multiple myeloma and lxr ligands : a matter of life and death ?
DESCRIPTION
PIBIC PROGRESS – 6 June 2013. Multiple Myeloma and LXR ligands : a matter of life and death ?. AIDA PANICCIA PhD Student Russo’ s Lab Cancer Gene Therapy Unit Division of Molecular Oncology. Multiple Myeloma (MM). - PowerPoint PPT PresentationTRANSCRIPT
Multiple Myeloma and LXR ligands:
a matter of life and death?
AIDA PANICCIAPhD StudentRusso’s Lab
Cancer Gene Therapy UnitDivision of Molecular Oncology
PIBIC PROGRESS – 6 June 2013
2
Multiple Myeloma (MM)
INCURABLE
B-cell neoplasia, characterized by the accumulation of malignant plasma cells in the bone marrow (BM)
•
Sympthoms: osteolytic bone destruction, renal failure, anemia, hypercalcemia
•
Treatment: - bortezomib, thalidomide, lenalidomide- autologous stem cell trasplantation
•
3
BM new vessels
VEGF
IGF-1
IL6TNFa
Collagen fibres
MM CELL
BM STROMAL CELL
cFLIP
FADD
Pro-caspase 8
Fas
MAPK-pathwayPI3-K/AKT-pathway
JACK-STAT
FibronectinICAM-1
LFA-1
VLA-4
VCAM
LXR LIGANDS?/OXYSTEROLS
Aim of the study
4
CHOLESTEROL ESTER
OXYSTEROLS
oxydized fatty acidis
RXR LXR
ABCG1 gene
ABCG1LXR ligands are products of cholesterol oxidation (OXYSTEROLS)
LXR is a nuclear receptor
LXR/LXR ligand signalling
FUNCTIONS1) regulation of cholesterol and fatty acids metabolism2) modulation of immune response
-
-
5
LXR-mediated immunosuppression
Tumor cellsMyeloyd dendrtic cells
CCR7
Lymph node
LXRLXR ligands/Oxysterols
Villablanca et al. Nat Med 2010
6
Myeloma and LXR ligands
LXR LIGAND PRODUCTION - Players- Regulation
LXR LIGAND EFFECTS- Indirect effects- Direct effects
THERAPEUTIC STRATEGIES BASEDON LXR SIGNALLING INACTIVATION
7
- No direct assay
- Indirect measurement by evaluating the effects on myeloyd dendritic cells
Tumor cells Myeloyd dendrtic cells
LXR ligands/Oxysterols
CCR7
Lymph node
LXR ACTIVATION(ABCG1 induction)
CCR7 INHIBITION
LXR
LXR ligand measurement
8
LXR ligand production: myeloma cells
MM cells (primary and cell lines) produce LXR ligands
CELL LINES
*******
**
ABCG
1 IN
DUCT
ION
LXR ACTIVATION
PRIMARY CELLS%
OF
CCR7
INHB
ITIO
N
CCR7 INHIBITION
** **
9
Acetyl-CoA
HMG-CoA
Mevalonic Acid
Squalene
2,3-Monoepoxysqualene
Lanosterol
Cholesterol
2,3;22,23-Diepoxysqualene
24(S),25-Epoxylanosterol
24(S),25-Epoxylcholesterol
LXR
Strategies to inactivate LXR/LXR ligands
10
Acetyl-CoA
HMG-CoA
Mevalonic Acid
Squalene
2,3-Monoepoxysqualene
Lanosterol
Cholesterol
2,3;22,23-Diepoxysqualene
24(S),25-Epoxylanosterol
24(S),25-Epoxylcholesterol
LXR
SULT2B1b-SO3H
-SO3H
-SO3H-SO3H -SO3H
-SO3H Inactivates LXR ligands by sulfurylation
Strategies to inactivate LXR/LXR ligands
**
LXR ACTIVATION
ABCG
1 IN
DUCT
ION
11
BM new vessels
VEGF
IGF-1
IL6TNFa
Collagen fibres
MM CELL
BM STROMAL CELL
cFLIP
FADD
Pro-caspase 8
Fas
MAPK-pathwayPI3-K/AKT-pathway
JACK-STAT
FibronectinICAM-1
LFA-1
VLA-4
VCAM
LXR LIGANDS?/OXYSTEROLS
Aim of the study
12
Stromal cells within lymphoid organs produce LXR ligands, which are able to position B cells within follicles (Yi et al. Immunity 2012)
BM stromal cells (primary and cell lines) produce LXR ligands
LXR ligand production: BM stromal cells
CELL LINES
*****
ABCG
1 IN
DUCT
ION
PRIMARY CELLS
** *****
ABCG
1 IN
DUCT
ION
LXR ACTIVATION LXR ACTIVATION
13
Pro-inflammatory stimuli may further increase the production of LXR ligands in tumor microenvironment
LXR ligand production regulation: pro-inflammatory stimuli
***
ABCG
1 IN
DUCT
ION
LXR ACTIVATION
**
ABCG
1 IN
DUCT
ION
LXR ACTIVATION
CELL LINES PRIMARY CELLS
14
LXR LIGAND EFFECTS- Indirect effects- Direct effects
THERAPEUTIC STRATEGIES BASED ON LXR SIGNALLING INACTIVATION
LXR LIGAND PRODUCTION - Players- Regulation
Myeloma and LXR ligands
MM microenviroment and LXR ligands
MM CELL
BM STROMAL CELL
LXR LIGANDS/OXYSTEROLS
plasmacytoid DCs
CXCR4
CXCR3
myeloid DCs
CCR7
Villablanca et al. Nat Med 2010
Raccosta et al. Manuscript under revision
Neutrophils
CXCR21) INDIRECT EFFECTS
Paniccia and Russo. Unpublished observations
2) DIRECT EFFECTSMM survival anddrug sensitivity
IFNgTNFa
16
Acetyl-CoA
HMG-CoA
Mevalonic Acid
Squalene
2,3-Monoepoxysqualene
Lanosterol
Cholesterol
2,3;22,23-Diepoxysqualene
24(S),25-Epoxylanosterol
24(S),25-Epoxylcholesterol
LXR
SULT2B1b-SO3H
-SO3H
-SO3H-SO3H -SO3H
-SO3H Inactivates LXR ligands by sulfurylation
Strategies to inactivate LXR/LXR ligands
WORKING HYPOTHESISLXR ligand deprivation can affect MM viability
17
LXR ligand effects on MM viabilityMM1S MOCK(LXR LIGAND PRODUCERS)
10% FBS
90
MM1S SULT2B1b(LXR LIGAND
NON PRODUCERS)
64.2
live cells
PI
Annexin V
early apoptotic cells
late apoptoticcells
1% FBS
1782.1
MM cells transduced with the LXR ligand-inactivating enzyme SULT2B1b are more prone to undergo apoptosis
LXR ligand effects on MM viability
***
***
10% FBS 1% FBS
10% FBS 1% FBS
***
** ***
***
10% FBS 1% FBS
******
10% FBS 1% FBS
19
LXR involvement:LXR target genes analysis
** ***
LXR signalling is not active in SULT2B1b-MM cells
ABCG1 EXPRESSIONMM1S
SREBP1c EXPRESSIONMM1S
20
LXR engagement partially rescues SULT2B1b-MM cells from apoptosis in serum starvation conditions
MM1S-MOCK(LXR LIGAND PRODUCERS)
MM1S-SULT2B1b(LXR LIGAND
NON PRODUCERS)
*****
VIABILITYLXR SINTHETIC AGONIST TREATMENT
LXR involvement:LXR agonist treatment
21
Acetyl-CoA
HMG-CoA
Mevalonic Acid
Squalene
2,3-Monoepoxysqualene
Lanosterol
Cholesterol
2,3;22,23-Diepoxysqualene
24(S),25-Epoxylanosterol
24(S),25-Epoxylcholesterol
LXRshLXR
Strategies to inactivate LXR/LXR ligands
LXRb silencing
***
LXRa silencing
***
22
LXRa or LXRb?
LXRa signalling sustains myeloma cell growth
SCRAMBLEshLXRb
SULT2B1b
shLXRa
shLXRa/shLXRb
PROLIFERATION MM1S
23
LXRa or LXRb?
SCRAMBLE
SULT2B1b
shLXRa
PROLIFERATION UTMC2
SCRAMBLE
SULT2B1b
shLXRa
LXRa signalling sustains myeloma cell growth
PROLIFERATIONOPM2
LXRa signalling and cell cycle progression
LXRa signalling abrogation induces G0/G1 cell cycle arrest
CELL CYCLE ANALYSISMM1S
24
25
Acetyl-CoA
HMG-CoA
Mevalonic Acid
Squalene
2,3-Monoepoxysqualene
Lanosterol
Cholesterol
2,3;22,23-Diepoxysqualene
24(S),25-Epoxylanosterol
24(S),25-Epoxylcholesterol
LXRa
Strategies to inactivate LXR/LXR ligands
LXRaantagonist
Geranyl-geraniol
26
Acetyl-CoA
HMG-CoA
Mevalonic Acid
Squalene
2,3-Monoepoxysqualene
Lanosterol
Cholesterol
2,3;22,23-Diepoxysqualene
24(S),25-Epoxylanosterol
24(S),25-Epoxylcholesterol
LXRa
Strategies to inactivate LXR/LXR ligands
LXRaantagonist
Geranyl-geraniol
The LXR antagonist geranyl-geraniol (GGOH) mimics the effects of
SULT2B1b and shLXRa
VIABILITYGERANYL-GERANIOL TREATMENT
***
MM1S-MOCK(LXR LIGAND PRODUCERS)
MM1S-SULT2B1b(LXR LIGAND
NON PRODUCERS)
27
LXR LIGAND EFFECTS- Indirect effects- Direct effects
THERAPEUTIC STRATEGIES BASED ON LXR SIGNALLING INACTIVATION
LXR LIGAND PRODUCTION
- Players- Regulation
Myeloma and LXR ligands
LXR ligand deprivation and drug sensitivity
LXR ligand deprivation makes MM cells more sensitive to drugs
DELTA 47
**
DELTA 47
**
UTMC2
n.s.
OPM2
*
MM1S
*
28
29
Acetyl-CoA
HMG-CoA
Mevalonic Acid
Squalene
2,3-Monoepoxysqualene
Lanosterol
Cholesterol
2,3;22,23-Diepoxysqualene
24(S),25-Epoxylanosterol
24(S),25-Epoxylcholesterol
LXR
Strategies to inactivate LXR/LXR ligands
Zaragozic Acid
Squalene synthaseinhibitor
UT
ZA 20mM
PROLIFERATIONUTMC2 + ZA
VIABILITYUTMC2 + ZA
5-8 weeksFACS analysis and Immunohistochemistry on peripheral blood and BM to evaluate:
1. % of engraftment Mock vs SULT2B1b2. Analysis of microenvironment (immune cells,
endothelial cells)
I.V. injection of
NSG (RAG2-/-gc-/-)immunodeficient mice
In vivo analysis of the properties of LXR ligand-producing MM cells
MOCK-transduced MM1S
SULT2B1b-transduced MM1S
30
Analysis of BM 6 weeks post injection
MM1S-MOCK
hCD38
mC
D45
MM1S-SULT2B1b
31
32
LXR LIGAND EFFECTS
THERAPEUTIC STRATEGIES BASED ON LXR LIGAND
INACTIVATION
LXR LIGAND PRODUCTION
Myeloma and LXR ligands:conclusions
- MM cells and BM cells produce LXR ligands- Pro-inflammatory stimuli (TNFa/IFNg) can foster LXR ligand production
- LXR ligands sustain MM cell survival via LXRa signalling
- LXR ligand deprivation makes MM cells more sensitive to drugs
33
LXR LIGAND EFFECTS
THERAPEUTIC STRATEGIES BASED ON LXR LIGAND
INACTIVATION
To investigate the effect of pro-inflammatory stimuli on LXR ligand-producing enzymes production in stromal cells and MM cells
To test the combination of the LXR ligand-production inhibitor Zaragozic Acid with myeloma-specific agents myeloma-specific CTLs in vitro and in vivo
LXR LIGAND PRODUCTION
Myeloma and LXR ligands:future plans
Chip assays to identify LXRa-dependent genes possibly involved in MM cell proliferation/survival
34
AKNOWLEDGEMENTSCancer Gene Therapy Unit
Laura RaccostaDaniela MaggioniMarta MorescoHelios Racalde
Raffaella FontanaMatias Soncini
Noemi Di MeglioAndrea Musumeci
Functional Genomics of Cancer UnitGiovanni Tonon
Leukemia Immunotherapy UnitAttilio BondanzaBarbara CamisaFabiana Gullotta
Pathology UnitMaurilio Ponzoni
FACS FacilityIvan Muradore
Simona Di Terlizzi
Vincenzo Russo