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CLU inhibition using OGX-011 synergistically enhances Zol activity in osteosarcoma

CLU inhibition using OGX-011 synergistically enhances Zol activity in osteosarcoma. François Lamoureux , Marc Baud’huin , Benjamin Ory , Martin E. Gleave , Dominique Heymann and Françoise Rédini. INSERM UMR957 Medicine Faculty , Nantes, FRANCE. Vicious cycle. Tumor

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CLU inhibition using OGX-011 synergistically enhances Zol activity in osteosarcoma

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  1. CLU inhibition using OGX-011 synergisticallyenhancesZolactivity in osteosarcoma François Lamoureux, Marc Baud’huin, Benjamin Ory, Martin E. Gleave, Dominique HeymannandFrançoise Rédini INSERM UMR957 MedicineFaculty, Nantes, FRANCE

  2. Vicious cycle Tumor proliferation Boneresorption ZoledronicAcid (ZOL) Zoledronicacid • 3rd generationnitrogencontainingbisphosphonate (highaffinity for HAP). • Inhibitsboneresorptionmediated by osteoclasts. • InhibitsFarnesylDiphosphateSynthase (mevalonatepathway) : inhibitsprenylation of smallGTPases. • Direct antitumoraleffectin vitro and in vivo (breast, prostate, pancreas, lung cancer, osteosarcoma). • Othereffects : inhibition of angiogenesis and cell migration. • Clinical use : • Osteoporosis • Multiple myeloma • Prostate and breastbonemetastases • Osteosarcoma : OS2006 protocol

  3. Clusterin (CLU) • CLU is a stress-induced cytoprotectivechaperone activated by varied treatment stressors: • Chemo & Radio-therapy • Targeted therapies (Herceptin, Velcade, Hsp90 inhibitors, etc) • CLU expression isdirectlyregulated by HSF-1 • CLU confers broad spectrum treatment resistance when highly expressed: • Blocks apoptosis: Inhibits activated Bax • Enhances survival signaling pathways: Enhances NF-kB activity and AKT signaling Chi et al, Expert Opin. Investig. Drugs 2008

  4. Rationale: ZoledronicacidinducesClusterin expression in OS celllines and in a OS xenograft model CT Zol HOS osteosarcomaxenograft model treatedwith 100μg/kg Zol CLU MG63 200µm 200µm CLU mRNA expression Zol (μM) ZOL induces/increased CLU expression in human OS cells (HOS, SaOS2, MG-63, U2OS) CLU expression Zol (µM) 0 3.1 6.2 12.5 25 50 60kDa Clu 40kDa Vinculin

  5. CLU and resistance to Zoledronicacid Zol-resistant MG63 osteosarcomacellsoverexpress CLU CLU *** *** * *** 60kDa Cellviability (%) 40kDa 60kDa CLU 40kDa Actin Zol (μM) CLU overexpressionprotects OS cellsfrom ZOL inducedcelldeath MG63 ** MG63 resistzol CLU ** * Vinculin

  6. Antisense Clusterin: OGX-011 Product Description A O O OCH2CH2OCH3 H H OCH2CH2OCH3 OCH2CH2OCH3 O O P - S O C O O O P - S O G O O O P - S O T O O O P - S O A O O O P - S O 2nd generation 21-mer MOE gapmer antisense oligonucleotide • Advantages of 2'MOE analogues • Increased potency and resistance to degradation • Tissue half life ~ 7 days • Facilitates more convenient dosing regimen (once-weekly infusion) P-S MOE MOE Control SCR B OGX-011 0 8 16 32 64 128 256 8 16 32 64 128 256 (nM) Clusterin CLU Knockdown by OGX-011 Vinculin

  7. Dose-effectcurve 1.0 0.8 0.6 Effect 0.4 0.2 0 200 400 600 800 1000 Dose CLU knockdown using OGX-011 synergisticallyenhances ZOL activity to inhibit OS cellproliferation Chou-Talalaymethod: Constant ratio OGX-011ASO / ZOL for 48h OGX-011 ASO targets CLU …and to induceapoptosis (HOS) OGX-011 ZOL OGX-011 + ZOL Antagonism *** Combination Index(CI) Caspase 3/7 activity (%) * Synergism

  8. CLU knockdown re-sensitizes OS tumorcells to zoledronicacid MG63 resistant and sensitive cells were treated twice with 300nM OGX-011 or control ScrB ASO + ZOL for 48h.

  9. OGX-011 does not affect ZOL inducedbone protection in the HOS xenograft model ScrB + Zol OGX-011 + Zol ScrB • Treatment • 100 μg/kg Zol (3 times/week) • 20mg/kg ASO (ScrB or OGX-011): IP injection everyday the first week, then3 times /week n.s. 35 *** 30 25 20 BV/TV (%) 15 10 5 0 ScrB ScrB + Zol OGX-011 + Zol TRAP staining 200µm 200µm 200µm ScrB + Zol ScrB OGX-011 + Zol

  10. OGX-011 potentiates ZOL activity in HOS xenograft model OGX-011 combinedwith ZOL delaystumorgrowth in OS xenograft model and enhancessurvival rate p<0.001 Survival rate (%) Tumor volume (mm3) *** Days Days *** % positive cells *** ScrB ScrB + Zol OGX-011 + Zol % positive cells Clu ScrB ScrB + Zol OGX-011 + Zol Ki67 200µm 200µm 200µm 200µm 200µm 200µm

  11. CONCLUSIONS Vicious cycle Boneresorption Tumor proliferation OGX-011 combined with ZOL: 1) Decreases Stress-induced CLU expression induced by ZOL 2) Decreases osteosarcoma cell growth in vitro and in vivo 3) Proof of principle to combine OGX-011 (phase II/III clinical trial in solid tumors) and zoledronic acid (phase III clinical trial in OS in France) in osteosarcoma treatment. Zoledronicacid Clu OGX-011 Tumor Cellssurvival

  12. Acknowledgments Lamoureux F. Ory B. Baud’huin M. Heymann D. Gleave M.E. Zoubeidi A. Sources of funding Experimental therapeutic unit (Nantes)

  13. de-differentiated parostealosteosarcoma extraosseous osteosarcoma CLU in sarcoma Mass Spectrometry Analyses fromtumor tissue OS, ES and synovial sarcoma: 100% positive for Hsp90 8 of 14 OS were positive for CLU Resultswereconfirmed by immunohistochemistry (TMA included 75 specimens) Rao et al. Human Pathology, 2012 Hsp90 Clu osteosarcoma samples displaying cytoplasmic and nuclear immunopositivity • Clusterinplays a role in resistance to doxorubicin in OS cells • (Lourda et al. Int. J. Cancer 2006)

  14. CLU Expression in humanosteosarcoma 10/15

  15. Effect of Clu inhibition using OGX-011 in OS xenograft model • HOS model: • -2M HOS cells/ Nude mouse • -Treatmentstartedwhentumoris palpable • 20mg/kg ASO (ScrB or OGX-011): • IP injection everyday the first week, • and then 3 times /week No effect of OGX-011 ASO on tumor progression

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