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A Phenotypic Screening Assay For Diamond Blackfan Anemia (DBA) Candidate Drugs
JOHAN FLYGARE
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Early erythroid progenitor
(BFU-E)
Epo-dependent erythroid precursor
(CFU-E)
Hematopoietic stem cell
(HSC)
Red blood cell (RBC)
Epo
Normal erythropoiesis (red blood cell production).
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Early erythroid progenitor
(BFU-E)
Epo-dependent erythroid precursor
(CFU-E)
Hematopoietic stem cell
(HSC)
CFU-Es generate < 60 RBCs in response to erythropoietin
BFU-Es generate millions of RBCs
Red blood cell (RBC)
Epo
Normal erythropoiesis (red blood cell production).
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Early erythroid progenitor
(BFU-E)
Epo-dependent erythroid precursor
(CFU-E)
Hematopoietic stem cell
(HSC)
Erythropoietin-based drugs are used to treat anemia
Red blood cell (RBC)
Epo
Normal erythropoiesis (red blood cell production).
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Early erythroid progenitor
(BFU-E)
Epo-dependent erythroid precursor
(CFU-E)
Hematopoietic stem cell
(HSC)
Red blood cell (RBC)
Epo
New drugs needed for anemias caused by lack ofEpo-responsive progenitors
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Early erythroid progenitor
(BFU-E)
Epo-dependent erythroid precursor
(CFU-E)
Hematopoietic stem cell
(HSC)
Red blood cell (RBC)
Patients with Diamond-Blackfan anemia (DBA) lack Epo-responsive precursors
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Diamond-Blackfan anemia (DBA) is a congenitalhypoplastic anemia
"Hypoplastic anemia"(Diamond and Blackfan Am J Dis Child, 1938)
Louis K. Diamond (1902 - 1999) Kenneth Blackfan (1883 - 1941)
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The bone marrow failure syndromeDiamond-Blackfan Anemia (DBA)
• Rare disorder, approx 1 / 200 000 world-wide
• Infant with reticulocytopenic anemia:
• Reticulocytes
• MCV • HbF
• eADA
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The bone marrow failure syndromeDiamond-Blackfan Anemia (DBA)
• Very few red cell precursors in normocellularmarrow
(adults often develop hypoplastic bone marrow)
• Congenital anomalities
• Family history (≈45% are inherited autosomaldominant with incomplete penetrence)
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Current treatments for DBA are ineffective and/or associated with severe adverse effects
• Prednisone (severe corticosteroid side effects!)- 60-80% respond initially (unknown mechanism)- 40% remain on corticosteroid therapy
• Transfusions and chelation(iron overload can become lethal)
• Hematopoietic stem cell transplant (GVHD, graft failure, infectons etc. )
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DBA genetics: Ribosomal protein gene mutationsGene Chromosome Inheritance Frequency in DBARPS19 19q13 Autosomal Dominant ≈ 25%RPL5 1p22.1 Autosomal Dominant ≈ 9%RPL11 1p36 Autosomal Dominant ≈ 6%RPL26 17p13 Autosomal Dominant ≈ 6%RPS10 6p Autosomal Dominant ≈ 3%RPS24 10q22 Autosomal Dominant ≈ 3%RPL35A 3q29 Autosomal Dominant ≈ 2%RPS7 2p25 Autosomal Dominant <2%RPS17 15q Autosomal Dominant <2%RPS26 12q Autosomal Dominant <2%RPL19 17q12 Autosomal Dominant <2%RPL15 3p24 Autosomal Dominant <2%RPS29 14q Autosomal Dominant <2%RPL31 2q11 Autosomal Dominant <2%Gata1 X X-chromosome linked recessive <2%Unknown ≈ 35%
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Ribosomal protein haploinsufficiency in DBA
DBA ribosomal protein mutationsaffect only one of the alleles = haploinsufficient levels of oneribosomal protein
RPS19 mutated in 25 % of patients
Eukaryotic ribosome
60S
40S
28S rRNA5S rRNA
5.8S rRNA≈49 ribosomal proteins
18S rRNA≈33 ribosomal proteins
RPS19
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DBA disease model needed to determine whyribosome protein deficiency causes anemia.
BFU-E ErythroblastHSC
Pathophysiology?
RBC
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Good disease model systems needed in order todevelop new drugs for DBA
BFU-E ErythroblastHSC
Pathophysiology?
Drugs?
RBC
Epo
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Doxycycline-inducible mouse model for RPS19-deficient DBA
RPS19-shRNA
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Doxycycline-inducible mouse model for RPS19-deficient DBA
Doxycycline
RPS19-shRNA
miR30-RNA
SA-2xpA tetO CMV RPS19-miR30-shRNA SV40pACol1A1locus
More Doxycycline = Less RPS19Breakdown of RPS19 mRNA
Less RPS19 protein
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Doxycycline-inducible Rps19 shRNA mouse tomodel DBA
Doxycycline
Anemia
In vitro proliferation defect
Jaako et al. Blood 2011
Severe bone marrow failure
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In vivo: mild anemia to severe bone marrowfailure depending on doxycycline dose.
Peripheral blood after 17 days with different doxycyclin doses in drinking water
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In vivo: DBA mouse responds to corticosteroidtreatment similar to patients.
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DBA mouse studies to test hypotheses regardingDBA disease mechanisms
BFU-E ErythroblastHSC
Pathophysiology?
RBC
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Hypothesis 1: “Nucleolar stress” leading to p53 activation causes anemia in DBA.
Holmberg Olausson et al. Cells 2012, 1(4), 774-798
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Hypothesis confirmed: removal of p53 rescuesanemia in DBA.
Graph from Jaako et al Blood 2011
(mild RPS19 deficiency)(severe RPS19 deficiency)
P53 KOwt
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Hypothesis 2: DBA is in part caused by reducedprotein translation.
Normal cells RPS19-deficient (DBA) cells
40S
60S
80S Polysomes 40S
60S
80S Polysomes
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If DBA is caused by inefficient protein translation, try to give Leucine to patients.
• Hematologic response in one DBA patient reported (Pospisilova D et al. Haematologica 2007; 92(5):e66-67)
• An “essential” amino acid – not stored and must be supplied by a dietary source
• One of the branched chain amino acids (BCAA)• BCAAs are the only amino acids not metabolized by the liver• Leucine has been studied for its ability to promote muscle mass.
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L-leucine improves the anemia in Rps19-deficient mice
******
n=10-38
± L-leucine
Controls & [D/D]Day 0
DoxycyclineDay 14
Blood analysis
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Leucine is currently tested DBA patients in Europeand USA.
• Hematologic response in one DBA patient reported (Pospisilova D et al. Haematologica 2007; 92(5):e66-67)
• 4/5 others with partial responses (Pospisilova, ICC 2008)
• US pilot study opened June 2013
Adrianna Vlachos, [email protected]
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DBA disease mechanisms:
BFU-E ErythroblastHSC RBC
Nucleolar stress p53
Reduced protein
translation
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Develop assay based on mouse model toscreen for new drugs and drug targets?
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In vitro: Healthy progenitors generates >20-fold more erythroblasts than Rps19-deficient cells
Medium containing SCF and Epo
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Phenotypic Screening to identify DBA modifyinggenes and candidate drugs
Compound screens
RNAi screens
BFU-E CFU-Es
DBA-modifying genes
DBA-modifying compounds
Pathobiology
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Phenotypic Screening to identify DBA modifyinggenes
RNAi screens
CFU-Es
DBA-modifying genes?
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Identify drug target/disease modifier genes through shRNA screens
Day 0 Day 3 Day 7
BFU-E cells from DBA mouse
Genome-wide shRNA library
= shRNA silencing of a specific gene that rescues erythroid proliferation in DBA
700-gene custom shRNA library
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>700 genes targeted (5 hairpins /gene)
• Target genes picked based on:– Expressed in erythroid cells (Flygare et al. Blood 2011).– Upregulated in Rps19-deficient mouse (Pekka Jaako, unpublished).– Upregulated in DBA patient cells (Gazda et al Stem Cells 2006).
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RNAi screen of genes rescuing defective proliferationin RPS19-deficient cells
Day 7End point
Day 0Lentiviral
transduction+ Dox administration
Day 3Starting-point
3500 hairpin library
Deep sequencing of integrated shRNA clones
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Cells transduced with ≈100 hairpins increase>50% in the bulk population over time
p53 among top hits indicates a satisfactory screen setting
Top enriched genes (≥2 shRNA/gene) were chosen for validation
n=10
Day 7 representation (log2 scale)
Day
3 re
pres
enta
tion
(log2
sca
le)
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Increase in GFP positive (silenced target) cells over time demonstrates rescued proliferation
Luciferase control, hairpin #1Luciferase control, hairpin #2Target X, hairpin #1Target X, hairpin #2
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Ongoing: validation of potential drug targetsidentified through shRNA screens
RNAi screens
CFU-Es
Several DBA-modifying genes identified!
Ongoing:
Transplant hematopoietic stem cells with silenced target gene to DBA mice
Cross DBA mice with target gene KO mice
Novel understanding of pathobiology
Novel therapeutic appraoches?
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Phenotypic Screening to identify candidate DBA drugs
Compound screens
BFU-E CFU-EsDBA-modifying compounds
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CellTiter-Glo® Luminescent Cell Viability Assay(ATP content), readout for rescued proliferation
• Develop an in vitro assay to screen multiple libraries
Cell lines, sorted primary cells, different read outs etc.Cell numbers, culture time, induction time : Z factor
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Compound screening logistics
1. Isolation of livers from embryos of 13 days pregnant transgenic mice
2. Isolation of cells expressing c-KIT, a strongly expressed surface marker of hematopoietic stem cells
3. Express shipment on ice to CBCS (KI) twice weekly –375 miles in 4.5 hours
LUND STOCKHOLM
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Small molecule screens: >12 000 compounds screened at 2 and 10uM.
Hit validation + analogues
Understand structure-activity relationship an identify target
DBA mechanisms and drug candidate er
ythr
oid
prol
ifera
tion
resc
ue
Healthy controlDBA + drugDBA control
176249081
Conc [M]1x10-7 1x10-6 1x10-5
%A
ctiv
atio
n
-30
-20
-10
0
10
20
30
40
Siva et al. ASH poster 2013
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Most hits in the screen reduce apoptosis in RPS19-deficient erythroid cells.
Compounds 1-21 are verified hits from the screen
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The mechanism is not through increasing RPS19 expression
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Several hits have known targets of which somehave been identified in other screening assays.
• Example: SB431542 (ALK4,5,7 inhibitor). Also identified in a screen on human DBA patient iPS cells by George Daley, Harvard Medical school.
SB431542
20uM 10uM 5uM 1uM
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SB431542 (ALK4,5,7 inhibitor) maintainsprogenitor cells and rescues apoptosis
Control SB431542 M SB431542
Hypothesis: TGF-B is a potential DBA therapy
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However, no correlation between drugs inhibitingAlk5 and drugs rescuing DBA…
Compound Described Target Rescues proliferation arrest induced by TGF-B Rescues DBA
SB 431542 TGF-b type I receptor/Alk5, and its relatives ALK4 and ALK7 ✔ ✔
SB 505124 TGF-b type I receptor/Alk5 ✔
SB 525334 TGF-b type I receptor/Alk5 ✔
Repsox TGF-b type I receptor/Alk5 ✔
GW 788388 TGF-b type I receptor/Alk5 ✔
LY 364947 TGF-b type I receptor/Alk5 ✔
SD 208 TGF-b type I receptor/Alk5 ✔ ✔
LY 210976 TGF-β receptor type I/II inhibitor ✔
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Several hits are likely kinase inhibitors.Do they have a common target kinase?
Send top hits for KINOMEscan® profiling
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KinomeScan (468 kinases) on the two mostpotent hits reveal two common target kinases.
Compound A
Compound B
Hypothesis: The two commonlytargeted kinases arepotential DBA drugtargets
% re
mai
ning
act
ivity
% re
mai
ning
act
ivity
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Ongoing: Validate mechanism and in vivo efficacyof novel candidate DBA drugs
Compound screens
BFU-E CFU-Es
Potential DBA-drugs identified!
Ongoing:
Knock down target genes
Treat DBA mice with compounds
Novel understanding of pathobiology
Novel DBA drugs?
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Summary: prolifertion-based phenotypic screensidentified novel strategies for DBA therapy
Compound screens
RNAi screens
BFU-E CFU-Es
DBA-modifying genes
DBA-modifying compounds
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Acknowledgements
Lund University• Sara Sjögren• Kavitha Siva• Stefan Karlsson• Pekka Jaako• Fredrik Ek• Roger Olsson
Freiburg University • Marcin WlodarskiBroad Institute (shRNA
screen)• David Root • John Doench
Karolinska Institute (drug screen)• Lars Johansson• Thomas Lundbäck• Hanna Axelsson• Anna Lena Gustavsson
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