and George Grigoriadis
Hudson Institute of Medical Research, Clayton, Victoria, Austral ia
The myelodysplastic syndromes (MDS) are a group of clonal haematopoietic stem cell diseases characterised by cytopenia(s), dysplasia in one or more myeloid cell lineages, ineffective haematopoiesis and an increased risk of developing acute myeloid leukaemia. In low-to-intermediate risk MDS the bone marrow is composed of a mixture of defective and normal haematopoietic stem and progenitor cells. It has been postulated that the presence of cytokines such as IL-17, TNFα and IFNγ in the bone marrow environment of MDS patients is pro-inflammatory that in turn suppresses haematopoiesis from normal stem cells whilst promoting the survival of abnormal clones culminating in the evolution of the disease. MDS patients exhibit a varying degree and number of cytopenias that often require blood transfusions. Some studies have suggested an adverse impact of transfusion dependence and iron overload on survival in low-to-intermediate risk MDS necessitating iron chelation therapy (ICT).
We have observed improvement in haematopoietic parameters in a proportion of patients with low-to-intermediate risk MDS treated with the oral iron chelator deferasirox (DFX). Our experimental results suggest that the haematopoietic response following treatment is likely to be a consequence of two distinct functions of DFX. Firstly, DFX inhibits NF-κB activity resulting in dampened inflammation in the bone marrow milieu. Secondly, iron chelation leads to mitochondrial dysfunction resulting in apoptosis of malignant clones whilst sparing normal haematopoietic stem cells in part via inhibition of autophagy. Our study provides for the first time a mechanistic basis for improved haematopoiesis in MDS patients treated with ICT.
USING A NOVEL ENZYMATIC TAGGING APPROACH TO IDENTIFY REGULATORS OF BAK
Jonathan Bernardini, Jarrod Sandow, Iris Tan and Grant Dewson
Cell Signall ing & Cell Death Division, The Walter & Eliza Hall Institute
Bak is a critical effector of intrinsic apoptotic cell death, responsible for permeabilising the mitochondrial outer membrane during apoptosis. The ‘fine tuning’ of Bak by other members of the Bcl-2 family acts as a molecular switch that commits the cell to death. We have shown that Bak and Bax reside in large multisubunit complexes in mitochondria that comprise non-Bcl-2 proteins including VDAC2. The components of these complexes represent novel regulators of Bak and Bax function. To identify novel putative regulators of apoptosis we established an enzymatic tagging approach to interrogate the interacting partners of the death effector Bak.
Proximity-induced biotinylation utilises a mutant version of the bifunctional ligase/repressor (BirA) from E. coli that is able to non-specifically conjugate proximal proteins within 10-20 nm with a biotin moiety. Biotinylation modifications are rare and extremely stable in mammalian cells, making them ideal for high purity enrichment for downstream analysis. We then analysed these samples by mass spectrometry to identify putative interacting partners of Bak in the presence or absence of an apoptotic stimulus. Our studies have revealed a number of candidate proteins that selectively interact with Bak in a healthy cell and are subsequently lost after induction of apoptosis. These proteins represent potential novel regulators of Bak and reveal a potential interaction between the apoptotic machinery and the machineries controlling mitochondrial dynamics and mitochondrial turnover. We intend to further validate these proteins with a series of biochemical assays coupled with CRISPR/Cas9 gene editing including assaying native complex formation and cell death in response to apoptotic stimuli. This proximity-dependent enzymatic tagging approach is a robust, sensitive system that can be adapted to interrogate a host of protein-protein interactions, including those such as membrane proteins or transient interactions that are difficult to examine by conventional immunoprecipitation.
NLRP3 INFLAMMASOME IS ACTIVATED DOWNSTREAM OF CYTOPLASMIC LPS RECOGNITION BY BOTH CASPASE-4 AND CASPASE-5
D Boucher1, PJ. Baker2,4, D. Bierschenk1, C. Tebartz3,4, PG. Whitney3,4, DB. D’Silva2,4, MC. Tanzer3,4, M. Monteleone1, AAB. Robertson1, MA. Cooper1, S. Alvarez-Diaz2,4, MJ. Herold2,4, S. Bedoui3,4, SL. Masters2,4 , K. Schroder1
1. Institute for Molecular Bioscience, The University of Queensland 2. The Walter and Eliza Hall Institute of Medical Research 3.The Peter Doherty Institute for Infection and Immunity, Melbourne 4.The University of Melbourne, Parkvil le, New South Wales, Austral ia
Objectives: In mice, the direct recognition of cytoplasmic lipopolysaccharide (LPS) by the inflammatory protease caspase-11 elicits pyroptosis and subsequent NLRP-3 dependent secretion of IL-1β. Human orthologs of caspase-11, the caspase-4 and 5, also directly recognise cytoplasmic LPS but the downstream signalisation leading to IL-1β secretion remain uncharacterised. This study aims to elucidate the downstream signaling of caspase-4 and 5 in response to intracellular gram-negative bacterium.
Methods: Using the nuclease Cas9, we engineered the human monocyte-like cell line THP-1 and deleted the inflammatory caspase-1, 4 or 5. The cell lines generated were used to assess the individual and combined implication of those proteases into pyroptosis and IL-1β secretion after challenge with intracellular LPS or Salmonella Typhimurium. Their contribution was also measured in primary macrophages using silencing RNA.
Results: We found that the deletion of caspase-4 protects against cell death and IL-1β production following transfection of LPS into the cytoplasm. Although deletion of caspase-5 did not confer protection against transfected LPS cell death, it reduces IL-1β production. Silencing RNA targeting caspase-4 and 5 also attenuate IL-1β production in HMDM whereas only caspase-4 silencing has an effect on cell death following infection. Using quantitative PCR, we also report that infection with Salmonella strongly induce caspase-5 mRNA expression in human derived primary macrophage (HMDM). Finally, inhibition of NLRP3 using its specific inhibitor MCC950 show a strong reduction of IL-1β release in response to cytoplasmic LPS, positioning this inflammasome downstream of caspase-4 and 5 signaling.
Conclusions: Our results identify the NLRP3 inflammasome as the specific platform for IL-1β maturation, downstream of cytoplasmic LPS detection by caspase-4/5, and show that both proteases are functionally important for appropriate responses to intracellular Gram-negative bacterium.
DEREGULATION OF TNF EXPRESSION CAUSES POLYARTHRITIS AND HEART VALVE DISEASE
Derek Lacey, Peter Hickey, Benedicta D. Arhatari, Lorraine O’Reilly, Leona Rohrbeck, Helen Kiriazis, Xiao-Jun Du, Philippe Bouillet
Molecular Genetics of Cancer Division, The Walter & Eliza Hall Institute of Medical Research
BPSM1 mice are the result of a spontaneous dominant mutation that leads to a severe symmetrical, erosive chronic poly-arthritis, reminiscent of human RA, which primarily affects the peripheral joints with a high load factor. On the BALB/c genetic background, most of the mutant mice die suddenly between 90 and 160 days of age from aortic root aneurysm that follows a dramatic inflammation, thickening and fibrosis of the aortic and mitral valves The disease is caused by the insertion of a small interspersed element (SINE) in the 3’ untranslated region (3’UTR) of TNF. This retrotransposon provides a new polyadenylation signal to TNF mRNA and removes the normal negative regulation imposed by the 3’UTR, causing a large accumulation of a shorter mRNA that still encodes the same TNF protein in myeloid cells.
The CCCH zinc finger-containing protein Zfp36 regulates TNF expression via its AU-rich elements (ARE). We have investigated the regulatory potential of 50 other CCCH-containing ZPF on the 3’UTR of TNF and identified 5 new regulators and a new regulatory element within TNF 3’UTR. All of these constitute new targets to try and manipulate TNF expression pharmacologically.
HEART DISEASE IN ARTHRITIS My heart is aflame!
Is this conflagration love Or necroptosis?
STRUCTURAL INSIGHTS INTO BAK ACTIVATION AND OLIGOMERISATION
Jason M Brouwer1,2*, Dana Westphal1,2, Grant Dewson1,2, Adeline Y Robin1,2, Rachael T Uren1,2, Ray Bartolo1, Geoff V Thompson1, Ahmad Z Wardak1, Ruth M Kluck1,2, Peter M Colman1,2 and Peter E Czabotar1,2
1Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkvil le, Victoria 3052, Austral ia 2Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3052, Austral ia *[email protected]
Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting in mitochondrial outer membrane permeabilisation and subsequent cell death. Crystal structures by Czabotar et al. (2013) provided novel insights into BH3-only induced Bax activation and oligomerisation, namely the separation of the core and latch domains, followed by core domain dimerisation. Here we aimed to provide complementary studies on the related protein Bak. We present the crystal structures of Bak core-latch domain swapped dimers and demonstrate their dissociation upon Bak activation. A second crystal structure of the Bak core domain provides the first high-resolution details for this key dimerisation unit upon which the larger Bak oligomer builds. Cellular assays, guided by the presented crystal structures, confirm the physiological relevance of these key events in the intrinsic apoptotic pathway Brouwer et al, 2014). These studies confirmed an analogous mechanism for activation and dimerisation of Bak and Bax in response to BH3-only peptides. More recently we have performed structural studies on the direct interaction of BH3-only peptides with Bak. We have gained insight into the differences between interactions of BH3-only proteins with Bak compared to the pro-survival proteins; this may inform the design of novel therapeutics to manipulate cell death.
References Brouwer JM et al (2014). Bak Core and Latch Domains Separate during Activation, and Freed Core Domains Form Symmetric Homodimers. Mol Cell 55: 938-946 Czabotar PE et al (2013). Structural Transitions Activating Bax for Apoptosis. Cell 152: 519-31.
NECROPTOSIS INDUCED BY SMAC-MIMETIC AND CASPASE INHIBITOR OVERCOMES RESISTANCE AND TREATS ACUTE MYELOID LEUKAEMIAS
G Brumatti, C Ma, N Lalaoui, N Nguyen, M Navarro, J Richmond, M Ghisi, JM Salmon, N Silke, SP Glaser, E de Valle, R Gugasyan, MA Gurthridge, SM Condon, RW Johnstone, R Lock, G Salvesen, A Wei,
DL Vaux, PG Ekert, J Silke
The Walter & Eliza Hall Institute of Medical Research
Resistance to chemotherapy is a major problem in cancer treatment and frequently associated with failure of tumor cells to undergo apoptosis. Novel therapies that induce alternative death pathways, such as RIPK3/MLKL dependent necroptosis, may be the new strategy to overcome chemoresistance. Due to limitations of the compounds used, necroptosis induction has not been, to date, investigated in vivo as a therapeutic strategy for cancer. Birinapant, a clinical Smac-mimetic, mimics the interaction between Inhibitor of APoptosis proteins (IAPs) and Smac/DIABLO, thereby relieving IAP mediated caspase inhibition and promoting apoptosis of cells. Using a range of mouse models of Acute Myeloid Leukemia (AML) we found that AMLs differ in their sensitivity to birinapant induced apoptosis and pre-treatment with a clinical caspase inhibitor augments, rather than prevents, birinapant killing. Encouragingly, from a clinical perspective, even AMLs with resistance to birinapant or to chemotherapies such as Cytarabine, were readily killed by the combination of birinapant plus caspase inhibitor (bir/C8i). Using genetic knockout mouse models, we showed that deletion of caspase-8 sensitised AML to birinapant, whereas combined loss of caspase-8 and MLKL or RIPK3 prevented bir/C8i induced death, showing that inhibition of caspase-8 sensitises AML cells to birinapant-induced necroptosis. Remarkably however, loss of MLKL alone did not prevent a slower caspase dependent bir/C8i induced death. Our data demonstrates the anti-leukemic efficacy and tolerance of the bir/C8i combination in vivo, validating the activation of necroptosis as a new therapeutic opportunity in AML.
C A179L, A PROMISCUOUS ANTI-APOPTOTIC PROTEIN FROM AFRICAN SWINE FEVER VIRUS
Sofia Caria*, Suresh Banjara, Mark Hinds, Marc Kvansakul
La Trobe Institute for Molecular Science, La Trobe University *[email protected]
Viruses from different families have been able to ensure their survival by hijacking their host’s programmed cell death or apoptosis signalling. The proteins that regulate apoptosis belong to the Bcl-2 protein family.
Several viral anti-apoptotic proteins have been characterised and their host cell partners identified. The host cell partners are the effector proteins, Bak and Bax, as well as BH3-only proteins. Interestingly, anti-apoptotic proteins tend to be selective for their partners, binding only some of the members of the family.
A179L is an African swine fever virus anti-apoptotic Bcl-2 protein homolog. In this project we aim to characterise A179L binders and determine crystal structures of their complexes. This enables us to shed light on the A179L mechanism of action.
Our ITC data shows that, unlike other anti-apoptotic Bcl-2 proteins, A179L is able to bind to all the tested BH3-only and effector peptides. This makes A179L the first pan pro-death Bcl-2 inhibitor identified to date. A179L crystal structures were determined in complex with Bax and Bid BH3 peptides with a resolution of 2.9 and 2.5 Å, respectively. Analysis of those structures gives insight into the structural characteristics of A179L that may explain its promiscuity
NEUTROPHILS RESIST INFLAMMASOME-DEPENDENT CELL DEATH AND RIPK3-DEPENDENT INFLAMMATION
Kaiwen W. Chen1, Dave Boucher1, Kate E. Lawlor2, Motti Gerlic3, Amanda C. Stanley1, Jelena S. Bezbradica1, Seth Masters2, Matthew J. Sweet1, Katryn J. Stacey4, James E. Vince2, Kate Schroder1
1Institute for Molecular Bioscience, The University of Queensland 2The Walter and Eliza Hall Institute of Medical Research 3Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 4School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia 4072, Austral ia.
Objectives: The NLRP3 inflammasome is a multi-protein signalling complex that assembles in the cytosol upon sensing microbial or endogenous danger signals, and is best characterised in macrophages. NLRP3 triggers oligomerisation of the adaptor ASC, which in turn enables the recruitment and activation of pro-caspase-1 and pro-caspase-8 in macrophages. Active caspase-1 drives the maturation and secretion of specific proinflammatory cytokines (e.g. interleukin (IL)-1β), and initiates pyroptosis, while caspase-8 triggers apoptosis. Recent studies demonstrate that chemical inhibition or genetic ablation of inhibitor of apoptosis proteins (IAPs), triggers various RIPK3-dependent cell death pathways and activates the NLRP3 inflammasome or caspase-1-independent IL-1β maturation in macrophages. We previously demonstrated that neutrophils resist pyroptosis downstream of NLRC4 activation. Here, we investigated whether neutrophils similarly avoided NLRP3 and RIPK3-dependent cell death and inflammation.
Methods: Purified neutrophils were stimulated with either the NLRP3 agonist, nigericin, or the smac-mimetic, Compound A, and quantified for NLRP3 and RIPK3-dependent outputs such as ASC oligomerisation, pyroptosis, apoptosis, necroptosis and cytokine secretion.
Results: The kinetics of ASC oligomerisation was delayed in neutrophils as compared to macrophages, leading to a corresponding delay in caspase-1 processing and IL-1β maturation. Surprisingly and unlike macrophages, chronic nigericin stimulation did not trigger caspase-1-dependent pyroptosis, caspase-8 cleavage or caspase-8-dependent apoptosis in neutrophils. Further, unlike in
macrophages, suppression of inhibitory IAP signals did not trigger NLRP3/caspase-1 activation or caspase-1-independent IL-1β cleavage in LPS-primed neutrophils.
Conclusion: We previously demonstrated that neutrophils resist NLRC4 and AIM2-dependent pyroptosis. Our data here indicate that neutrophils also resist all known forms of NLRP3-dependent cell death, suggesting that their avoidance of cell death pathways downstream of inflammasomes is an important and universal feature of neutrophils. Surprisingly, unlike in macrophages, RIPK3-dependent cell death did not drive inflammatory responses in neutrophils. Collectively, our data indicate that the interregulation of cell death and inflammatory pathways are uniquely specialised in neutrophils.
IDENTIFICATION OF SRC KINASE AND AKT AS MAJOR DETERMINANTS GOVERNING NEURONAL CELL SURVIVAL IN EXCITOTOXICITY BY SYSTEMS BIOLOGY AND CELL BIOLOGY APPROACHES
Ashfaque Hoque, M Iqbal Hossain, Carli L Roulston, Ching-Seng Ang, Heung-Chin Cheng*
Department of Biochemistry and Molecular Biology, Bio21 Institute, and Department of Medicine, St. Vincent’s Hospital, University of Melbourne, Parkvil le, Victoria 3010 *[email protected]
Background: Excitotoxicity resulting from over-stimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke and neurodegeneration. The over-stimulated glutamate receptors exert their neurotoxic effects in part by over-activation of calpains which cause neuronal death by catalysing limited proteolysis of specific cellular proteins. These cellular proteins in turn modulate the phosphorylation states of cellular proteins governing cell survival and cell death to induce neuronal death.
Aims: We use biochemical approaches and systems biology to identify key signalling proteins that mediate the neurotoxic signals emanating from the over-stimulated glutamate receptor and the over-activated calpains.
Results: We discovered that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpain near the N-terminus to generate a truncated Src fragment. A cell membrane-permeable fusion
peptide derived from sequence around the calpain cleavage site in Src effectively prevents calpain from cleaving Src in neurons and protects against neuronal death induced by glutamate over-stimulation. Furthermore, expression of recombinant Src fragment was sufficient to induce neuronal death. We then used systems biology approach to identify cellular proteins that operate in parallel and down-stream of the truncated Src fragment to induce neuronal death in excitotoxicity. This approach involves the use of quantitative stable isotope dimethyl labelling to explore the changes in phosphoproteome of neurons within 15 min following glutamate over-stimulation. Our results revealed that glutamate over-stimulation as early as 15 min induces significant changes in the phosphorylation states of specific cellular proteins governing signalling pathways controlling mitochondrial dysfunction, axonal guidance, cell survival and death. Examples of these phosphoproteins include Erk1/2 and protein substrates of the pro-survival kinase. Further biochemical studies revealed that the neurotoxic truncated Src fragment induces neuronal death in part by suppressing the activity of Akt.
Conclusions: Besides unveiling a new function of Src, our discoveries of the neurotoxic action of the truncated Src fragment and the phosphoproteins affected by glutamate over-stimulation suggest new avenues to decipher the mechanism of neuronal death in excitotoxicity.
References: 1. Hossain MI et al (2013) A Truncated Fragment of Src Protein Kinase Generated by Calpain-Mediated Cleavage is A Mediator of Neuronal Death in Excitotoxicity. J Biol Chem 288, 9696-9709 2. Hossain et al (2015) Dual role of Src kinase in governing neuronal survival. Brain Research, 1594, 1-14
Equipment broken Abandoned, wait ing for love
When wil l the day come?
STAPHYLOCOCCUS AUREUS PANTON-VALENTINE LEUKOCIDIN (PVL) TRIGGERS CASPASE 1-INDEPENDENT CELL DEATH IN MAMMALIAN MACROPHAGES
SH Chow1*, P Baker2, SL Masters2, JE Vince2, AAB Robertson3, MA Cooper3, K Gabriel1, T Naderer1
1Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Austral ia 2The Walter and Eliza Hall Institute of Medical Research 3Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Austral ia *[email protected]
Background: Methicillin-resistant Staphylococcus aureus (MRSA) are able to cause skin-infections but also life-threatening necrotizing pneumonia in otherwise healthy individuals. Nearly all of these community-acquired MRSA (CA-MRSA) strains express the secreted pore-forming leukotoxin Panton-Valentine leukocidin (PVL). PVL specifically kills macrophages triggering caspase-1 and IL-1β activation, which leads to inflammatory lung responses. However, how PVL kills macrophages remains unknown as mice and its immune cells remain resistant.
Aims: To characterise the cytotoxic activities of extracellular PVL on human THP-1 macrophages.
Results: We show that PVL is highly cytotoxic to human macrophages and that this depends on a functional PVL complex, as the subunits LukS-PV or LukF-PV show little affect. In addition we show PVL killing depends on C5a receptor expression, which is upregulated in human THP-1 monocytes to macrophage differentiation, correlating with killing. To gain a more dynamic view how PVL kills, we followed the interaction of PVL with macrophages using live-cell imaging. This shows that PVL causes mitochondrial damage prior to cell death. PVL toxicity can be blocked by inhibiting intracellular potassium efflux and cathepsin B activity in human THP-1 macrophages. However, while PVL triggers caspase-1 activation and subsequently induces inflammatory response in THP-1 macrophages, loss of caspase-1 does not prevent PVL-mediated cell death.
Conclusion: These observations suggest that PVL kill macrophages by activating host cell death factors. We are now using genetic approaches to delineate the host death signalling pathways and to determine their role in PVL positive MRSA infections.
CLEAVAGE AND SECRETION OF INTERLEUKIN-1β IN THE ABSENCE OF CELL DEATH
Stephanie Conos1*, James Vince1, David L. Vaux1, Lisa M. Lindqvist1
Cell Signall ing & Cell Death Division, The Walter & Eliza Hall Institute of Medical Research *[email protected]
Background: Interleukin-1β (IL-1β) activation by caspase-1 is required for host protection against microbial infection, but it can also drive pathology in auto-inflammatory diseases. Host, environmental, and pathogen derived danger molecules activate specific inflammasome sensor proteins. These trigger formation of a functional inflammasome that activates caspase-1, which processes IL-1β into its mature, biologically active, form. Active caspase-1 can also induce a lytic cell death known as pyroptosis, whether caspase-1 activation of IL-1β and pyroptosis can be functionally separated remains controversial.
Aims: The aims of this study are to study the functions of caspase-1 in the absence of inflammasome signalling and to determine whether caspase-1 induced cell death is necessary for the secretion of IL-1β.
Results: We created a lentiviral flag-tagged caspase-1-gyrase-GFP fusion construct (FCasp1GG). Expression of the caspase-1 fusion protein was induced by addition of doxycycline. The divalent antibiotic coumermycin was used to dimerise the gyrase domains, causing caspase-1 to auto-activate.
Mouse embryonic fibroblast (MEF) cell lines engineered to constitutively express inactive precursor IL-1β were stably infected with the inducible FC1GG construct. Upon caspase-1 induction and dimerization, the cells, which express no other inflammasome components, secreted cleaved IL-1β. Notably, caspase-1 dependent IL-1β cleavage and secretion occurred in the absence of caspase-1-mediated cell death.
Conclusions: We have shown that caspase-1 can cleave IL-1β and cause its secretion from cells in the absence of specialised upstream inflammasome machinery. We have also shown that IL-1β is actively secreted from cells as a result of caspase-1 activation, and that this can occur in the absence of cell death.
STRUCTURAL INVESTIGATION OF BAX OLIGOMERISATION
Angus D Cowan*,1,2, Peter M Colman1,2 and Peter E Czabotar1,2
1Structural Biology Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkvil le, Victoria 3052, Austral ia. 2Department of Medical Biology, University of Melbourne, Parkvil le, Victoria 3052, Austral ia. *[email protected]
The Bcl-2 protein family regulates the intrinsic apoptotic pathway and the critical step of mitochondrial outer membrane permeabilisation (MOMP). MOMP releases Cytochrome c and other molecules from the mitochondrial intermembrane space, leading to the formation of the apoptosome and caspase activation1. Bax and Bak are structurally and functionally homologous pro-apoptotic Bcl-2 proteins that facilitate MOMP1. BH3-only proteins bind to and activate Bax/Bak, inducing homodimerisation and oligomerisation2. Bax/Bak oligomers are able to disrupt the mitochondrial outer membrane resulting in MOMP. BH3-only protein binding and activation causes exposure of the Bax/Bak BH3 alpha helix and dissociation of Bax/Bak into two distinct domains known as the "core" and "latch" domains3,4. The structures of both homodimerised Bax and homodimerised Bak core domains, with a GFP fusion tag to aid crystallisation, have been solved3,4. The core domains dimerise through a symmetrical interface involving the reciprocal exchange of the BH3 alpha helix of one Bax/Bak molecule into the hydrophobic groove of the partner Bax/Bak molecule. To further investigate the Bax core domain, we have expressed it as a GST fusion protein and removed the GST tag. We have solved the structure of a core domain oligomer and will discuss its implications.
References [1] Youle RJ, Strasser A (2008). The BCL-2 protein family: opposing activities that
mediate cell death. Nat Rev Mol Cell Biol 9, 47-59. [2] Kuwana T et al (2005) BH3 Domains of BH3-Only Proteins Differentially
Regulate Bax-Mediated Mitochondrial Membrane Permeabilization Both Directly and Indirectly. Mol Cell 17, 525-535.
[3] Czabotar PE et al (2013) Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis. Cell 152, 519-531.
[4] Brouwer JM (2014) Bak Core and Latch Domains Separate during Activation, and Freed Core Domains Form Symmetric Homodimers. Mol Cell 55, 1-9.
HUMAN RHINOVIRUS 3C PROTEASE CLEAVES RIPK1, AN IMPORTANT INTERMEDIATE IN EXTRINSIC APOPTOSIS
Sarah Croft*, Erin Walker, Reena Ghildyal
Centre for Research in Therapeutic Solutions, University of Canberra *[email protected]
Background: Human Rhinovirus (HRV) is a human pathogen of significant medical importance, being a major cause of upper respiratory tract infections and causing the majority of the virus-induced asthma exacerbations. One cellular response to viral infection is the initiation of apoptosis, apoptotic signals is propagated via caspase cascades that lead to cell death, thereby reducing HRV viral replication which relies on cellular machinery.
Aims: In this study, we investigated whether HRV could modulate apoptosis, a key antiviral innate immune response, and induce a cellular environment conducive to viral replication. We investigated the HRV mediated cleavage of RIPK1, an extrinsic apoptosis adaptor protein and aimed to determine the effects of this cleavage on viral replication.
Results: We have used HRV16 infected cells, cells treated with chemical inducers and inhibitors of extrinsic apoptosis, and in vitro protease cleavage assays to show that HRV16 3C protease cleaves a key intermediate in extrinsic apoptosis. RIPK1was cleaved by caspase 8, as expected, during chemical induction of extrinsic apoptosis. RIPK1 was also cleaved in HRV infection albeit at a different cleavage site. Interestingly, caspase 8 activation, which is associated with extrinsic apoptosis, was required for optimal HRV 3C protease mediated cleavage of RIPK1. This was potentially achieved by increasing the accessibility of the HRV 3C cleavage site within RIPK1.
Conclusion: The caspase 8 mediated RIPK1 cleavage product has a pro-apoptotic function, and further cleavage of this pro-apoptotic product by HRV 3C may provide a mechanism by which HRV regulates apoptosis.
D
CAR T CELLS INFLICT SEQUENTIAL KILLING OF MULTIPLE TUMOUR TARGET CELLS
Alexander J Davenport1-3*, Misty R Jenkins 1,2, Ryan S Cross2,4, Carmen S Yong 1,2, David S Ritchie1-3,, Joseph A Trapani1,2, Michael H Kershaw1,2, Phillip K Darcy1,2§, Paul J Neeson1,2§
1Cancer Immunology Research, Peter MacCallum Cancer Center 2Sir Peter MacCallum Department of Oncology. 3The ACRF Translational Research Laboratory 4Differentiation and Transcription Laboratory, Peter MacCallum Cancer Center, East Melbourne.
Introduction: Adoptive therapy with chimeric antigen receptor (CAR) T cells has shown enormous promise clinically. However, important areas of CAR-T cell biology that have not been explored kinetics of: CAR T cell activation, immune synapse formation and tumor cell killing.
Method: We developed a novel transgenic mouse (CAR.OT-I), where CD8+ T cells co-expressed clonogenic T cell receptors detecting the ovalbumin peptide SIINFEKL, and a scFv specific for human HER2. Seven Day activated CAR.OT-I T cells were co-cultured with SIINFEKL-pulsed or HER2-expressing tumour cells and visualized in real time using time-lapse microscopy.
Results: Engagement via CAR or TCR did not affect cell death kinetics, except that the time from recognition to detachment was reduced when the CAR was engaged. We showed, for the first time, that a subset of individual CAR.OT-I cells can kill multiple tumour cells sequentially (‘serial killing’). At low E:T ratios, tumor cell killing rate was similar via TCR or CAR ligation over the first 24 hours of co-incubation. However, after 24 hours, tumour cell death mediated through CAR became attenuated. This was due to CAR down-regulation throughout the time-course.
Conclusion: Our study shows visual evidence (movies) that CAR T cells can serially kill multiple tumour targets in quick succession and provides important insights into CAR-T/tumour cell interactions, with implications for single- or dual-receptor-focused T cell therapy.
DECIPHERING THE ROLE OF CASPASE-2 IN PROTECTING AGAINST GENETIC INSTABILITY AND TUMORIGENESIS
Sawati Dawar, Yoon Lim, Joey Puccini, Loretta Dorstyn, Sharad Kumar
Centre for Cancer Biology, University of South Austral ia, Adelaide, SA 5001, Austral ia
Caspase-2, one of the most evolutionarily conserved of caspases, has been shown to be involved in apoptosis induced by various stimuli including cytoskeletal disruption and DNA damage. Our studies have shown that loss of caspase-2 causes extensive aneuploidy and enhances tumorigenesis in Eμ-Myc and Atm-/- mice. As such, caspase-2 deficient mice develop normally but show premature ageing traits and when challenged by certain stressors, succumb to enhanced tumor development accompanied by aneuploidy. However, the molecular mechanism(s) by which caspase-2 mediates these functions is currently unclear. This is partly due to a lack of known substrates that define its function in these pathways.
In order to define its role in preventing chromosomal instability, we have established an ex vivo system for aneuploidy where primary splenocytes from caspase-2 mice were exposed to anti mitotic drugs and followed up by live cell imaging. Our data shows that loss of caspase-2 leads to significantly enhanced aneuploidy and this is partly due to reduced death of cells that carry chromosomal defects. Acute knockdown of caspase-2 as oppose to chronic absence in the cells showed similar results. We propose that caspase-2 is required for apoptosis to delete aberrant cells in response to stress and this may explain its function in the suppression of tumorigenesis.
Bak and Bax foxtrot Caspase 8 taps Bak’s shoulder.
Asks “May I cut in?”
BIM IS CRITICAL FOR DNA DAMAGE-INDUCED APOPTOSIS TO PREVENT LYMPHOMA DEVELOPMENT AND MEDIATE THE KILLING OF CHEMORESISTANT (P53-DEIFICENT) TUMOURS
Alexis R Delbridge*, A Strasser
Molecular Genetics of Division, The Walter & Eliza Hall Institute of Medical Research *[email protected]
While a large body of knowledge has accumulated in relation to the p53-dependent DNA damage-induced apoptotic pathway, comparatively little is known about the p53-independent apoptotic pathways that can be activated by DNA damage. This is in spite of the fact that such pathways they are known to contribute significantly to the efficacy of anti-cancer therapy and they may also be important for tumour suppression to prevent the accumulation of tumour-promoting mutations in nascent tumour cells.
In order to address this we have investigated the role of the pro-apoptotic BH3-only proteins in the response of p53-deficient thymic lymphoma derived cell lines to DNA damage induced by γ-irradiation or chemotherapeutic drugs. Our findings identify Bim as a critical initiator of apoptosis induced by DNA damage inducing drugs in the absence of p53. Moreover this novel apoptotic pathway also appears to play a role in the suppression of lymphoma development following activation in response to aberrant levels of RAG activity. We suspect that Bim may play a crucial surveillance role in pre-leukaemic cells, acting to eliminate cells in response to dangerous levels of DNA damage.
Consistent with this hypothesis, our data show that the additional loss of Bim markedly accelerates lymphoma development in p53-/- and p53+/- mice, but in a strictly RAG1-dependent manner.
INVESTIGATING THE EARLY STEPS OF BAX ACTIVATION FOR APOPTOSIS: HOW DOES THE PROPOSED BAX ‘REAR’ SITE CONTRIBUTE?
Michael A Dengler*, Leonie Gibson, Jerry M Adams
Molecular Genetics of Division, The Walter & Eliza Hall Institute of Medical Research *[email protected]
Background: Bax is a crucial pro-apoptotic member of the apoptosis-regulating Bcl-2 family of proteins. Structural transitions convert it from an inert cytosolic monomer into a killer that oligomerises on the mitochondrial outer membrane (MOM) and perforates it. However, the initial trigger activating Bax in the cytosol remains uncertain. Certain apoptosis-initiating BH3-only proteins (Bid, Bim) can drive later steps by binding to a Bax surface groove resembling that in pro-survival relatives, but these ‘activators’ have been proposed to trigger Bax translocation to the MOM instead by engaging a novel Bax ‘rear’ site. That site is not well characterised, however, and how it contributes remains uncertain.
Aims: We want to determine whether binding of a BH3 peptide to the proposed Bax rear site is necessary or sufficient to induce Bax activation.
Results: As the affinity of BH3 peptides for the rear site may be very low, we attempted to cross-link BH3 peptides from Bim or Bid to Bax via a cysteine placed near its rear site. Indeed, cysteine-tagged Bid or Bim BH3 peptides were readily cross-linked near the proposed Bax ‘rear’ site, and the cross-linking efficacy strongly depended on the position of the cysteine tags, indicative of specificity. However, other BH3 peptides such as Bad that do not directly activate Bax could also be cross-linked, suggesting that ‘rear’ site binding might be insufficient to trigger Bax activation. Furthermore, a Bid BH3 peptide cross-linked to the Bax ‘rear’ site did not increase Bax translocation to the MOM or induce oligomerisation.
Conclusion: Our findings indicate that Bax may indeed have a weak alternative binding site for BH3-only proteins, but its function remains elusive, because an activator BH3 linked to this site did not trigger Bax activation.
NEISSERIA CAUSES MITOCHONDRIA DAMAGE IN MACROPHAGES VIA PORB SECRETION ON EXTRACELLULAR VESICLES
Pankaj Deo*, Jhih-Hang Jiang, Eva Heinz, and Thomas Naderer
Department of Biochemistry and Molecular Biology, Monash University *[email protected]
Background: Neisseria gonorrhea causes gonorrhoea in more than 80 million people by evading innate and adaptive immunity. While high numbers of neutrophils and macrophages are recruited to urethral infection they fail to control Neisseria. Recent studies suggest that innate immune responses may actually promote bacterial replication and disease (1). The molecular host-pathogen interactions, however, remain unknown. Neisseria express a number of enzymes and toxins, which are thought to be important for virulence. In particular, we and others have recently shown that toxin PorB is targeted to mitochondria to modulate host cell death pathways (2-4). However, PorB is the major protein of the outer membrane of Neisseria and it is unclear how it targets host mitochondria pathways. We have now identified that Neisseria gonorrhea secretes large number of extracellular vesicles, ranging from 20-200 nm in diameter that contain PorB.
Aims: Aims of this project are to investigate whether extracellular vesicles are the major secretion system for the pathogenic form of PorB and whether vesicles derived PorB induces apoptosis in macrophages.
Results: We have performed extensive proteomic analysis to show that PorB is the major protein on purified vesicles from Neisseria. After phagocytosis of these extracellular vesicles by bone marrow derived macrophages from mices, PorB traffics to the mitochondria as determined by super resolution microscopy. PorB targeted to mitochondria release cytochrome C, activate caspase-3 and cell death. We use live-cell imaging to show that Neisseria secreted extracellular vesicles induces extensive macrophage blebbing.
Conclusion: We have identified a novel mechanism for how extracellular bacteria control innate immune responses. Neisseria secretes its major virulence factor, PorB toxin, via extracellular vesicles which enables effective targeting of macrophage mitochondria. We predict that this is a major strategy how Neisseria controls the innate immune response.
MECHANISM OF BIM-MEDIATED APOPTOSIS DURING SEPSIS-INDUCED LYMPHOPENIA
Marcel Doerflinger*, Christina Nedeva*, Boris Reljic, Marco Herold, Marc Pellegrini and Hamsa Puthalakath
Department of Biochemistry and Genetics, La Trobe University, *mdoerfl [email protected]
Sepsis is a significant cause of death in non-coronary ICUs and incidents are estimated to further increase due to an aging population and an increase in comorbidities. The majority of deaths (more than 80%) occur during the prolonged lymphopenia stage when the patients succumb to nosocomial infections. The apoptotic protein Bim plays a central role in sepsis-mediated lymphocyte death in experimental rodent models and human patients. However, the molecular mechanisms leading to Bim induction remain elusive.
We show that systemic ER stress induces Bim transcription and translation leading to cell death during sepsis and therefore presents a target for therapeutic intervention. To this end, we biochemically purified a form of the ER chaperone BiP secreted by activated macrophages - central mediators of inflammation during sepsis - responsible for Bim induction in target cells leading to apoptosis. We developed an ELISA to detect secreted BiP as a diagnostic marker in mice undergoing polymicrobial sepsis and in human sepsis patients.
To identify the upstream mediators responsible for Bim induction, we undertook a genome-wide CRISPR screen and identified a novel protein named “Function Unknown Membrane Receptor” (FUMR) as the receptor that mediates this cell death. CRISPR-mediated knock out of this gene in cell lines and in mice (FUMR-/-) confirmed the role of this receptor in BiP-mediated cell death. Currently we are trying to understand the structural basis of this interaction with the eventual aim of developing novel therapeutics for treating sepsis.
Experimental drug therapies for sepsis are at cross roads with more than 30 drug trials failing in the last 25 years. Current thinking in the field is that the lymphopenia stage holds the key and therefore, our findings have a great significance for developing novel therapies.
DEXAMETHASONE RESISTANT SCREEN USING CRISPR/CAS9 GUIDE RNA LIBRARY IN MOUSE LYMPHOMA CELL LINES
Li Dong* and David Vaux
Cell Signall ing & Cell Death Division, The Walter & Eliza Hall Institute *[email protected]
Background & Aims: Past studies have shown that there are at least six unique genes required for dexamethasone (Dex) induced killing in mouse lymphoma cells line WEHI7 cells. While they showed that the first gene was the glucocorticoid receptor, they were not able to identify the other genes. Here we used CrispR technology to identify the genes involved dexamethasone-induced killing in WEHI7 cells.
Results: Bax and Bak deletion generated by CrispR/Cas9 prevented most of dexamethasone induced cell death. We used a mouse CrispR guide RNA library containing 87,000 sgRNAs to generate knockout and performed dexamethasone resistant screens in WEHI7 cells. All the Dex resistant colonies we got from Dex screen were glucocorticoid receptor mutant cells.
Conclusion: Our results are consistent with previous research showing that glucocorticoid receptor is essential for Dex induced cell death and cell cycle arrest.
F
MINDBOMB-2 IS A NOVEL RIPK1 INTERACTING E3 UB-LIGASE THAT PROTECTS CELLS FROM THE CYTOTOXIC EFFECTS OF TNF
Rebecca Feltham1*, Tencho Tenev, Conor Kearney, Gianmaria Liccardi, Celia Monteiro Domingues, Otto Morris, Timo Glatter, Seamus Martin, and Pascal Meier
1The Walter and Eliza Hall Institute *[email protected]
Background: RIPK1 is a key regulator of TNF-induced apoptosis and necroptosis and RIPK1-mediated inflammation and programmed cell death are finely balanced to control a variety of physiological and pathological conditions. Despite evidence that TNF signals either cell survival or cell death, the mechanisms that switch between these distinct biological outcomes remain elusive. A better understanding of the complex relationship between cell death and inflammation is critical because these biological events lie at the heart of many human diseases.
Aim: We aimed to address how the decision is taken between cytokine induced pro-inflammatory signalling and cell death, and what underlying mechanisms limit these responses.
Results: Using an affinity purification approach to isolate novel regulators of RIPK1, and complex-II (RIPK1/FADD/caspase-8) formation, we identified Mindbomb-2 (MIB2) as a novel regulator of TNF signalling, selectively regulating the pro-apoptotic effects of TNF without inhibiting the NFκB pathway. MIB2 is an E3 ligase that interacts directly with RIPK1 and targets RIPK1 for ubiquitylation. MIB2 is recruited to the TNF-R1 signalling complex-I, and functions to selectively inhibit TNF-induced complex-II formation and cell death. Abrogation of MIB2 function, either via mutation of the interaction surface between MIB2 and RIPK1 or disruption of MIB2’s E3 ligase activity, results in increased complex-II formation, caspase-8 activation and cell death.
Conclusion: Our data demonstrates that MIB2 acts at the level of the TNF receptor complex to regulate RIPK1 recruitment to complex-II in a process that is dependent on MIB2’s E3 activity, and independent of the activation of NFκB.
Cell death Cell – you poor bastard:
One for al l and al l against the weakest
G
THE TYPE III EFFECTOR NLEB FROM ENTEROPATHOGENIC E. COLI ANTAGONISES DEATH RECEPTOR SIGNALLING
Cristina Giogha1*, Michelle Kelly1, Tania Wong Fok Lung1, Aleksandra Bankovacki2, Giuseppe Infusini2, Andrew I. Webb2, John Silke2, Andreas Strasser2, Jaclyn S. Pearson1, Elizabeth Hartland1
1Peter Doherty Institute for Infection and Immunity 2The Walter and Eliza Hall Institute *[email protected]
Background: Enteropathogenic Escherichia coli (EPEC) is an extracellular intestinal pathogen which utilises a type III secretion system (T3SS) to translocate virulence (effector) proteins directly into host cells. In recent years, several of these effectors have been implicated in the subversion host cell signaling. Non-LEE encoded effector B (NleB) is one such effector which inhibits death receptor signaling within host cells to block apoptosis. This allows EPEC to persist, replicate and disseminate to other hosts.
Aim: To understand the molecular mechanism of inhibition of host cell death by NleB.
Results: We identified the death domain containing adaptor protein FADD, which is recruited to both TNFR1 and FAS, as a binding partner of NleB. NleB was previously discovered to have homology to the GT-8 family of glycosyl transferases and contains a DxD catalytic signature that is essential for it’s function. We showed that NleB adopts a novel enzymatic activity and modifies FADD in an unexpected manner, by transferring a sugar to a conserved arginine residue within the death domain of FADD rather than targeting serines, threonines or asparagines as other glycosyltransferases do. This novel modification is not removed by commonly used deglycosylating enzymes and appears to be very stable. The conserved arginine residue plays a critical role in FADD dimerization and FAS-FADD interactions. Mutation of this residue prevents NleB mediated glycosylation of FADD while mutation of a nearby serine residue does not.
Conclusions: Our research identifies a novel biochemical modification which inhibits host cell apoptotic signaling, and provides insight into the mechanism of pathogenesis by bacterial gut pathogens.
MOLECULAR CONTROL OF FOXP3+ REGULATORY T CELL SURVIVAL AND DEATH DURING CHRONIC VIRAL INFECTION
Charis E Teh1,2, Simon Preston1,2, Gregor Ebert1,2, Andreas Strasser1,2, Marc Pellegrini1,2, Daniel HD Gray1,2 *
1The Walter and Eliza Hall Institute of Medical Research 2University of Melbourne, Department of Medical Biology *[email protected]
Background: Foxp3+ regulatory T (Treg) cells have emerged as a crucial population that maintains the balance between tissue-damaging and protective effects in the immune system. The immunosuppressive functions of Treg cells are important in controlling autoimmunity and chronic viral infections; however, the death and survival programs that control the Treg cell pool during infection are unknown. In this study, we examined how two major pathways of apoptosis, the intrinsic and extrinsic apoptotic pathways, impinge upon Treg cell homeostasis and function under steady-state conditions and during chronic inflammatory conditions.
Aims: To address this issue, we generated mice with a Treg cell-specific deletion of key mediators of the intrinsic (BaxΔFoxP3Bak-/-) or extrinsic (Casp8ΔFoxP3) apoptotic pathways.
Results: Unexpectedly, Treg cell-specific ablation of both pathways, individually, resulted in a marked increase in FoxP3+ Treg cells with an effector phenotype (CD62LlowPD1hiICOShi) in steady state conditions, suggesting dual control mechanisms maintain Treg cell homeostasis. Infection with the fast-replicating chronic lymphocytic choriomeningitis virus (LCMV) Docile strain triggered a massive expansion in the Treg cell population in wild-type and BaxΔFoxP3Bak-/- mice eight days post-infection. By contrast, Treg cell-specific ablation of Caspase 8 resulted in a striking loss of Treg cell number and increased CD8+ and CD4+ cell activation, indicating a pro-survival role for this caspase in Treg cells only during chronic inflammation.
Conclusion: Taken together, these data reveal that the intrinsic and extrinsic apoptotic pathways are both vital for Treg homeostasis in the steady state. By contrast, these pathways have divergent roles in the control of Treg cell function under chronic inflammatory conditions. Understanding how these pathways alter Treg cell homeostasis and the immune response is pivotal because drugs that target these pathways are currently in clinical trials for treatment of cancers and autoimmune disease.
CHEMOPROTEOMIC APPROACH TO IDENTIFY A NOVEL EFFECTOR OF THE NECROPTOTIC SIGNALING PATHWAY DOWNSTREAM OF MLKL
Christoph Grohmann*, Mark Van Delft, Joanne Hildebrand, Maria Tanzer, James Murphy, John Silke and Guillaume Lessene
The Walter and Eliza Hall Institute of Medical Research *[email protected]
Necroptosis has been identified as an alternative process of cell death functioning when apoptosis is inhibited or defective. Several tissue inflammatory diseases have been linked to a dysregulation of necroptosis, and activation occurs through a well-orchestrated signaling cascade. However apart from RIP3 and its substrate MLKL, its effectors have only partly been identified to this point.
Using a phenotypic screen, we identified small molecule 1 as an inhibitor of the necroptotic pathway, acting downstream of RIP3/MLKL with a hitherto unknown effector. This unidentified protein represents a potential therapeutic target for treatment of diseases related to necroptosis.
Herein, we describe the synthesis of chemical probes of inhibitor 1 for target identification studies of the necroptotic signaling cascade downstream of MLKL using a chemoproteomic approach and first preliminary results are presented.
H
ACTIVATION OF THE PSEUDOKINASE MLKL UNLEASHES THE FOUR-HELIX BUNDLE DOMAIN TO INDUCE MEMBRANE LOCALIZATION AND NECROPTOTIC CELL DEATH
Joanne M Hildebrand1, 2,*, Maria C Tanzer 1, 2, Isabelle S Lucet1, 2, 3, Sukhdeep K Spall1, 2, Sam N Young1, 2, Warren S Alexander1, 2, Peter E Czabotar1, 2, Andrew F Wilkes5, Guillaume Lessene1, 2, 4, JM Murphy,1, 2 John Silke1, 2
1Walter and Eliza Hall Institute of Medical Research, 2University of Melbourne Dep. Medical Biology, 3Dep. Biochem. and Mol. Biology, Monash University, 4Dep. Pharmacology and Therapeutics, University of Melbourne, 5SynThesis MedChem *jhi [email protected]
Necrotic cell death has long been regarded as an uncontrolled consequence of chemical or mechanical insult. Recent work has revealed it to be intricately programmed and deliberate in certain scenarios like infection. This programmed necrosis or ‘necroptosis’ is triggered by a cascade of intracellular signals culminating in the phosphorylation of the pseudokinase Mixed Lineage Kinase domain-Like (MLKL). How MLKL goes on to cause cell death remains a strong topic of contention, with proposed mechanisms ranging from ion channel modulation to direct physical phospholipid bilayer disruption. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain an N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high molecular weight, membrane localized protein complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, while MLKL mutations in the other cluster translocated to membranes but failed to kill; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. MLKL is a compelling drug target for the treatment of necroptosis related disease because it is not required for normal development and homeostasis and belongs to the relatively divergent pseudokinase family that can theoretically be targeted more specifically than the larger and more conserved kinase family.
To die or not to die? - Accept your destiny
and may the Fas be with you
ACTIVATED BAK BINDING TO MCL-1 OR BCL-XL UNDERLIES DIFFERENTIAL SENSITIVITY TO THE BH3-ONLY PROTEINS BID AND BIM
Colin Hockings*, Grant Dewson, Ruth Kluck
The Walter and Eliza Hall Institute, Parkvil le, Victoria, Austral ia *[email protected]
The Bcl-2 apoptotic switch is the key decision point in the intrinsic pathway of apoptosis, an important anti-cancer mechanism. It has not been clear to what extent the prosurvival Bcl-2 family members such as Mcl-1 and Bcl-xL act by binding BH3-only proteins such as Bid and Bim (MODE 1) or binding activated effector proteins Bak or Bax (MODE 2). We were able to compare the contributions of MODE 1 and MODE 2 sequestration by measuring both Bak activation and cytochrome c release. We found that Mcl-1 caused profound resistance to Bid indirectly by sequestering Bak in MODE 2. In contrast, Bcl-xL caused resistance to Bid mainly via MODE 1. Both Mcl-1 and Bcl-xL caused resistance to Bim through MODE 1 as well as MODE 2 sequestration. Bim, but not Bid, was able to overcome MODE 2 caused by Mcl-1, and so was able to cause MOMP in the presence of Mcl-1. In contrast, Bid was able to overcome MODE 1 sequestration by Bcl-xL. These findings show that prosurvival proteins, even at relatively low concentrations, can cause resistance to BH3-only proteins via MODE 2, in both mitochondrial assays and cultured cells. In particular, the ability of Mcl-1 to cause profound resistance to Bid by binding Bak may allow some cancer cells to resist death signalling from immune cells or anti-cancer treatments such as TRAIL agonists.
I FERROPTOSIS REGULATOR GPX4 OR VITAMIN E IN HEART PREVENTS CARDIO SUDDEN DEATH IN MICE
Hirotaka Imai*, Tomoko Koumura and Masaki Mastuoka
School of pharmaceutical Sciences, Kitasato University *[email protected]
Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme that could directly reduce phospholipid hydroperoxide using glutathione. GPx4 is a regulator of novel iron dependent cell death (ferroptosis) by which anti cancer drug, Erastin induce non-apoptotic cell death in mutated Ras dependent cancer cells. We previously reported testis and photoreceptor specific GPx4 KO mice and hole GPx4 KO mice. Depletion of GPx4 in testis and retina induced cell death of spermatogenic cells and photoreceptor cells and became male infertility and blindness. To clarify the relationship between endogenous generation of phospholipid peroxidation and heart disease, we made heart specific GPx4 KO mice. Heat specific GPx4 KO mice died at 18.5 dpc by cardio sudden death without activation of caspase3 and DNA fragmentation. We detected generation of phospholipid hydroperoxide in heart of 17.5 dpc pups. Administration of vitamin E to mothers completely rescued the lethality of 18.5dpc heart specific GPx4 KO pups. Heart specific GPx4 KO mice normally could grow by eating vitamin E added diet. However change of vitamin E added diet to normal diet induced cardio sudden death until 10 days in adult heart specific GPx4 KO mice. Oxylipidomics analysis showed that enrichment of phospholipid hydroperoxide in heart of vitamin E decreased heart specific GPx4 KO mice. These results demonstrate that endogenously generation of phospholipid hydroperoxide by imbalance of vitamin E or GPx4 cause cardio sudden death by ferroptosis like cell death.
Cherry blossoms A springtime Melbourne whodunit
Which protein ki lls cells?
J
HSP90 ACTIVITY IS REQUIRED FOR TRANSLOCATION OF MLKL TO MEMBRANES AND THE INDUCTION OF NECROPTOTIC CELL DEATH
Annette V Jacobsen1*, Kym Lowes1, Maria Tanzer1, Isabelle S Lucet1, Emma J Petrie1, Mark F van Delft1, Joanne Hildebrand1, David Huang1, John Silke1, Guillaume Lessene1, James Murphy1
1The Walter and Eliza Hall Institute of Medical Research *[email protected]
Background: Necroptosis is a form of programmed cell death implicated in a range of inflammatory and autoimmune diseases. It is initiated by signalling through death receptors or pattern recognition receptors, leading to activation and phosphorylation of the pseudokinase, mixed lineage kinase domain-like (MLKL), the most downstream known essential effector of necroptosis, by receptor interacting protein kinase 3 (RIPK3). RIPK3-mediated activation of MLKL induces its oligomerisation and translocation to the plasma membrane leading to destabilisation and eventual lysis of the cell. However, the mechanisms by which this occurs and the interactions involved are poorly characterised.
Aims: To identify novel participants in the necroptosis pathway using screening methods and validate any “hits” using cell-based techniques.
Results: Using a phenotypic screen, we identified a range of heat shock protein 90 (HSP90) inhibitors that provided protection against cell death mediated by an activated mutant MLKL. We found inhibition of HSP90 to confer only a modest reduction of MLKL protein levels in both human and mouse cell lines. Interestingly, we found that HSP90 inhibition restrict the ability of an activated, phosphomimetic MLKL mutant to translocate to membranes and form high molecular weight complexes. In contrast, HSP90 inhibitors conferred no protection against cell death caused by the N-terminal executioner domain of MLKL, indicating that the HSP90 “client” is the MLKL pseudokinase domain.
Conclusion: These results suggest a role for HSP90 in controlling MLKL activation during necroptosis, and expand on its previously known functions in this pathway through interaction with RIPK3 and its partner kinase RIPK1.
IDENTIFICATION OF THE CRITICAL P53 TUMOR SUPPRESSION MECHANISMS IN VIVO
Ana Janic1*, Elizabeth Valente1, Lujambia A2, Liz Milla1, Stephen Wilcox1, Scott Lowe2, Liam O’Connor1, Marco Herold1, Andreas Strasser1
1 The Walter and Eliza Hall Institute of Medical Research, Austral ia 2 Memorial Sloan Kettering Cancer Center, New York, USA *[email protected]
The tumor suppressor p53 is mutated in ~50% of human cancers. Accordingly, mice lacking functional p53 are tumor prone and their cells are markedly resistant to DNA damage and certain other cytotoxic insults. p53 is a transcription factor that can activate a broad range of cellular effector functions. It has been postulated that p53 suppresses tumorigenesis mainly by inducing apoptosis with possible contributions by cell cycle arrest and senescence. This appears, however, unlikely, given that no spontaneous tumors arose in mice lacking Puma, Noxa and p21, the essential mediators of p53-induced apoptosis and cell cycle arrest/senescence, respectively, or in mice bearing mutations in p53 that impair its transcriptional activation of these genes. To better define the mechanisms of p53-mediated tumor suppression we screened an shRNA library to identify p53 targets that when knocked-down in haematopoietic stem/progenitor cells (HSPCs) lacking the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence (Puma-/-p21-/-) can promote lymphoma/leukemia development upon transplantation into recipient mice. This library was also screened for shRNAs that could further accelerate lymphoma development driven by deregulated MYC expression even when pro-apoptotic PUMA was absent (Em-Myc;Puma-/- HSPCs). These screens yielded p53 target genes implicated in the control of DNA repair (Mlh1) cell proliferation (Rfwd2, TGFa, Cav1) and some with poorly defined functions (Zmat3, Crip2, Ctsf). Knockdown of several of these genes promoted tumor development only when p53-mediated apoptosis, cell cycle arrest and senescence were also impaired (i.e. in Puma-/-p21-/- HSPCs) but knockdown of Mlh1 even caused lymphoma/leukemia in a wt background. These findings reveal that several p53-regulated processes must fail to cause spontaneous tumor development and that control of DNA repair is the most important tumor suppressive function of p53.
MONITORING THE PROGRESSION OF CELL DEATH AND DISASSEMBLY OF DYING CELLS BY FLOW CYTOMETRY
Lanzhou Jiang*, Rochelle Tixeira, Sarah Caruso, Georgia K Atkin-Smith, Amy A Baxter, Stephanie Paone, Mark D Hulett, Ivan KH Poon
The La Trobe Institute for Molecular Science (LIMS) *[email protected]
The use of annexin V and propidium iodide/7AAD stains to measure cell death by flow cytometry has been considered the gold standard by most investigators. However, this widely used method often makes the assumption that there are only three types of particles in a sample, that is viable, apoptotic and necrotic cells. In order to study the progression of cell death in greater details, in particular how apoptotic cells undergo fragmentation to generate membrane-bound vesicles known as apoptotic bodies, we have recently established a new flow cytometry-based protocol to accurately and rapidly measure the cell death process. This protocol utilises a combination of annexin V and TO-PRO-3 (a commercially available nucleic acid-binding dye that stains apoptotic and necrotic cells differentially), and a logical five-step analytical approach to distinguish six types of particles in a sample, including apoptotic bodies and cells at three different stages of cell death.
L
TARGETING P38 OR MK2 ENHANCES THE ANTI-LEUKEMIC ACTIVITY OF SMAC-MIMETICS
Najoua Lalaoui1,2*, Kay Hänggi3, Gabriela Brumatti1,2, Diep Chau1,2, Nhu-Y Nguyen4, Margherita Ghisi5, Stephen M Condon6, Ricky Johnstone5, Andrew Wei4, Lynn W Wong3, John Silke1,2
1The Walter and Eliza Hall Institute of Medical Research 2University of Melbourne, Melbourne, Austral ia, 3University of Zürich, Switzerland, 4The Alfred Hospital and Monash University, Melbourne, Austral ia 5Peter MacCallum Cancer Centre, Melbourne, Austral ia 6TetraLogic Pharmaceutical Corporation, Phoenixvi l le, PA, USA *[email protected]
Birinapant is a smac-mimetic evaluated in phase II trials for the treatment of cancer. Smac-mimetics antagonize Inhibitor of APoptosis (IAP) proteins and simultaneously induce TNF secretion to render cancers cells sensitive to TNF induced killing. To enhance smac-mimetic efficacy, we screened kinase inhibitors for their ability to increase TNF production of smac-mimetic treated cells. We found that all tested p38 inhibitors increased TNF induced by smac-mimetic. Unexpectedly, and even though p38 is required for Toll-Like Receptors to induce TNF, loss of p38 or the downstream kinase MK2 increased induction of TNF by smac-mimetic as well as enhanced smac-mimetic killing. Hence, we show that p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Interestingly, the killing by smac-mimetic plus p38 inhibitors required caspase-8 and RIPK1 activities but not RIPK3 and neither MLKL. Importantly, clinical p38 inhibitors overcame resistance of different primary murine AML models to birinapant in vivo and sensitized AML human samples to birinapant. This study provides an exciting new therapeutic strategy for the use of birinapant in the clinic and new insights in the role of IAPs in cytokine regulation.
Appointment with death On a precipice
Facing death. By caspases? Or pseudokinase?
RIPK3 PROMOTES CELL DEATH AND NLRP3 INFLAMMASOME ACTIVATION IN THE ABSENCE OF MLKL
Kate Lawlor*, John Silke, David Vaux, James Vince
The Walter & Eliza Hall Institute of Medical Research *[email protected]
RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3–caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1β inflammatory responses independent of MLKL and necroptotic cell death1. 1. Lawlor KE 2015 Nature Communications 6:6282
CHARACTERISING INTERFACES REQUIRED FOR BAK APOPTOTIC ACTIVITY BY FUNCTIONAL SCREENING
Xiang Mark Li1,2, Grant Dewson1,2
Cell Signall ing & Cell Death Division, The Walter & Eliza Hall Institute of Medical Research
BAK and BAX are the pivotal effectors of the intrinsic apoptosis pathway. Accumulating evidence suggests that BAK and BAX undergo a series of conformational changes upon activation that facilitate homo-oligomerisation to the putative apoptotic pore. However, the interfaces involved in the activation and oligomerisation of BAK and BAX are unclear. For instance, it is well accepted that BH3-only proteins bind to BAK/BAX but the interface(s) responsible for the initial BAK/BAX activation is still in debate. In addition, BAK and BAX oligomerisation requires the multimerisation of BAK/BAX homodimers through an unknown secondary interface, the structural determination of which is considered the “holy grail” of apoptosis research. Identifying the important interfaces responsible for BAK/BAX function would potentially allow the therapeutic manipulation of BAK/BAX to either stop BAK/BAX activation to prevent cell death for example following ischemic stroke or to activate BAK/BAX as an approach to combat cancer.
We have developed a simple screening system that allows us to determine the interfaces required for BAK function.
As proof-of-concept, we have exploited this approach to confirm the importance of a canonical hydrophobic surface groove in both BAK activation by BH3-only proteins and also oligomerisation. This screening approach will functionally map interfaces required for BAK function and can be extended to explore other proteins of interest Life, energy, movement
Destiny cal ls. .. RIP Kinase kinase kinase
SINGLE-CELL ANALYSIS REVEALS THE DIGITAL RESPONSE OF INFLAMMASOME-CASPASE-1 SIGNALING
Ting Liu1*, Yoshifumi Yamaguchi1,2, Yoshitaka Shirasaki3,4, Osamu Ohara3,5, and Masayuki Miura1,6
1Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo 2PRESTO, Japan Science and Technology Agency 3Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences ( IMS-RCAI) 4Department of Biological Sciences, Graduate School of Science, The University of Tokyo 5Department of Human Genome Research, Kazusa DNA Research Institute 6CREST, Japan Agency for Medical Research and Development
Background: Caspase-1, a member of cysteine protease family caspases, regulates the secretion of pro-inflammatory cytokines like interleukin-1β (IL-1β) as well as inflammatory cell death termed pyroptosis in response to various kinds of stimuli. The activation of caspase-1 is regulated by the intracellular multiple protein complexes called ‘the inflammasomes’. Although the dynamics of caspase-1 activation, IL-1β secretion, and cell death have been examined intently with bulk assays in population-level studies, they remain poorly understood at the single-cell level because of technical limitations.
Aims: Our purpose is to reveal the relationship between caspase-1 activation and its outputs (IL-1β secretion and cell death) in macrophages at the single-cell resolution.
Results: By conducting single-cell imaging with FRET sensor for caspase-1 activation, we found that caspase-1 exhibits all-or-none (digital) activation at the single-cell level, with similar activation kinetics irrespective of the type of inflammasome or the intensity of the stimulus. Concurrent live-imaging of caspase-1 activation and IL-1β release demonstrated that dead
macrophages containing activated caspase-1 secrete IL-1β in a digital manner, which identified these macrophages as the main source of IL-1β within cell populations.
Conclusion: Our results highlight the value of single-cell analysis in understanding the pathology of caspase-1-related inflammatory diseases. In addition, combining our single-cell imaging system with siRNA or chemical library would provide us a critical opportunity to uncover the molecular mechanisms of pyroptosis and unconventional secretion downstream of caspase-1 activation.
References: Liu, T., et al., Cell Reports, 8, 974-982, 2014
Nine circles of hell eight t imes Microsoft Word
and one times Excel
IMPACT OF LOSS OF NF-kB FAMILY MEMBERS ON SLE-LIKE AUTOIMMUNE DISEASE CAUSED BY MUTATIONS IN FAS
Jun Ting Low*, Peter Hughes1, Ann Lin, Reema Jain, Daniel Gray, Steve Gerondakis2, Andreas Strasser and Lorraine A O’Reilly
The Walter and Eliza Hall Institute of medical Research, 1 Department of Nephrology, The Royal Melbourne Hospital, 2Department of Biochemistry and Molecular Biology, Monash University. *j [email protected]
Background: Defects in apoptosis can cause autoimmune disease. Loss-of-function mutations in the 'death receptor' FAS impair the deletion of autoreactive and chronically stimulated lymphocytes in the periphery, leading to progressive lymphadenopathy and systemic lupus erythematosus-like autoimmune disease in mice (Faslpr/lpr mice: homozygous for the lymphoproliferation inducing spontaneous mutation) and humans. The REL/nuclear factor-κB (NF-κB) transcription factors regulate a broad range of immune effector functions. Defects in their function have been implicated in various autoimmune diseases. Some current autoimmune diseases therapies dampen the activity of the entire NF-kB system, and this can cause adverse side effects, such as immune-suppression. Therefore a more targeted therapy is warranted.
Aim: Compound mutant mice were generated to investigate the individual functions of the NF-κB family members NF-κB1, NF-κB2 and c-REL in lymphadenopathy and the autoimmune pathologies of Faslpr/lpr mutant mice.
Results: Loss of individual transcription factors resulted in amelioration of many classical autoimmune disease features, including hyper-gammaglobulinaemia, anti-nuclear autoantibodies and autoantibodies against tissue-specific antigens. Interestingly, Faslpr/lprnfkb2-/- mice presented with a dramatic acceleration of lymphadenopathy accompanied by severe lung pathology. Faslpr/lprnfkb1-/- mice exhibited the combined pathologies caused by defects in FAS-mediated apoptosis and premature ageing due to loss of NF-kB1. Remarkably, only c-REL deficiency substantially reduced immune complex-mediated glomerulonephritis and significantly extended the lifespan of Faslpr/lpr mice.
Conclusions: Different NF-κB family members exert distinct roles in diverse autoimmune and lymphoproliferative pathology development that arise in Faslpr/lpr mice, suggesting that pharmacological targeting of c-REL should be considered as a potential strategy for therapeutic intervention in autoimmune diseases.
M
THE ROLE OF RIP1 IN THE DEVEOPMENT OF ACUTE MYELOID LEUKEMIA
Chunyan Ma*, Gabriela Brumatti and John Silke
Cell Signall ing & Cell Death Division, The Walter & Eliza Hall Institute of Medical Research *[email protected]
Background: Leukemias carrying mixed lineage leukemia (MLL) rearrangements are a subtype of acute leukemias, associated with high-risk pediatric, adult and therapy-induced leukemia. MLL-ENL is one of the most common MLL translocated leukemias in the patients. RIP1 is an important gene involved in many pathways. It has been reported that RIP1 is highly expressed in several solid tumors and associated with short survival. However the role of RIP1 in haematological malignancies has yet to be explored.
Aims: The aim of this study is to explore the role of RIP1 in Acute Myeloid Leukemia (AML)
Results: We generated MLL-ENL leukemia mice models by transducing fusion cDNA using retroviral infection into E14 fetal liver cells. Surprisingly, we saw a much faster disease onset in RIP1 knockout MLL-ENL compared to wild type mice. No differences in blood profile, histology, myeoloid markers or cell cycling were observed. The mix of wild type support cells could slow down the fast onset of RIP1 knockout tumors. However, when we retransplanted these leukemias, RIP1 knockout MLL-ENL clearly had a prolonged disease onset compared to wild type. Interestingly, these successfully retransplanted RIP1 knockout tumor took a normal range of disease onset time to get further retransplanted.
Conclusion: RIP1 knockout AML took a longer time to be retransplanted compared with wild type. In vivo selection could select tumors more easily to be further retransplanted.
Spring Goddess IDUN: the wife of Smac Mimetic;
apple of God's eye
N INDUCING INTRINSIC APOPTOSIS ELIMINATES INTRACELLULAR LEGIONELLA
Mary Speir, Kate Lawlor, Stefan Glaser, Guillaume Lessene, James Vince and Thomas Naderer*
Monash University and The Walter and Eliza Hall Institute *[email protected]
Background: Programmed cell death is an important innate immune response in controlling persistent intracellular pathogens. Several pathogens, however, have evolved mechanisms to evade or utilize macrophage cell death pathways, which promote egress and dissemination. Thus disease depends on the mode and timing of host cell death. Whether inducing host cell death to control intracellular pathogens can be utilized therapeutically remains thus unresolved.
Aims: The aim is to determine whether intracellular bacterial infections can be eliminated by inducing apoptosis of macrophages.
Results: Legionella reside primarily in alveolar macrophages and evade apoptotic and pyroptotic cell death to promote replication. We now demonstrate that intracellular Legionella replication is prevented by the genetic deletion, or BH3-mimetic drug targeting, of the host cell anti-apoptotic protein, BCL-XL. Loss of BCL- XL in myeloid cells in vitro and in vivo did not impact on macrophage viability or animal health, but strikingly, induced rapid intrinsic apoptosis of macrophages upon infection with virulent Legionella strains. A single dose of BCL-XL-targeted BH3-mimetic therapy, or myeloid cell BCL-XL deletion, significantly limited Legionella replication and bacterial burdens in the lungs of mice, thereby preventing lethal bacterial infection.
Conclusion: These results demonstrate that the repurposing of anti-cancer drugs, such as BH3-mimetic compounds, to target specific host pro-survival factors, as opposed to bacterial molecules, represents a novel and promising strategy for the treatment of intracellular microbial pathogens.
APOPTOTIC EPITHELIAL CELLS CONTROL REGULATORY T CELL EXPANSION VIA CD300A ON DENDRITIC CELLS
Yoshiyuki Nakamura1*, Chigusa Nakahashi-Oda1 and Akira Shibuya1-3
1Department of Immunology, Faculty of Medicine, 2Life Science Center of Tsukuba Advanced Research All iance (TARA), 3Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), University of Tsukuba, Tsukuba, Japan *[email protected]
Background: CD300a is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containg immunoglobulin-like receptor and mediates an inhibitory signal in myeloid cells. We have recently identified phosphatidylserine (PS) as a ligand for CD300a in myeloid cells. Because the epithelium shows rapid turnover and generates numerous apoptotic cells even in normal physiological conditions, we speculated that CD300a-expressing cells adjacent to epithelial tissues may modulate the immune responses for maintenance of the epithelial tissue homeostasis.
Aims: To clarify the regulatory role of CD300a in the homeostasis of colon tissues.
Results: Here we show that CD300a is highly expressed on CD11b+ dendritic cells (DC) in the colon. CD300a on the CD11b+ DC were found to directly bind PS on epithelial apoptotic cells. To explore the physiological role of CD300a in the colon, we investigated a colitis model by administrating dextran sulfate sodium (DSS). Cd300a–/– mice showed significantly milder change of body weight, colon length and epithelial morphology compared with wild-type (WT) mice. We found that regulatory T (Treg) cell population was increased in Cd300a–/– mice compared with WT mice in specific-pathogen free conditions, but not in germ-free conditions. Cd300a–/– mice treated with anti-CD25 monoclonal antibody for depletion of Treg showed comparable reduction of body weight to WT mice. Quantitative RT-PCR analyses demonstrated that interferon-β (IFN-β) expression was significantly higher in Cd300a–/– CD11b+ DC. In addition, Cd300a–/– mice treated with IFN-β mAb showed comparable Treg cell population and body weight loss in DSS-induced colitis to WT mice.
Conclusion: These results indicated that apoptotic epithelial cells inhibit commensal-mediated signal for IFN-β production by CD11b+ DC via CD300a, resulting in suppression of Treg cell expansion and regulating the immune responses in colon.
ENGULFMENT OF DEAD CELLS IN THE EVENT OF A MYOCARDIAL INFARCTION
Michio Nakaya*
Kyushu University *[email protected]
Myocardial infarction (MI) is considered a major health problem worldwide. After MI, cardiomyocytes receiving blood supply from the occluded blood vessels undergo rapid death. In the process, the dead cells release noxious intracellular contents that can induce secondary cell death and an inflammatory response. Therefore, speedy removal of these cells by phagocytes can prevent the expansion of the damaged area. However, specific cells and molecules responsible for the engulfment of dead cells in the event of a MI remain largely unknown.
To identify the molecules involved in the engulfment of dead cells after MI, first we performed a microarray experiment using RNA obtained from the heart tissues of mice, 3 days after a sham operation or permanent occlusion of the left anterior descending artery to induce MI. The microarray analysis revealed that milk-fat globule-epidermal growth factor-8 (MFG-E8), an engulfment-related gene, was remarkably upregulated after the occlusion. MFG-E8 was reported to bind specifically to both phosphatidylserine exposed on the dead cells and integrin avb3 on phagocytes and promote the engulfment of dead cells.
In this study, I have discussed the roles of MFG-E8 and MFG-E8- producing cells in the event of a myocardial infarction.
DOSE-DEPENDENT EFFECTS OF MIR-155 IN ACUTE MYELOID LEUKEMIA
Narayan N.1,2, Morenos L.3, Brumatti G. 1, Goodall G.J. 4, Ekert P.G. 2,3
1 Walter and Eliza Hall Institute 2 University of Melbourne 3 Murdoch Children’s Research Institute 4Centre for Cancer Biology (Austral ia)
MicroRNAs are a class of small, non-coding RNA molecules that repress their mRNA targets, and regulate critical cellular processes including cell survival, proliferation and differentiation. A subset of microRNAs have roles in regulating developmental processes such as hematopoiesis and a select few contribute to the development of hematological malignancies and other cancers.
MiR-155, encoded by the B-cell integration cluster gene, has an established role in promoting the development of lymphoid cancers. However, the role of miR-155 in acute myeloid leukemia (AML) is unclear, with conflicting data supporting both an oncogenic and a tumour suppressor role for miR-155 in AML. Enforced expression of miR-155 in murine AML cell lines and AML models was used to establish the role of miR-155 in the biology of AML.
Our data demonstrates that enforced very high expression of miR-155 in AML cell lines results in reduced proliferation and colony formation that could be interpreted as a tumour suppressor function. However, critical long-term assays of cells transduced with miR-155 showed that cells expressing intermediate levels of miR-155 are positively selected over time, and this is accompanied by a restoration in clonogenic potential. In vivo, enforced expression of miR-155 in murine AML models showed no differences in disease onset and latency compared to controls, but mice overexpressing the miR-155 had increased tumour burden. Moreover, the same selection phenomenon in favour of intermediate miR-155 expression level was observed. Importantly, data from pediatric AML samples suggests elevated miR-155 is detectable in human AML and may be associated with worse clinical outcome. Thus, we show that the apparent tumour suppressor effects of enforced miR-155 are likely to be an artifact of unphysiologically high levels of expression and at more physiological levels miR-155 may promote disease progression in AML.
TRAIL and fibroblasts Ale and neurons
We kil l , converge, converge and kil l
VDAC2 IS A VITAL COMPONENT OF THE BAX CELL DEATH PATHWAY
Robert Ninnis*, Stephan Ma, Iris Tan, Laura Dagley, Andrew Webb, Grant Dewson
The Walter & Eliza Hall Institute of Medical Research *[email protected]
Certain aspects of how apoptosis is controlled remain unclear. A detailed understanding of how each step of this critical pathway is regulated will identify new therapeutic targets and aid the design of compounds that can directly modify the apoptotic pathway either negatively or positively for therapy. The pro-apoptotic Bcl-2 family proteins, Bax and Bak, are the critical effectors of the intrinsic apoptosis pathway and once activated they oligomerise leading to mitochondrial outer membrane (MOM) permebilisation, releasing pro-apoptotic proteins into the cytosol. Recent evidence suggests that in non-apoptotic cells, Bax and Bak exist in equilibrium between the cytosol and MOM. What determines their relative distribution is important as it governs the response of a cell to apoptotic stimuli. Prior to an apoptotic stimulus, Bak and Bax associates with voltage-dependent anion channel (VDAC)2 forming a high molecular weight complex. Our data suggests that interaction with this complex is an important determinant of the targeting of Bax and Bak to the MOM. We find that in the absence of interaction with VDAC2, Bax apoptotic function is severely compromised. We have utilised crosslinking mass spectrometry and affinity purification to identify novel components of this important mitochondrial complex, each of which represents a novel targets to manipulate apoptosis. Our studies reveal new insight into how Bax/Bak are trafficked and inserted into the MOM. Understanding of how these proteins function in this important complex will allow the development of small molecules that can modulate the function of these proteins for the treatment of diseases with unregulated cell death.
O
LOSS OF THE NF-ΚB FAMILY MEMBER C-REL PREVENTS AUTOIMMUNITY CAUSED BY DEFECTS IN THE FASL/FAS CELL DEATH PATHWAY
Lorraine. A. O’Reilly*, Peter Hughes1, Ann Lin, Paul Waring2, Reema Jain, Daniel H. D. Gray, Steve Gerondakis3 and Andreas Strasser
The Walter and Eliza Hall Institute, Royal Melbourne Hospital, Melbourne University, 3Monash University. *oreil [email protected]
Background: FASL/FAS-induced apoptosis prevents autoimmune disease and lymphadenopathy. Mutant mice lacking membrane-bound FASL (FasLΔm/Δm), which is essential for FAS-induced apoptosis, develop lymphadenopathy and systemic autoimmune disease with immune-complex mediated glomerulo-nephritis. Prior to disease onset, FasLΔm/Δm mice contain abnormally increased numbers of leukocytes displaying activated NF-kB and elevated NF-kB-regulated cytokines. This indicates that NF-kB-driven inflammation may be a key pathological driver in this multi-faceted autoimmune disease.
Aims: Overall inhibition of NF-kB signalling leads to immunosuppressive complications making it difficult to use such therapies long-term. We therefore tested the hypothesis that inhibition of select NF-κB family members could inhibit autoimmune pathology in FasLΔm/Δm mutant mice without causing immunosuppression.
Results: We deleted the c-Rel or NF-κB2 genes in FasLΔm/Δm mice and examined the health of the compound mutant mice long-term. We found that loss of NF-kB2 reduced the levels of inflammatory cytokines and autoantibodies but the impact on animal survival was minor due to a, surprisingly, accelerated and exacerbated lympho-proliferative disease affecting many organs. In contrast, loss of c-REL significantly extended lifespan coincident with striking reductions in classical parameters of autoimmune pathology (hypergammaglobulineamia, anti-nuclear auto-antibodies (ANA), glomerulonephritis) and elevated cytokine levels.
Conclusion: Combined with the recent discovery of links between polymorphisms in c-Rel and increased susceptibility to certain human autoimmune diseases, our findings suggest that targeting c-REL constitutes an attractive strategy to alleviate or prevent autoimmune pathologies, while avoiding the major side-effects associated with pan-NF-κB inhibition.
IMPAIRED AUTOPHAGY AND CHRONIC PANCREATITIS
Masaki Ohmuraya*, Kazuya Sakata, and Ken-ichi Yamamura. Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan
E-mail: [email protected] Background & Aims: Mutations in serine protease inhibitor Kazal type 1 (SPINK1) are associated with human chronic pancreatitis (CP). Genetic deletion of Spink3, mouse homolog of SPINK1, causes postnatal lethality precluding mechanistic investigations into the effects of SPINK deficiency. Here we have developed Spink3SPINK1/- transgenic mice (termed “SPINK1-in”) in which one ablated Spink3 allele is replaced by knocked-in SPINK1. This partial restoration of SPINK function rescues mice from lethality; but SPINK1-in mice progressively develop spontaneous CP. Methods: We used Cre-Lox technology to generate Spink3SPINK1/- mice and their controls, Spink3SPINK1/+. Pancreas damage and pancreatitis responses were analyzed using light, fluorescence, and electron microscopy, immunoblotting, real-time PCR, and enzymatic assays. Results: In contrast to Spink3SPINK1/+ and Spink3SPINK1/SPINK1, SPINK1-in mice within 4 weeks developed pancreas damage the earliest manifestations of which were impaired autophagy and increased trypsin activity. This resulted in accumulation of large vacuoles (with features of autolysosomes and crinophagy), macrophage-type inflammation, intralobular fibrosis with activated stellate cells, and acinar cell death. We also found pancreas damage (i.e., pancreas fibrosis) was prevented by Ripk3 deletion, suggesting that Ripk3-dependent necroptosis is critical factor for onset of CP. Conclusions: SPINK1-in mice represent a novel, clinically relevant, genetic model of human CP, revealing the mechanisms whereby SPINK insufficiency causes CP. The results identify new targets of SPINK regulating autophagic pathways and necroptosis in pancreas.
P REGULATION OF AXON REGENERATION BY DYING CELL-RECOGNITION SIGNALLING
Strahil Iv. Pastuhov*, Naoki Hisamoto, Kunihiro Matsumoto
Graduate School of Science, Nagoya University *pastuhov.strahil . [email protected]
Background: Understanding the mechanism of axon regeneration is an important first step in the developing of therapies for treating spinal cord injuries and other cases of nerve damage. When an axon is severed, the proximal fragment, which is connected with the cell body, can begin to regenerate by forming a growth cone at its tip. Conversely, the separated from the cell body distal fragment undergoes Wallerian degeneration and ultimately disappears. The JNK MAP kinase cascade and related signalling cascades were recently demonstrated to regulate axon regeneration in various species but many details about this signalling are missing.
Results: We discovered that in C. elegans the CED complex CED-2/CED-5/CED-12 (CrkII/DOCK180/ELMO), which during development regulates engulfment of apoptotic cells, also activates the JNK MAP kinase cascade to promote axon regeneration. Some evidence suggests that upstream of the CED complex function the alpha integrin subunit INA-1 and its associated non-receptor tyrosine kinase SRC-1, which also regulate engulfment of apoptotic cells.
Conclusion: These data suggest that the signalling cascade regulating the recognition of dying cells is co-opted to regulate axon regeneration as well. Now we are trying to identify the nature of the dying signal that promotes axon regeneration and to establish whether it is sent by the degenerating distal fragment of the severed axon.
Necrosis- programmed? Ridiculed and abused
Now loved, even by WEHI
UNDERSTANDING THE ROLE OF APOPTOSIS AND NECROPTOSIS IN BACTERIAL GUT INFECTION
Jaclyn S. Pearson1*, John Silke2,3 and Elizabeth L. Hartland1
1Department of Microbiology and Immunology, University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Austral ia 2The Walter and Eliza Hall Institute of Medical Research 3Department of Medical Biology, University of Melbourne *[email protected]
Background: Enteropathogenic E. coli (EPEC) is an extracellular gastrointestinal pathogen that intimately adheres to intestinal enterocytes and translocates a diverse repertoire of virulence (effector) proteins directly into enterocytes using a type III secretion system (T3SS). The effector proteins subvert a number of innate immune signaling pathways, including NF-κB activation and death receptor-mediated apoptosis and necroptosis. This suggests that these pathways are important for host immune defence.
Aims: In this project we aim to identify host immune defence pathways that are important for fighting bacterial gut infection and to use transcriptomic analysis of infected mouse colonic tissue to understand why mice deficient in these immune defence pathways suffer more severe disease, similar to that seen in inflammatory bowel diseases.
Results: Using the EPEC-like mouse pathogen C. rodentium, we showed that mice lacking key signaling proteins for apoptosis and necroptosis were highly susceptible to infection with C. rodentium. For example, mice deficient in Fas-mediated apoptotic signaling (Faslpr/lpr or Fasgld/gld) suffered severe diarrhoea and increased gut pathology compared to wild type C57BL/6 mice during C. rodentium infection. Mice deficient in apoptotic and necroptotic signaling pathways (Ripk1-/-/Ripk3-/-
/Casp8-/-) suffered even more severe pathology, with diarrheoa, severe weight loss and tissue damage during C. rodentium infection. The underlying basis of this increase in disease severity is unknown.
Conclusion: The characterisation of T3SS effector targets and activities allows the identification of enterocyte defence pathways important for pathogen resistance and clearance as the pathogen acts to avoid immune activation. We believe that our in vivo model of gut infection is an excellent model for understanding the underlying basis of inflammatory bowel diseases.
DELETION OF cFLIP IN THE EPIDERMIS RESULTS IN TNFR1-DEPENDENT AND INDEPENDENT LETHAL DERMATITIS
Xuehua Piao1, Sanae Miyake1, Sachiko Komazawa-Sakon1, Masato Koike2, Yasuo Uchiyama2, and Hiroyasu Nakano1
1Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo 143-8540, Japan. 2Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Previous studies have shown that tamoxifen-inducible epidermis-specific Cflip-deficient (CflipE-KO) mice develop severe dermatitis and dermatitis is attenuated by the blockade of tumor necrosis factor (TNF)α. However, it is unclear whether an TNFα-independent signal might be involved in the development of dermatitis in CflipE-KO mice. Here we show that CflipE-KO;Tnfrsf1a-/- mice were born at the expected Mendelian ratio, but still developed severe dermatitis and succumbed soon after birth. Apoptotic keratinocytes were scattered in the epidermis, and numbers of proliferating basal cells were increased in CflipE-KO;Tnfrsf1a-/- mice compared to Tnfrsf1a-/- mice. In sharp contrast, CflipE-KO;Tnfrsf1a+/- mice died in utero due to massive apoptosis of keratinocytes. Expression of a late differentiation marker, loricrin along with early differentiation markers, keratin 5 (K5) and keratin 14 (K14), in the epidermis of CflipE-KO;Tnfrsf1a-/- mice were comparable to those of Tnfrsf1a-/- mice at birth. Intriguingly, expression of loricrin, but not K5 or K14, were abrogated in the epidermis of CflipE-KO;Tnfrsf1a-/- mice at postnatal day 5. Together, these results suggest that cFLIP suppresses TNFR1-dependent and independent apoptosis of keratinocytes to maintain normal epidermal differentiation.
Bcl-2 to cell Never say die You bastard!
THE INTRINSIC PATHWAY OF APOPTOSIS PLAYS A ROLE IN THYMIC PROGENITOR FITNESS
Policheni A*, Grabow S, Bouillet P, Strasser A, Gray, D.
The Walter & Eliza Hall Institute of Medical Research *[email protected]
Background: Cell competition is a process whereby tissue integrity is maintained on the basis of cellular fitness. A number of studies have highlighted the importance of cell competition in T cell development and malignancy. Disruption of thymic cell competition can be achieved by grafting lobes from wildtype mice into immunodeficient mice, creating a situation where host progenitors are incapable of displacing donor. The ensuing reliance upon ‘old’ thymic progenitors for T cell development eventually leads to leukemia (T-ALL) development. It is currently unknown what processes governs competitive fitness or displacement.
Aim: To determine whether the intrinsic pathway of apoptosis in thymic progenitors plays a role in cell competition and whether its dysregulation can promote leukaemogenesis.
Results: We tested whether reducing cell survival diminishes progenitor fitness by grafting lobes of Bcl2-/- mice into immunodeficient mice (Rag2-/-
/gc-/-). Surprisingly, we found that these mice succumb to T-ALL at an accelerated rate (average latency of 100 days and 100% penetrance) as compared to Rag2-/-/gc-/- mice grafted with wildtype thymi (average latency of 250 days). Furthermore, we have identified a cellular phenotype associated with “winner” and “loser” thymic progenitors that will aid dissection of the cellular processes leading to T-ALL in situations of disrupted cell competition.
Conclusion: The dysregulation of the intrinsic pathway of apoptosis in thymic progenitors alters cell competition and subsequently leukaemia development.
References: Martins et al. 2014. Cell competition is a tumour suppressor mechanism in the thymus. Nature.
POSSIBLE SYNERGISTIC ROLES OF THE ENTEROPATHOGENIC ESCHERICHIA COLI TYPE III EFFECTORS NLEB AND NLEF
Georgina L. Pollock, Jaclyn S. Pearson, Elizabeth L. Hartland
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Austral ia *[email protected]
Background: During infection, the extracellular enteric bacterial pathogen enteropathogenic Escherichia coli (EPEC) translocates virulence (effector) proteins directly into the cytosol of infected enterocytes via a type III secretion system (T3SS). Once inside the host cell, these effector proteins subvert various immune signalling pathways including NF-κB mediated inflammation and death-receptor-induced apoptosis. One such effector protein is the Non-LEE encoded effector NleB, which inhibits extrinsic apoptotic signalling via the Fas death receptor. NleB transfers GlcNAc to Arg117 in the death domain of FADD and inhibits Fas ligand (FasL) stimulated caspase-8 cleavage. Another effector secreted by the T3SS is NleF. Previous studies have shown that NleF binds to and inhibits the activity of caspase 4, 8 and 9 in vitro.
Aim: We aim to understand contribution of the Non-LEE encoded effector NleF to EPEC pathogenesis.
Results: Here we demonstrate that a doxycycline inducible HeLa cell line expressing NleF inhibited FasL-induced cleavage of caspase-3 and RIPK1, which are both downstream of caspase-8 activation. However, unlike NleB, NleF had no effect on FasL driven caspase-8 cleavage. Hence, during EPEC infection, NleF may act synergistically with NleB to block caspase-8 dependent cell death pathways by acting downstream of NleB.
Conclusions/Future work: In future work we aim to understand the respective contributions of NleB and NleF to EPEC pathogenesis in vivo, in particular, how they work together to inhibit death receptor signalling. This work will advance our understanding of bacterial immune evasion strategies as well as the role of extrinsic cell death pathways in innate immunity.
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SOLUBLE CD52 INDUCES APOPTOTIC CELL DEATH IN THE INNATE IMMUNE SYSTEM
Maryam Rashidi*, Esther Bandala-Sanchez, Yuxia Zhang, Kate Lawlor, John Wentworth, James Vince, Leonard C. Harrison
The Walter & Eliza Hall Institute of Medical Research *[email protected]
Background: CD52 is a glycosyl-phosphatidylinositol (GPl)-anchored cell surface glycopeptide, but its physiological role has not been well defined. We have recently shown that CD52 released from activated T cells suppresses T-cell function by binding to the inhibitory sialic acid-binding immunoglobulin-type lectin 10 (Siglec-10) receptor. In the present study, we demonstrate that soluble CD52 can induce cell death in innate immune cells.
Results: Exposure of innate immune cells to low concentrations (10 μg/ml) of CD52-Fc potently inhibited NF-κB signaling and cytokine production in response to a range of inflammatory stimuli, including both TLR and TNFR ligands, without evidence of cell death. However, at higher concentrations (30 μg/ml and above) we observed that CD52-Fc induced rapid (within 24 h) cell death in human monocytes and mouse bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs). Cell death was caspase-dependent because the pan-caspase inhibitor Q-VD-OPh inhibited CD52-Fc-induced cell death. In addition, high-dose CD52-Fc induced cleavage of caspases 8 and 9, and its ability to induce cell death was significantly decreased in caspase-8-deficient BMDMs. CD52-Fc-induced cell death was not altered in Bak/Bax-deficient BMDMs. When cell death was inhibited by Q-VD-OPh, CD52-Fc still suppressed cytokine production, demonstrating that suppression of cytokine production by CD52-Fc is separable from and not due to its ability to cause cell death.
Conclusion: Our findings demonstrate that, in addition to suppressing NF-kB signaling and cytokine production, soluble CD52-Fc induces cell death in myeloid cells through the extrinsic apoptotic pathway, suggesting that CD52-Fc may have therapeutic potential for the treatment of myeloproliferative disorders.
HEPATOCYTE GROWTH FACTOR RENDERS BRAF MUTANT HUMAN MELANOMA CELL LINES RESISTANT TO PLX4032 BY DOWN-REGULATING THE PRO-APOPTOTIC BH3-ONLY PROTEINS PUMA AND BIM
Leona Rohrbeck1,2*, Andrew J Kueh1,2, Lin Tai1, Guillaume Lessene1,2, Andreas Strasser1,2 and Marco J Herold1,2
1 The Walter and Eliza Hall Institute of Medical Research 2 Department of Medical Biology, University of Melbourne *[email protected]
A large proportion of melanomas harbor the activating BRAFV600E mutation that renders these cells dependent on MAPK signaling for their survival. While the highly specific and clinically approved BRAFV600E kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we found that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its close relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very strong, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is secretion of Hepatocyte Growth Factor (HGF) from the tumor microenvironment or the cancer cells themselves that activates its receptor cMET, which is expressed on the surface of many melanoma cells. Interestingly, we found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through down-regulation of PUMA and BIM rather than increasing the expression of pro-survival proteins. Our results suggest that resistance to PLX4032 might be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signaling cascades.
STRUCTURE-GUIDED DEVELOPMENT OF NOVEL SMALL MOLECULE INHIBITORS OF BCL-XL
Michael J. Roy*, Amelia Vom, Peter M. Colman, Peter E. Czabotar and Guillaume Lessene
The Walter and Eliza Hall Institute, The University of Melbourne *[email protected]
Interactions between members of the BCL-2 family of proteins control the life/death fate of cells by regulating apoptosis. It is now well established that the ability to evade apoptosis is a genetic feature of many cancers, often via the over-expression of pro-survival members of the BCL-2 family, such as BCL-2, BCL-XL or MCL1. Such changes are not only a key step in the progression to cancer, but also an important mechanism by which cancer cells can become resistant to standard anti-cancer therapies.
Small-molecules able to mimic the activity of pro-apoptotic BH3-only proteins to reactivate apoptosis hold therefore significant potential as novel targeted chemotherapeutics, either as single agents in certain tumours, or to sensitise cancers to existing therapies. Whilst the development of small-molecules to block protein-protein interactions remains incredibly challenging, nonetheless a few validated ‘BH3 mimetic’ drugs have reached clinical and pre-clinical development [1].
Previous work at WEHI has led to the development of small molecules possessing a benzoylurea core which are able to mimic a-helical BH3 peptides and which bind to pro-survival BCL-XL with low micromolar binding affinity [2]. Co-crystal structures for a number of these compounds in complex with BCL-XL have been obtained and have opened the way for further structure-based medicinal chemistry efforts with the aim of improving the affinity and selectivity of this series.
This presentation will outline a number of successes, challenges, and new insights gained during this ongoing structure-guided medicinal chemistry campaign, including discovery of a novel 400 nM inhibitor of pro-survival BCL-XL.
[1] Roy MJ, Vom A, Czabotar PE, and Lessene G, ‘Cell death and the mitochondria: therapeutic targeting of the BCL-2 family-driven pathway’, Br J Pharmacol. (2013) 171(8), 1973.
[2] Brady, RM et al., ‘De-Novo Designed Library of Benzoylureas as Inhibitors of BCL-XL: Synthesis, Structural and Biochemical Characterization’ J Med Chem (2014) 57(4), 1323.
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MITOCHONDRIAL DAMAGE ELICITS A VIRAL RNA-DEGRADING INNATE IMMUNE RESPONSE
Tatsuya Saitoh*
Division of Molecular Genetics, Institute for Enzyme Research, Tokushima University, Japan *[email protected]
Innate immune system senses RNA virus by pattern-recognition receptors (PRRs) and protects hosts from virus infection. PRRs mediate the production of immune modulatory factors and the direct elimination of RNA virus. Here we show an unexpected role of mitochondria in PRR-mediated antiviral response. 2,3,7,8-tetrachlorodibenzo-p-dioxin-induible poly (ADP-ribose) polymerase (TIPARP), a CCCH-type zinc-finger containing protein, is a novel PRR that binds to sindbis virus (SINV) RNA via its zinc-finger domain. TIPARP recruits the exosome complex and induces degradation of SINV RNA. TIPARP basally localizes in the nucleus, but it accumulates in the cytoplasm after SINV infection, thereby targeting cytoplasmic SINV RNA. Redistribution of TIPARP is induced by reactive oxygen species (ROS)-dependent oxidization of the nuclear pore that affects the cytoplasmic-nuclear transport. Bax and Bak1, Bcl-2 family members, mediate mitochondrial damage to generate ROS after SINV infection. Thus, mitochondrial damage triggers RNA virus elimination that is mediate by TIPARP, a unique viral RNA sensing PRR
Disappearing pipettes Where are you my right hand fr iend?
Heart breaks for your loss
OLFACTORY INPUT-DEPENDENT SPATIAL REGULATION OF NEURONAL TURNOVER IN THE ADULT MOUSE OLFACTORY BULB
Masato Sawada and Kazunobu Sawamoto*
Nagoya City University *[email protected]
Background: Throughout life, new neurons are added and old ones eliminated by cell death in the adult mouse olfactory bulb. Previous studies suggested that olfactory experience controls the process by which new neurons are integrated into mature circuits. The mechanisms regulating this neuronal turnover, which underlies the plasticity and stability of the adult neural circuitry, are not fully understood, largely because of the difficulty of monitoring neurons over time in living adult mammals.
Aims: To investigate the mechanisms of neuronal turnover directly in vivo, we used a three-time-point two-photon laser-scanning microscopy (2PLSM) imaging technique.
Results: Using 2PLSM and sensory manipulations in adult live mice, we found that the neuronal turnover was dynamically controlled by olfactory input in a neuronal subtype-specific manner. Olfactory input enhanced this turnover, which was characterized by the reiterated use of the same positions in the glomeruli by new neurons.
Conclusion: This study suggests that the olfactory-input-dependent mechanisms of neuronal turnover control the balance between the structural plasticity and stability of glomeruli, thereby enabling active remodeling of the neuronal circuitry and maintenance of the glomerular structure, depending on the olfactory input.
Reference: Sawada, M., Kaneko, N., Inada, H., Wake, H., Kato, Y., Yanagawa, Y., Kobayashi, K., Nemoto, T., Nabekura, J. and Sawamoto, K. (2011). Sensory input regulates spatial and subtype-specific patterns of neuronal turnover in the adult olfactory bulb. The Journal of Neuroscience 31(32), 11587–96. doi:10.1523/JNEUROSCI.0614-11.2011
BFL-1/A1: THE FORGOTTEN PRO-SURVIVAL SIBLING OF THE BCL-2 FAMILY
Robyn L Schenk1,2*, Lin Tai1, Andreas Strasser1,2 & Marco J Herold1,2
1The Walter and Eliza Hall Institute of Medical Research 2Department of Medical Biology, University of Melbourne *[email protected]
Background: The BCL-2 family proteins are the critical regulators of apoptosis. The family members are classified as either pro-survival or pro-apoptotic proteins, and it is the balance between these two sub-groups that determines whether a cell will live or die. BFL-1, or A1 as it is known in mice, is one of the pro-survival members, but compared to its pro-survival siblings BCL-2, BCL-XL, BCL-W and MCL-1, little is understood about BFL-1/A1’s function in vivo. Generation of an A1 knockout mouse model has been difficult to achieve, due to the presence of three functional isoforms in mice (A1-a, A1-b and A1-d) that are in close proximity to one another in the gene locus, and have other functional genes interspersed between them. However, our lab has successfully generated a complete A1 knockout mouse model.
Aim: To characterize the role of the pro-survival protein BFL-1/A1 in vivo using the novel knockout mouse model.
Results: A1 in mice is a haematopoietic-specific protein, and thus I have analysed various immune cell subsets from wild-type and knockout haematopoietic organs. Overall, the knockout mice appear normal, with the exception of the B1-B cells, which were significantly reduced in the lymph nodes of knockout mice, and regulatory T cells (Tregs), which were decreased in the thymus and spleen. Furthermore, there was a significant decrease in the expression of the Treg-specific transcription factor FOXP3 in these organs.
Conclusion: At steady-state, mice lacking A1 appear relatively normal. There are some differences observed in B1-B cells and Tregs which warrant further investigation.
SINGLE-CELL MEASUREMENT OF IL-1b SECRETION DYNAMICS DURING PYROPTOTIC PROGRAMED CELL DEATH
Yoshitaka Shirasaki1,2,3*, Mai Yamagishi1,2,3, Nobutake Suzuki1,2, Miki Okamura1, Ting Liu4, Yoshifumi Yamaguchi4, Masayuki Miura4, Osamu Ohara3, and Sotaro Uemura1,2
1Department of Biological Sciences, Graduate School of Science, The University of Tokyo
2ImPACT Program of Council for Science, Technology and Innovation (Cabinet Office, Government of Japan)
3Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences ( IMS-RCAI) 4Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo *[email protected]
Background: Inflammatory cytokines play a key role in the initiation of inflammation. However, their secretion dynamics remains unclear especially at the single cell resolution because any conventional method cannot follow the real-time secretion process at the single-cell level. For example, intracellular staining can quantify pre-secreted cytokines within each single cell. On the other hand, enzyme-linked immunospot assay can detect secreted cytokines. Although both methods allow us to observe cytokines from a large number of single cells at once, they could offer data just as a snapshot of the accumulated amounts. To overcome this issue, we have developed real-time single-cell secretion imaging platform.
Aim: Reveal the dynamics of IL-1β production from individual cells during the pyroptotic cell death.
Results: Some research suggests that IL-1β has secreted through membrane pores that were formed by Caspase-1 via inflammasome activation. We verify this hypothesis by our real-time secretion assay coupled with monitoring of cell membrane integrity. First, we checked the correlation between IL-1β and loss of cell membrane integrity from human peripheral monocytes stimulated with LPS/ATP. We found that most of the IL-1β secreting cells were stained with the dead cell staining, SYTOX blue reagent. We next checked the chronological order and found that IL-1β secretion followed the loss of cell membrane integrity. Then we
mathematically analysed the IL-1b secretion signal and found that IL-1b showed burst secretion at the single-cell level.
Conclusion: Non-classical secretion of IL-1b during the pyroptotic cell death was transiently and temporally associated with loss of membrane integrity in individual cells.
References Shirasaki, Y., et al., Scientific Reports, 4, 4736, 2014 Liu, T., et al., Cell Reports, 8, 974-982, 2014
BIOPHYSICAL CHARACTERISATION OF THE TSR DOMAINS OF BAI 1 PROTEIN
Niccolay Madiedo Soler*, Ivan Poon and Marc Kvansakul
La Trobe University *[email protected]
Apoptosis, also called programmed cell death, occurs throughout life as a normal part of development, helping to maintain tissue homeostasis and the good function of living organisms. This process ensures the disposal of damaged, aged and infected cells in a well-organised manner. However, studies have shown that under failed or defective clearance, uncleared apoptotic cells contribute to a wide range of diseases processes linked to inflammatory diseases, autoimmunity and cancer.
Brain-specific angiogenesis inhibitor 1 (BAI1) protein has been proposed to play an important role in the clearance of apoptotic cells by the binding of phosphatidylserine (PS) through the thrombospondin type 1 repeats (TSR) domains in its extracellular region. Also, BAI1 protein has been suggested to promote the activation of the actin cytoskeleton of the phagocytes inducing the engulfment of dying cells (Park et al, 2007).
The aim of this project is to characterise how BAI1 TSRs mediate binding to PS on apoptotic cells, as well as identify whether all BAI1 TSRs participate in the binding to PS.
We have designed constructs for the expression and purification of the different TSRs in bacteria. However, expression tests have shown insolubility of expressed proteins. Purifications of soluble TSRs have shown low yield and instability which make challenging the concentration of purified TSRs for crystal trials.
DISTINCT ROLES FOR IAP FAMILY MEMBERS IN INNATE IMMUNE SIGNALLING
Che Stafford1*, John Silke1, Ueli Nachbur1
Cell Signall ing & Cell Death Division, The Walter & Eliza Hall Institute of Medical Research *[email protected]
Signaling via innate immune receptors triggers the activation of a range of transcription factors and the production of pro-inflammatory cytokines. Intracellular NOD1 and NOD2 receptors detect bacterial peptidoglycan and their activation is associated with Th17 differentiation. Receptor Interacting Kinase 2 (RIPK2) is activated downstream of the NOD receptors and recruits members of the Inhibitor of Apoptosis family (IAPs) to the signaling complex. However the exact identity and function of individual IAPs at the NOD signaling complex are a point of major dispute. Here we show that XIAP is required for NOD signaling, while cIAPs are dispensable using genetic, biochemical and pharmacological approaches. XIAP coordinates the temporal activation of signaling events by addition of K63 ubiquitin chains on RIPK2, which is essential for the orchestrated activation of transcription factors and the production of cytokines. cIAPs, on the other hand, regulate the strength of downstream signals but signaling events also occur in the absence of cIAP. This study provides evidence that targeted inhibition of individual IAPs has the potential for therapeutic intervention of inflammatory diseases such as multiple sclerosis and inflammatory bowel disease.
T
TLR3 EXACERBATES RADIATION-INDUCED GASTROINTESTINAL SYNDROME BY INDUCING EXTENSIVE CRYPT CELL DEATH
Naoki Takemura,1,2* and Satoshi Uematsu1,2
1Department of Mucosal Immunology, School of Medicine, Chiba University; 2Division of Innate Immune Regulation, International Research and Development Center for Mucosal Vaccines, Institute of Medical Science, The University of Tokyo *[email protected]
Background: High-dose ionizing radiation induces severe DNA damage in the epithelial stem cells in small intestinal crypts and causes gastrointestinal syndrome (GIS). Although the tumor suppressor p53 is a primary factor inducing death of crypt cells with DNA damage, its essential role in maintaining genome stability means inhibiting p53 to prevent GIS is not a viable strategy.
Aims: We aimed to investigate the involvement of innate immunity in the pathogenesis of GIS.
Results: Among the mice deficient in Toll-like receptors (TLR), we found that Tlr3-/- mice exhibited significantly milder GIS symptoms, including mortality, diarrhea and weight loss, than did their wild-type mice. Tlr3-/- mice also showed markedly reduced radiation-induced crypt cell death in the small intestine and avoided villous destruction owing to re-epithelization from surviving crypts. Despite the overall reduction of crypt cell death, p53-dependent crypt cell death was not impaired in Tlr3-/- mice. p53-dependent crypt cell death caused leakage of cellular RNA, which induced extensive cell death via TLR3. An inhibitor of TLR3-RNA binding, (R)-2-(3-chloro-6-fluorobenzo [b] thiophene-2-carboxamido)-3-phenylpropanoic acid, ameliorated crypt cell death and GIS.
Conclusion: TLR3 functioned downstream of p53 and caused lethal disturbance of GI tract by inducing extensive crypt cell death following irradiation. Our findings provide a new framework to understand the pathogenesis of GIS and suggest blocking TLR3 activation as a novel therapeutic approach for treatment of GIS.
References: Takemura et al. Nat Commun. 2014 Mar 18;5:3492.
Bye bye dead cell How I wonder how you died Then your story I could tel l
DETERMINING THE MOLECULAR COMPONENTS OF APOPTOTIC CELL DISASSEMBLY
Tixeira R*1., Nedeva C1., Atkin-Smith G.S. 1, Phan T.K. 1,Puthalakath H.
1, Herold M. 2, Hulett M. 1 and Poon I.K.H. 1
1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Austral ia 2 The Walter and Eliza Hall Institute, Parkvil le *rt [email protected]
Background: Apoptotic cell death is an important process in maintaining homeostasis in the body. Efficient clearance of apoptotic cells is essential, whereby failure of this process has been linked to various diseases including autoimmune, cancer and inflammation. Efficient clearance of apoptotic cells involves the disassembly of apoptotic cells into smaller fragments known as apoptotic bodies. Various morphological changes including circular membrane bludges called blebs and string like membrane protrusion (apoptopodia) can facilitate formation of apoptotic bodies. Inhibitor based studies have shown that Rho Kinase 1 (ROCK1) and P21 activated kinase 2 (PAK2) are involved in membrane blebbing, while membrane protein Pannexin 1(PANX1) is a negative regulator of apoptotic body formation. Yet the importance of these regulators of apoptotic cell disassembly and their implication on cell clearance is not well understood.
Aims: Generate knockout cell lines for ROCK1, PAK2 and PANX1 to address their specific role in apoptotic cell disassembly and clearance.
Results: ROCK1, PAK2 and PANX1 genes were successfully disrupted in Jurkat cells using CRISPR technology. Functionally, ROCK1 is able to block blebbing but, is not necessary for apoptopodia formation. Loss of PAK2 did not show any changes to the apoptotic cell disassembly, while lack of functional PANX1 remarkably increased fragmentation into apoptotic body.
Conclusion: These results confirm previous inhibitor studies showing that both positive regulation by ROCK1 and negative regulation via PANX1 play a role in the controlled disassembly of dying cells, while PAK2 did not play a role. Future work using these gene disrupted cell lines can provide insight on the functional significance of apoptotic cell disassembly.
U A DISTURBANCE IN THE FORCE: BAK DIMERS RANDOMLY AGGREGATE TO RUPTURE THE MITOCHONDRIAL OUTER MEMBRANE DURING APOPTOSIS
Rachel T Uren*, Martin O’Hely, Sweta Iyer, Ray Bartolo, Amber E Alsop, Dana Westphal, Grant Dewson, Ruth M Kluck
The Walter & Eliza Hall Institute of Medical Research *[email protected]
Apoptotic pore formation by Bak and Bax involves major conformation change followed by dimerization via a reciprocal BH3:groove interaction. How dimers associate to high order oligomers to drive pore formation is not known, although linkage has identified several points at which dimers can associate. Crystal structures and cysteine scanning mutagenesis indicate that the core of the dimer (α2-α5 domains) dimer and α6 adopt an in-plane topology while α9 forms a transmembrane domain. To gain an overall view of Bak topology after pore formation we extended cysteine labelling and linkage to other Bak regions. Extensive cysteine linkage of the N- and C-extremities indicated these regions of the Bak dimer were highly mobile, whereas the core domain dimer was constrained. We identified a region centered on V61 in the flexible solvent-exposed N-terminus where cysteines could link completely between dimers but not within dimers, providing a novel measure of higher order oligomerization. The V61C:V61C’ linkage pattern indicated that dimers can aggregate in complexes greater than 16 Bak molecules, but aggregation occurs in an irregular and detergent-labile fashion rather than via a single protein:protein interface. Mathematical modelling of our linkage data also support random aggregation of dimers. We discuss how irregular association of dimers might porate the mitochondrial outer membrane.
V
MOLECULAR ARRANGEMENT OF THE APOPTOTIC CASPASE, PROCASPASE-8, AT A DEATH INDUCING COMPLEX
Parimala R Vajjhala1*, Alvin Lu2,3, Darren L Brown4, Siew Wai Pang1, Vitaliya Sagulenko1, David P Sester1, Simon O Cridland1, Justine M Hill1, Kate Schroder4, Jennifer L Stow4, Hao Wu2,3, Katryn J. Stacey1, 4
1School of Chemistry and Molecular Biosciences, The University of Queensland, 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 3Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, and 4Institute for Molecular Bioscience, The University of Queensland *[email protected]
Background: Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerisation of its pyrin domain (PYD) into filaments. ASC PYD has also been shown to recruit procaspase-8 to inflammasomes.
Aims: To investigate the interaction between procaspase-8 and ASC in inflammasomes and to gain insights into the molecular arrangement of procaspase-8 at inflammasomes that leads to its activation.
Results: ASC PYD interacts with the procaspase-8 tandem death effector domains (DEDs). The interaction optimally required both DEDs and represents an unusual heterotypic interaction between domains of the death-fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Furthermore, clustered ASC nucleates procaspase-8 death effector domain (DED) filaments in vitro and in vivo suggesting that procaspase-8 DED filaments are initiated from ASC PYD filaments. Finally, we observed condensation of procaspase-8 filaments containing the catalytic domain suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation.
Conclusion: The interaction of ASC and procaspase-8 via conserved death-fold interaction modes rationalizes crosstalk between inflammatory and apoptotic signalling pathways. Initiation of procaspase-8 filaments and condensation are likely to be important for procaspase-8 activation and procaspase-8 filaments may also be relevant to apoptosis induced by death receptors.
Cell death research
As painful as an error bar In three dimension
FOXO3 SUPPRESSES MYC-DRIVEN LYMPHOMAGENESIS
Cassandra J Vandenberg,1,2* Noboru Motoyama3 and Suzanne Cory1,2
1 Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research 2Department of Medical Biology, University of Melbourne, Victoria 3010, Austral ia 3Department of Human Nutrit ion, Sugiyama Jogakuen University School of Life Studies, 17-3 Hoshigaoka-motomachi, Nagoya, Japan
The PI3kinase/Akt pathway is frequently activated in cancer cells, leading to the cytoplasmic localisation of FoxO transcription factors and their consequent inactivation. Some of the transcriptional targets of FoxO proteins include pro-apoptotic BH3-only proteins Bim and Puma and cell cycle inhibitors p27Kip1 and p130Rb2, suggesting that loss of FoxOs should promote cell survival and proliferation. Consistent with this, combined deletion of FoxO1, 3 and 4 in haemopoietic cells has revealed they are tumour suppressors, with mice developing thymic lymphomas and haemangiomas.1 The FoxOs appear to act redundantly, as single FoxO null mice have relatively mild phenotypes. However, we hypothesized that loss of a single FoxO might cooperate in tumorigenesis when combined with deregulation of an oncogene. Using two different mouse models, we show that FoxO3 has a significant tumour suppressor function in the context of Myc-driven lymphomagenesis. Loss of FoxO3 significantly accelerated myeloid tumorigenesis in vavP-MYC10 transgenic mice and B lymphomagenesis in Eµ-myc transgenic mice. While no difference in cell cycling was observed, cells from pre-neoplastic mice lacking FoxO3 were found to have enhanced survival capacity in in vitro assays. 1 Paik et al., Cell 128, 309-323, 2007.
POST-TRANSLATIONAL REGULATION OF INTERLEUKIN-1β
Swarna Vijayaraj*, Laura Dagley, Andrew Webb, Kate Lawlor and James Vince
The Walter & Eliza Hall Institute of Medical Research
Background: IL-1β is a potent proinflammatory cytokine that requires caspase-1, a cysteine protease, to cleave its inactive precursor form, pro-IL-1β, to its active secreted p17 fragment(1). Excessive IL-1β secretion causes autoinflammatory diseases such as CAPS (Cryopyrin associated periodic syndromes) and gout (2). Despite its major role in inflammatory diseases, the post-translational regulation of IL-1β is poorly understood.
Aims: We aim to examine and define post- translational modifications of IL-1β.
Results: We observed endogenous IL-1β was ubiquitylated in response to both TLR4 ligand LPS and TLR 1/2 ligand pam3Cys. In accord with previous studies we observed NLRP3 ubiquitylation as well in response to TLR ligands mentioned above. Mass spectrometry analysis revealed ubiquitylation at K133 residue and phosphorylation at S134 of pro IL-1β. Interestingly, in vitro studies showed ubiquitylation of IL-1β construct where all the conserved lysine residues were mutated to alanine. This hints to a non-canonical ubiquitylation mechanism. With MG132, a proteasome inhibitor pro IL-1β ubiquitylation is stabilized suggesting ubiquitylation could be involved in the stabilization of the protein.
Conclusion: endogenous pro-ILβ undergoes post-translational modification by both ubiquitylation and phosphorylation.
References: 1. F. Martinon, A. Mayor, J. Tschopp, in Annual Review of Immunology. (Annual
Reviews, Palo Alto, 2009), vol. 27, pp. 229-265. 2. P. Menu, J. E. Vince, The NLRP3 inflammasome in health and disease: the good,
the bad and the ugly. Clinical and Experimental Immunology 166, 1 (Oct, 2011)..
EVOLUTION OF CELL DEATH RESPONSES TO CYTOSOLIC DNA
Nazarii Vitak1*, Karyn Johnson2, David Sester1 and Katryn Stacey1
1 School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Austral ia; 2 School of Biological Sciences, University of Queensland
Background: Eukaryotic cells sequester their DNA in the nucleus and organelles. The presence of DNA in the cytosol indicates a danger, such as infection, activity of retrotransposons or DNA damage. In mammals cytosolic DNA is recognized by AIM2, leading to a rapid lytic death of macrophages, termed pyroptosis. There is no data about such a system for invertebrates, and AIM2 itself is a mammalian-restricted protein.
Aim: We hypothesized that defence against cytosolic DNA is a feature of all eukaryotic cells, and fundamental to maintenance of genome integrity as well as recognition of infection.
Results: To investigate responses to cytosolic DNA in Drosophila or non-mammalian vertebrates which lack AIM2, we transfected both fruitfly and chicken cells with DNA via electroporation and assessed viability. Cytosolic DNA but not double stranded synthetic RNA was toxic for both Drosophila and chicken cells, and elicited rapid lytic cell death phenotypically like pyroptosis. Both single- and double stranded DNA induced cell death in fruitfly cells, in contrast to the mammalian AIM2 response that is mediated by only double stranded DNA. Further experiments showed the existence of a similar lytic pathway of cell death in mammalian non-macrophages that lack AIM2.
Conclusion: An ancient AIM2-independent mechanism of DNA recognition in the cytosol leads to rapid lytic cell death in insects, birds and mammals. Elucidation of the molecular basis for this DNA recognition will allow determination of whether it plays a role in defence against infection or in guarding genome integrity.
Y USING IN VIVO SHRNA LIBRARY SCREENS TO IDENTIFY NOVEL TUMOUR SUPPRESSORS
Bruce Yang*, Ana Janic, Stephen Wilcox, Marco Herold, Andreas Strasser
Molecular Genetics of Cancer Division, The Walter & Eliza Hall Institute of Medical Research *[email protected]
Background: The tumour suppressor gene p53 is the most frequently mutated gene in human cancers. P53 is a transcription factor that can be activated by diverse stimuli, including DNA damage, hypoxia and, importantly for tumour suppression, induction of certain oncogenes. Once activated, p53 can trigger a broad range of cellular responses, including cell cycle arrest, cellular senescence and cell death (apoptosis). The cyclin dependent kinase inhibitor (CDKI) p21 is critical for p53-mediated induction of cell cycle arrest and senescence, whereas the pro-apoptotic BH3-only BCL-2 family members PUMA and (to a lesser extent) NOXA are essential for p53-mediated apoptosis. Surprisingly, we found that mice lacking PUMA, NOXA and p21 are not cancer prone, despite the failure of cells derived from these mice to undergo p53 mediated apoptosis, cell cycle arrest and senescence. In contrast, all p53-deficient mice succumbed to lymphoma or other tumours between 120-250 days [1].
Aim: To identify novel p53 target genes or even p53 independent genes that function as tumour suppressors.
Results: An shRNA pool was introduced into wt or puma-/- p21-/- hematopoietic stem and progenitor cells, which were transplanted into irradiated recipient mice. After 350 days, about 30% mice from each group developed tumour, from which a few genes were selected, indicating their function in repressing tumorigenesis.
References 1. Valente, Liz J., et al. "p53 efficiently suppresses tumor development in the complete absence of its cell-cycle inhibitory and proapoptotic effectors p21, Puma, and Noxa." Cell Reports 3.5 (2013): 1339-1345.
CASPASE-8 AND RIP KINASE REGULATE RETINOIC ACID-INDUCED CELL DIFFERENTIATION
Masataka Someda, Shunsuke Kuroki, and Shin Yonehara*
Graduate School of Biostudies, Kyoto University *[email protected]
Caspase family members are involved in execution of apoptosis. Among them caspase-8 is unique with associated critical activities to induce and suppress death receptor-mediated apoptosis and necrosis, respectively. Caspase-8 inhibits necroptosis, a form of necrosis, through suppressing the function of receptor interacting protein kinase 1 (RIP1 or RIPK1) and RIP3 (RIPK3). Disruption of caspase-8 expression causes embryonic lethality in mice at embryonic day (E) 11.5, which is rescued by depletion of RIP3, suggesting that the defect of caspase-8-deficient embryos is traced back to its activity to suppress necroptosis.
To examine the role of caspase-8 in cell differentiation, we established ES and other types of cells with tetracyclin/doxicyclin-inducible (Tet-On) expression system of short hairpin RNA specific for caspase-8.
Induced knockdown of caspase-8 expression was shown to markedly enhance retinoic acid (RA)-induced differentiation of embryonic stem (ES) cells as well as RA-induced gene expression in various types of mouse and human cell lines. In addition, RA-dependent gene expression was strengthened in caspase-8-deficient mouse embryos around E 10.5. The marked enhancement of RA signaling was dependent on RIP1 and RIP3 but independent of MLKL, an essential molecule for execution of necroptosis. Knockdown of caspase-8 expression in ES cells induced complex formation of retinoic acid receptor (RAR) to RIP1 and RIP3 in nucleus, and RA treatment induced binding of the complex to retinoic acid response element (RARE) at the promoter region of RA-responsible genes. Furthermore, administration of RA antagonist to pregnant mice significantly suppressed the lethality of Caspase-8-deficient embryos at E 11.5.
Thus abnormality of Caspase-8-deficient embryo which is dependent on both RIP1 and RIP3, was shown to be induced by not only excess of necroptosis but also abnormal promotion of RA signaling, both of which are dependent on RIP1 and RIP3.
ACKNOWLEDGEMENTS
Jaci Hoysted - Organisation Lisa Trinh - Organisation
Catherine McLean - Meeting Booklet David Vaux - Rubber Chickens
Che Stafford - Chauffeur Boris Reljic - Chauffeur
Margs Brennan - Chauffeur & Organisation James Murphy - Organisation
Paul Ekert - Sommelier Amy Vaux, Ben Silke, Jonathan Bernardini - Photography
Iain McLean - Beer Dylan Silke - Barman
Denise Heckman, Zikou Liu - Reception Drew Berry - Illuminarium
James Murphy, Kate Stacey, Catherine McLean, David Vaux, Chunyan Ma, Diep Chau, Sharad Kumar, Maria Tanzer, Warren Pavey, Rebecca
Feltham, Jiang Lanzhou, Marcel Doerflinger, Joanne Hildebrand, Rachel Uren, Parimala Vajjhala, Strahil Pastuhov - Haiku (mostly)
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