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

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).

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

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  1. PIBIC PROGRESS – 6 June 2013 Multiple Myeloma and LXR ligands: a matter of life and death? AIDA PANICCIA PhDStudent Russo’s Lab Cancer Gene TherapyUnit DivisionofMolecularOncology

  2. Multiple Myeloma (MM) B-cell neoplasia, characterized by the accumulation of malignant plasma cells in the bone marrow (BM) Sympthoms: osteolytic bone destruction, renalfailure, anemia, hypercalcemia • Treatment: • bortezomib, thalidomide, lenalidomide • autologousstemcelltrasplantation INCURABLE

  3. Aim of the study MM CELL VEGF MAPK-pathway PI3-K/AKT-pathway IGF-1 Fas JACK-STAT BM new vessels Pro-caspase 8 cFLIP IL6 TNFa FADD LXR LIGANDS?/ OXYSTEROLS VLA-4 LFA-1 Collagenfibres VCAM Fibronectin ICAM-1 BM STROMAL CELL

  4. LXR/LXR ligandsignalling - LXR is a nuclearreceptor CHOLESTEROL ESTER - LXR ligands are products of cholesteroloxidation (OXYSTEROLS) ABCG1 oxydizedfattyacidis LXR RXR ABCG1 gene OXYSTEROLS FUNCTIONS 1) regulation of cholesteroland fattyacids metabolism 2) modulation of immune response

  5. LXR-mediatedimmunosuppression Myeloyddendrticcells Tumorcells Lymphnode CCR7 LXR LXR ligands/ Oxysterols Villablanca et al. NatMed 2010

  6. Myeloma and LXR ligands • LXR LIGAND PRODUCTION • Players • Regulation • LXR LIGAND EFFECTS • Indirecteffects • Direct effects THERAPEUTIC STRATEGIES BASED ON LXR SIGNALLING INACTIVATION

  7. LXR ligandmeasurement - No directassay - Indirectmeasurement by evaluating the effects on myeloyddendriticcells Lymphnode Myeloyddendrticcells Tumorcells CCR7 LXR LXR ligands/ Oxysterols CCR7 INHIBITION LXR ACTIVATION (ABCG1 induction)

  8. LXR ligand production: myelomacells LXR ACTIVATION CCR7 INHIBITION ** % OF CCR7 INHBITION ABCG1 INDUCTION ** ** *** ** ** CELL LINES PRIMARY CELLS MM cells (primary and cell lines) produce LXR ligands

  9. Strategies to inactivate LXR/LXR ligands Acetyl-CoA HMG-CoA Mevalonic Acid Squalene 2,3-Monoepoxysqualene Lanosterol 2,3;22,23-Diepoxysqualene Cholesterol 24(S),25-Epoxylanosterol 24(S),25-Epoxylcholesterol LXR

  10. Strategies to inactivate LXR/LXR ligands Acetyl-CoA LXR ACTIVATION HMG-CoA Mevalonic Acid Squalene ** ABCG1 INDUCTION 2,3-Monoepoxysqualene Lanosterol 2,3;22,23-Diepoxysqualene Cholesterol 24(S),25-Epoxylanosterol 24(S),25-Epoxylcholesterol -SO3H -SO3H -SO3H -SO3H -SO3H -SO3H LXR Inactivates LXR ligands by sulfurylation SULT2B1b

  11. Aim of the study MM CELL VEGF MAPK-pathway PI3-K/AKT-pathway IGF-1 Fas JACK-STAT BM new vessels Pro-caspase 8 cFLIP IL6 TNFa FADD LXR LIGANDS?/ OXYSTEROLS VLA-4 LFA-1 Collagenfibres VCAM Fibronectin ICAM-1 BM STROMAL CELL

  12. LXR ligand production: BM stromalcells • Stromal cells within lymphoid organs produce LXR ligands, which are able to position B cells within follicles (Yi et al. Immunity 2012) LXR ACTIVATION LXR ACTIVATION ABCG1 INDUCTION *** ABCG1 INDUCTION *** ** ** ** PRIMARY CELLS CELL LINES BM stromal cells (primary and cell lines) produce LXR ligands

  13. LXR ligand production regulation: pro-inflammatorystimuli LXR ACTIVATION LXR ACTIVATION ** *** ABCG1 INDUCTION ABCG1 INDUCTION CELL LINES PRIMARY CELLS Pro-inflammatorystimulimayfurtherincrease the production of LXR ligands in tumormicroenvironment

  14. Myeloma and LXR ligands • LXR LIGAND PRODUCTION • Players • Regulation • LXR LIGAND EFFECTS • Indirecteffects • Direct effects THERAPEUTIC STRATEGIES BASED ON LXR SIGNALLING INACTIVATION

  15. MM microenviroment and LXR ligands 2) DIRECT EFFECTS MM survival and drug sensitivity CXCR2 MM CELL Neutrophils IFNg TNFa Raccosta et al. Manuscript under revision LXR LIGANDS/ OXYSTEROLS 1) INDIRECT EFFECTS plasmacytoidDCs CXCR3 CXCR4 CCR7 BM STROMAL CELL myeloidDCs Paniccia and Russo. Unpublishedobservations Villablanca et al. NatMed 2010

  16. Strategies to inactivate LXR/LXR ligands Acetyl-CoA HMG-CoA Mevalonic Acid Squalene WORKING HYPOTHESIS LXR liganddeprivation can affect MM viability 2,3-Monoepoxysqualene Lanosterol 2,3;22,23-Diepoxysqualene Cholesterol 24(S),25-Epoxylanosterol 24(S),25-Epoxylcholesterol -SO3H -SO3H -SO3H -SO3H -SO3H -SO3H Inactivates LXR ligands by sulfurylation LXR SULT2B1b

  17. LXR ligand effects on MM viability MM1S MOCK (LXR LIGAND PRODUCERS) MM1S SULT2B1b (LXR LIGAND NON PRODUCERS) late apoptotic cells 10% FBS 64.2 90 live cells earlyapoptotic cells 1% FBS PI 82.1 17 Annexin V

  18. LXR ligand effects on MM viability *** *** 1% FBS 1% FBS 1% FBS 1% FBS 10% FBS 10% FBS 10% FBS 10% FBS ** *** *** *** *** *** MM cells transduced with the LXR ligand-inactivating enzyme SULT2B1b are more prone to undergo apoptosis

  19. LXR involvement: LXR target genes analysis SREBP1c EXPRESSION MM1S ABCG1 EXPRESSION MM1S ** *** LXR signalling is not active in SULT2B1b-MM cells

  20. LXR involvement: LXR agonist treatment VIABILITY LXR SINTHETIC AGONIST TREATMENT *** ** MM1S-MOCK (LXR LIGAND PRODUCERS) MM1S-SULT2B1b (LXR LIGAND NON PRODUCERS) LXR engagement partiallyrescues SULT2B1b-MM cells from apoptosisin serumstarvationconditions

  21. Strategies to inactivate LXR/LXR ligands Acetyl-CoA HMG-CoA Mevalonic Acid *** *** Squalene 2,3-Monoepoxysqualene Lanosterol 2,3;22,23-Diepoxysqualene LXRbsilencing LXRasilencing Cholesterol 24(S),25-Epoxylanosterol 24(S),25-Epoxylcholesterol shLXR LXR

  22. LXRa or LXRb? PROLIFERATION MM1S SCRAMBLE shLXRb SULT2B1b shLXRa/shLXRb shLXRa LXRasignalling sustains myeloma cell growth

  23. LXRa or LXRb? PROLIFERATION UTMC2 PROLIFERATION OPM2 SCRAMBLE SCRAMBLE SULT2B1b SULT2B1b shLXRa shLXRa LXRasignalling sustains myeloma cell growth

  24. LXRasignalling and cellcycleprogression CELL CYCLE ANALYSIS MM1S LXRasignallingabrogation induces G0/G1 cell cycle arrest

  25. Strategies to inactivate LXR/LXR ligands Acetyl-CoA HMG-CoA Mevalonic Acid Squalene 2,3-Monoepoxysqualene Lanosterol 2,3;22,23-Diepoxysqualene Cholesterol 24(S),25-Epoxylanosterol 24(S),25-Epoxylcholesterol Geranyl-geraniol LXRa LXRa antagonist

  26. Strategies to inactivate LXR/LXR ligands Acetyl-CoA VIABILITY GERANYL-GERANIOL TREATMENT HMG-CoA *** Mevalonic Acid Squalene 2,3-Monoepoxysqualene Lanosterol 2,3;22,23-Diepoxysqualene MM1S-MOCK (LXR LIGAND PRODUCERS) MM1S-SULT2B1b (LXR LIGAND NON PRODUCERS) Cholesterol 24(S),25-Epoxylanosterol 24(S),25-Epoxylcholesterol The LXR antagonistgeranyl-geraniol (GGOH) mimics the effects of SULT2B1b and shLXRa LXRa Geranyl-geraniol LXRa antagonist

  27. Myeloma and LXR ligands • LXR LIGAND PRODUCTION • Players • Regulation • LXR LIGAND EFFECTS • Indirecteffects • Direct effects THERAPEUTIC STRATEGIES BASED ON LXR SIGNALLING INACTIVATION

  28. LXR liganddeprivation and drugsensitivity n.s. ** ** DELTA 47 DELTA 47 UTMC2 * * MM1S OPM2 LXR liganddeprivationmakes MM cells more sensitive to drugs

  29. Strategies to inactivate LXR/LXR ligands PROLIFERATION UTMC2 + ZA Acetyl-CoA HMG-CoA UT Mevalonic Acid ZA 20mM Squalene Squalenesynthase inhibitor 2,3-Monoepoxysqualene Lanosterol 2,3;22,23-Diepoxysqualene VIABILITY UTMC2 + ZA Cholesterol 24(S),25-Epoxylanosterol 24(S),25-Epoxylcholesterol Zaragozic Acid LXR

  30. In vivo analysis of the properties of LXR ligand-producing MM cells NSG (RAG2-/-gc-/-) immunodeficient mice FACS analysis and Immunohistochemistry on peripheralblood and BM to evaluate: % of engraftmentMock vs SULT2B1b Analysis of microenvironment (immune cells, endothelialcells) 5-8 weeks I.V. injection of MOCK-transduced MM1S SULT2B1b-transduced MM1S

  31. Analysis of BM 6 weeks post injection MM1S-MOCK MM1S-SULT2B1b mCD45 hCD38

  32. Myeloma and LXR ligands: conclusions LXR LIGAND PRODUCTION • MM cells and BM cells produce LXR ligands • Pro-inflammatorystimuli (TNFa/IFNg) can fosterLXR ligand production LXR LIGAND EFFECTS • LXR ligandssustain MM cellsurvival via LXRasignalling THERAPEUTIC STRATEGIES BASED ON LXR LIGAND INACTIVATION - LXR liganddeprivationmakes MM cells more sensitive to drugs

  33. Myeloma and LXR ligands: future plans To investigate the effect of pro-inflammatory stimuli on LXR ligand-producing enzymes production in stromal cells and MM cells LXR LIGAND PRODUCTION • Chip assays to identify LXRa-dependent genes possibly involved in MM cell proliferation/survival LXR LIGAND EFFECTS THERAPEUTIC STRATEGIES BASED ON LXR LIGAND INACTIVATION To test the combination of the LXR ligand-production inhibitorZaragozic Acid with myeloma-specific agents myeloma-specific CTLs in vitro and in vivo

  34. AKNOWLEDGEMENTS FunctionalGenomics of Cancer Unit Giovanni Tonon LeukemiaImmunotherapy Unit Attilio Bondanza Barbara Camisa Fabiana Gullotta Pathology Unit MaurilioPonzoni FACS Facility Ivan Muradore Simona Di Terlizzi Cancer Gene TherapyUnit Laura Raccosta Daniela Maggioni Marta Moresco HeliosRacalde Raffaella Fontana Matias Soncini Noemi Di Meglio Andrea Musumeci Vincenzo Russo

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