1. 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

2. Zoledronic Acid (ZOL) 3rd generation nitrogen containing bisphosphonate (high affinity for HAP). Inhibits bone resorption mediated by osteoclasts. Inhibits Farnesyl Diphosphate Synthase (mevalonate pathway) : inhibits prenylation of small GTPases. Direct antitumoral effect in vitro and in vivo (breast, prostate, pancreas, lung cancer, osteosarcoma). Other effects : inhibition of angiogenesis and cell migration. Clinical use : – Osteoporosis – Multiple myeloma – Prostate and breast bone metastases – Osteosarcoma : OS2006 protocol Bone resorption Tumor proliferation Vicious cycle Zoledronic acid

3. Clusterin (CLU) Chi et al, Expert Opin. Investig. Drugs 2008 CLU is a stress-induced cytoprotective chaperone activated by varied treatment stressors: -Chemo & Radio-therapy -Targeted therapies (Herceptin, Velcade, Hsp90 inhibitors, etc) CLU confers broad spectrum treatment resistance when highly expressed: 1.Blocks apoptosis: Inhibits activated Bax 2.Enhances survival signaling pathways: Enhances NF-kB activity and AKT signaling CLU expression is directly regulated by HSF-1

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

5. CLU and resistance to Zoledronic acid CLU overexpression protects OS cells from ZOL induced cell death Zol-resistant MG63 osteosarcoma cells overexpress CLU CLU MG63MG63 resist zol Vinculin 60kDa 40kDa *** * Cell viability (%) Zol (μM) CLU Actin 60kDa 40kDa ** * CLU

6. Antisense Clusterin: OGX-011 Product Description MOE P-S 2 nd 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) CLU Knockdown by OGX-011 Clusterin Vinculin 0 8 16 32 64 128 256 8 16 32 64 128 256 (nM) Control SCR B OGX-011 6

7. CLU knockdown using OGX-011 synergistically enhances ZOL activity to inhibit OS cell proliferation 0 2004006008001000 0.2 0.4 0.6 0.8 1.0 Dose Dose-effect curve Effect Combination Index(CI) Antagonism Synergism OGX-011 OGX-011 + ZOL ZOL Caspase 3/7 activity (%) *** * OGX-011 ASO targets CLU …and to induce apoptosis (HOS) Chou-Talalay method: Constant ratio OGX-011ASO / ZOL for 48h

8. CLU knockdown re-sensitizes OS tumor cells to zoledronic acid 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 induced bone protection in the HOS xenograft model 200µm ScrB 200µm ScrB + Zol 200µm OGX-011 + Zol TRAP staining 0 5 10 15 20 25 35 ScrB + Zol OGX-011 + Zol BV/TV (%) n.s. *** 30 ScrB + Zol OGX-011 + Zol Treatment -100 μg/kg Zol (3 times/week) -20mg/kg ASO (ScrB or OGX-011): IP injection everyday the first week, then 3 times /week

10. OGX-011 potentiates ZOL activity in HOS xenograft model Tumor volume (mm 3 ) Days *** Ki67 ScrBScrB + ZolOGX-011 + Zol 200µm Clu Survival rate (%) Days p<0.001 OGX-011 combined with ZOL delays tumor growth in OS xenograft model and enhances survival rate % positive cells ScrB *** % positive cells *** ScrB + Zol OGX-011 + Zol

11. 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. CONCLUSIONS Bone resorption Tumor proliferation Vicious cycle Zoledronic acid Clu Tumor Cells survival OGX-011

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

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

15. Caseage (y)sexTumor locationPrimary/recurrenceHistologyResponse to chimio 155FsacrumPrimarygiant cell OSno chimio 256FsacrumRecurrencegiant cell OSno chimio 362MpelvisRecurrenceOSbad 438MscaphoidPrimarysynovialsarcoma 518MPrimaryOSgood 622MfemurPrimaryOSbad 726MfemurPrimaryOS 827MfemurRecurrenceOSbad 928MfemurRecurrenceOSbad 1080FfemurPrimaryOS on Pagetbad 1127FfemurPrimarylow grade OSno chimio 1218MPrimaryOSgood 1316MfemurPrimaryOSgood 14low grade OSno chimio 1516MhumerusPrimaryOSgood 10/15 CLU Expression in human osteosarcoma

16. Effect of Clu inhibition using OGX-011 in OS xenograft model HOS model: -2M HOS cells/ Nude mouse -Treatment started when tumor is 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