Multiple Myeloma and LXR ligands : a matter of life and death ?

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

•

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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

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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

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Myeloma and LXR ligands

LXR LIGAND PRODUCTION - Players- Regulation

LXR LIGAND EFFECTS- Indirect effects- Direct effects

THERAPEUTIC STRATEGIES BASEDON LXR SIGNALLING INACTIVATION

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- 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

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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

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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

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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

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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

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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

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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

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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

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LXR involvement:LXR target genes analysis

** ***

LXR signalling is not active in SULT2B1b-MM cells

ABCG1 EXPRESSIONMM1S

SREBP1c EXPRESSIONMM1S

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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

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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

***

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LXRa or LXRb?

LXRa signalling sustains myeloma cell growth

SCRAMBLEshLXRb

SULT2B1b

shLXRa

shLXRa/shLXRb

PROLIFERATION MM1S

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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

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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)

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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

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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

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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

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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

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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