1. LBH589 e altri inibitori delle istone-deacetilasi nel Mieloma Multiplo Claudia Polloni Clinica di Ematologia Azienda Ospedaliero-Universitaria Ospedali Riuniti Ancona

2. Introduction to Deacetylases (DACs) Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins, leading to regulation of gene transcription and other cellular processes Histones are one of the target proteins, which is why the class is sometimes referred to as histone deacetylases (HDACs) DACs also target non-histone proteins, which include transcription factors,  -tubulin, and HSP90

3. There are 4 Classes of DACs (I and II), Which Act on Different Target Proteins HDAC4 Class II DACs act on NON-HISTONE proteins located in the cytoplasm (e.g. HDAC6) Class I DACs act on HISTONES and TRANSCRIPTION FACTORS located in the nucleus There are 2 main classes of DACs HDAC1 HDAC2 HDAC3 HDAC8 HDAC5 HDAC7 HDAC9 HDAC6 HDAC10 HDAC7

4. Pan-DAC Inhibitors Target Both Classes of DACs, Modulating Histone and Non-Histone Proteins Specific DAC inhibitors may target Class I DACs only There are 2 main types of DAC inhibitors HDAC1 HDAC2 HDAC3 HDAC8 Pan-DAC inhibitors target both Class I and Class II DACs – interfering with both histone and non-histone proteins HDAC4 HDAC5 HDAC9 HDAC6 HDAC10 HDAC7

5. Isoenzyme-selectivity of pan-HDACi:

6. DNA Mutations/translocations Replication errors Genetic Variations and Epigenetic Changes Can Both Contribute to Oncogenesis GENETIC Chromatin EPIGENETIC Transformed cells Open/closed chromatin Enzyme modification errors Altered DNA/mRNA/proteins DNA sequence altered Altered mRNA/proteins DNA sequence not altered Oncogenesis Can be caused by: Abnormal modifications to histone proteins Abnormal DNA methylation Can be caused by: Abnormal modifications to histone proteins Abnormal DNA methylation

7. Altered Expression of DACs is Found in Several Malignancies DAC expression can increase cell-cycle progression and prevent cell death, which leads to increased cell proliferation DAC expression can correlate with estrogen and progesterone receptor expression DACs can be upregulated in malignant prostate cancer, with the highest levels found in HRPC DAC expression can be associated with tumor aggressiveness MULTIPLE MYELOMA BREAST CANCER PROSTATE CANCER GASTRIC CANCER

8. Histone  -tubulin HSP90 HIF-1  Pan-DAC Inhibition May Have Potential in Several Cancers DACs Hematologic & Solid Tumors Breast, Multiple Myeloma RCC, Melanoma CML, Breast, Prostate, NSCLC 50% of Cancers DAC Inhibitor p53

9. Epigenetic changes, such as histone modifications and DNA methylation, play key roles in chromatin structure and gene activity Altered patterns of epigenetic modifications are common in many human diseases, including cancer Silencing of tumor suppressor genes by abnormal histone modifications is a key feature of cancer cells DAC inhibitors were developed when they were found to reactivate genes that had been epigenetically silenced Epigenetic Changes Can Drive Cancer

10. Acetylation of Histones by HAT Allows Gene Expression Acetylation by histone acetyltransferases (HATs) allows transcription and gene expression Acetylated Histone Open chromatin Transcription factors can access DNA Deacetylated Histone Closed chromatin Transcription factors cannot access DNA HAT HISTONE ACETYLATION Ac: acetyl group Transcription factors –Ac Ac–

11. Deacetylation of Histones by HDAC Can Prevent Gene Expression Acetylation by histone acetyltransferases (HATs) allows transcription and gene expression Deacetylation by histone deacetylases (HDACs) can prevent transcription and gene expression HAT HISTONE ACETYLATION HISTONE DEACETYLATION HDAC Acetylated Histone Open chromatin Transcription factors can access DNA Deacetylated Histone Closed chromatin Transcription factors cannot access DNA Ac: acetyl group HDAC depicts a class I deacetylase Transcription factors –Ac Ac–

12. In Tumor Cells, Imbalanced HAT and HDAC Activity Can Result in Deregulated Gene Expression Tumor Cell Unchecked Cell Growth and Survival Decreased Tumor Suppressor Gene Activity (p21, p27) Increased HDAC Activity Decreased HAT Activity HDAC HAT Ac: acetyl group TF: transcription factors HDAC depicts a class I deacetylase TF –Ac Ac–

13. HDAC Inhibition Restores Gene Expression in Tumor Cells HDAC DAC Inhibition Increases Acetylation of Histones HAT DAC Inhibitor Increased Tumor Suppressor Gene Activity (p21, p27) Cell-Cycle Arrest and Differentiation Normalized Cell Ac: acetyl group TF: transcription factors HDAC depicts a class I deacetylase –Ac Ac– TF

14. DACs are Implicated in Cancer by Modulating Histone and Non-Histone Proteins Involved in Oncogenesis Non-histone proteins are implicated in multiple oncogenic pathways Histone p53 Histone proteins are implicated in epigenetic modifications that could cause cancer Proteins modulated by DACs DAC  -tubulin HSP90 HIF-1 

15. DAC Pan-DAC Inhibition Interferes with the Multiple Hallmarks of Cancer Proteins modulated by DACs DAC depicts individual deacetylases, e.g. HDAC1, HDAC4, HDAC6 Histone DAC  -tubulin HSP90 HIF-1  Cell-cycle arrest Apoptosis Cell motility and Invasion Cell proliferation and survival Angiogenesis Tumor effects DAC Inhibitor Tumor suppressor gene activity Loss of tumor suppressor function Microtubule depolymerization/ aggresome formation VEGF Oncoproteins Downstream effects p53

16. HSP90 HDAC6 DAC Inhibitor Growth and survival proteins Acetylated HSP90 – binding to growth and survival proteins prevented HSP90 Deacetylated HSP90 – binds growth and survival proteins HDAC6 Growth and survival proteins DAC Inhibition can Control Myeloma Cell Proliferation and Survival Through HSP90 DAC Activity through HSP90 Overexpression of growth and survival proteins Proliferation and survival DAC inhibition through HSP90 Overexpression of growth and survival proteins Proteins protected from degradation Proteins degraded Proliferation and survival

17. Acetylated α-tubulin HDAC6 DAC Inhibitor Deacetylated α-tubulin HDAC6 DAC Inhibition can Induce Apoptosis in Myeloma Cells Through the Aggresome Pathway Protein degradation Cell survival Apoptosis Aggresome formation DAC Activity through aggresomes DAC inhibition through aggresomes No aggresome formation Misfolded proteins recruited to aggresomes Protein degradation Accumulation of cytotoxic misfolded proteins

18. HDAC6 Acytelated α-tubulin DAC Inhibition can Synergize with Proteasome Inhibition to Induce Increased Apoptosis in Myeloma Cells Protein degradation Apoptosis Multiple Myeloma cell Protein degradation Proteasome Inhibitor (Bortezomib ) Proteasome Inhibitor (Bortezomib ) DAC Inhibitor Proteasome No aggresome formation Misfolded proteins Accumulation of misfolded proteins

19. DAC Inhibition Can Lead To Decreased Angiogenesis in Tumor Cells Through HIF-1  Implicated in RCC, melanoma and other solid tumors HIF-1  Deacetylated HIF-1α Stabilized HDAC4 HDAC6 Acetylated HIF-1α Destabilized Degraded DAC Activity HIF-1  DAC Inhibition HDAC4 HDAC6 DAC Inhibitor VEGF VEGF AngiogenesisAngiogenesis

20. DAC Inhibition can Induce Apoptosis in Myeloma Cells

21. Studi in corso: Tab 3 Hematology review

22. STUDIO DI FASE II, MULTICENTRICO, IN APERTO DI LBH589 ORALE IN ASSOCIAZIONE CON MELPHALAN, PREDNISONE E TALIDOMIDE (LBH-MPT) IN PAZIENTI CON MIELOMA MULTIPLO AVANZATO O REFRATTARIO LBH-MPT

23. Rationale LBH589-MPT Activty of LBH589 in solid tumors and hematologic malignancies Combination treatments standard for MM (MPT) Each drug has different mechanisms of action Safety will be closely evaluated

24. QTcF prol: 27% Nausea: 40% Diarrhea: 33% Vomiting: 33% Hypokalemia: 27% Anorexia: 13% Thrombocytopenia: 13% Safety of iv LBH-589

25. ARM ARM 1 (32 pts) 15 mg MWF (3 pts) 20 mg MWF (19 pts) 30 mg MWF(10 pts) ARM 3 (22 pts) 30 mg MWF eow(20 pts) 45 mg MWF eow(2 pts) $ ARM 5 (8 pts) 30 mg MT (3 pts) 45 mg MT ( 5 PTS) No grade 3 00 1 (diarrhea) 0 0 1 (fatigue) 1 (QTcF prol) 1 (PLTpenia) * No grade 4 00 1 (PLTpenia)* 00 Prot CLBH589B2101: safety of oral LBH589 Transient and reverseble; $ 1 pt grade 2 anemia and 1 pt grade 2 fatigue * Transient and reverseble; $ 1 pt grade 2 anemia and 1 pt grade 2 fatigue

26. Prot CLBH589B2101: cardiac toxicity ARM (dose) 30 mg 20 mg 30 mg QTc prol. 111 notes 4 days later, sepsis BBDmsec100 58 (QTc:503) 77 ARM (dose) 20 mg 30 mg FA22AFlutter10 1046 post dose ECG: median prolongation <10 msec T-wave flattening in 25 patients CK in 2 patients (not clinically relevant)

27. 6 cicli da 28 giorni 1 3 5 8 10 12 15 17 19 1 3 5 8 10 12 15 17 19 Melphalan 0,18 mg/kg Melphalan 0,18 mg/kg Prednisone 1,5 mg/kg LBH 589 per os Livello 0: 15 mg, Livello -1: 10 mg, Livello +1: 20 mg Talidomide 50 mg continuativamente TREATMENT G 1- 4

28. cicli da 28 giorni fino a progressione o tossicità intollerabile 1 3 5 8 10 12 15 17 19 1 3 5 8 10 12 15 17 19 LBH 589 per os alla dose utilizzata per LB-MPT Mantenimento: (pazienti con risposta ≥ malattia stabile) Prednisone 25 mg nei giorni 1, 3, 5 di ogni settimana fino a progressione

29. LBH-MPT: type of study and population Phase I-II, multicenter, non-comparative, non- randomized, open-label Adult patients with relapsed MM with any sign of PD during melphalan or thalidomide and who have not received melphalan or thalidomide in the last six months suitable for treatment or re-treatment with melphalan and thalidomide

30. Endpoints Primary The safety profile will be assessed by showing: –Any grade 3 non-hematologic toxicity –Grade 4 neutropenia ≥ a week, or any grade 4 hematologic toxicity except neutropenia The efficacy will be assessed by showing a significant PR rate Secondary - Determine the progression-free survival (PFS) - Determine the overall survival (OS) - Determine whether responses are associated with a prolongation of PFS, in comparison with that of non-responding patients. - Quality of Life assessment (QoL) - Assessment of common chromosomal abnormalities in multiplemyeloma by FISH

31. 19 pts Livello 0 19 pts livello +1 RP ≤ 4 Tox g 3-4 ≤ 3 STUDY DESIGN (Briant and Day method) RP ≥ 5 Tox g 3-4 >10 RP ≥ 5 Tox g 3-4 ≤10 19 pts livello -1 23 pts livello 0 RP ≤ 4 Tox g 3-4 ≥ 4 STOP 23 pts STOP STOP

32. Wolf et. Al, ASH 2008

34. Siegel, San Miguel et al, ASH 2009

37. Phase Ib study: Panobinostat combined with bortezomib ± dexamethasone in relapsed MM (B2207) Study design International, open-label, phase Ib, dose-escalation study Treatment 21-day cycles: –Escalating doses of oral panobinostat (cohorts 1–3: 10 mg, 20 mg, 20 mg) TIW –Escalating doses of bortezomib (cohorts 1–3: 1.0 mg/m 2, 1.0 mg/m 2, 1.3 mg/m 2 ) given on days 1, 4, 8, 11 –Optional dexamethasone (20 mg on day of and day after bortezomib) in cycle 2 onwards (i.e. after the DLT observation period) Patient characteristics (n=22) Median 3 previous lines of therapy (range 1–6), 11pts had previously received bortezomib Disease status at baseline: 10 relapsed and refractory, 11 relapsed, 1 unknown Median age 61 years (range 46–78), 19 had previously undergone SCT Sezer O et al. IMW 2009, Abstract 337 (data updated in oral presentation)

38. Safety of panobinostat combined with bortezomib ± dexamethasone in relapsed MM (B2207) MTD for panobinostat not reached at 20 mg TIW –Accrual ongoing for cohort 4 (panobinostat 30 mg, bortezomib 1.3 mg/m 2 ) Assessment of DLTs (cycle 1): –Cohort 1 (panobinostat 10 mg, bortezomib 1.0 mg/m 2 ): no DLTs –Cohort 2 (panobinostat 20 mg, bortezomib 1.0 mg/m 2 ): 1 DLT (grade 4 febrile neutropenia) –Cohort 3 (panobinostat 20 mg, bortezomib 1.3 mg/m 2 : no DLTs After 1035 post-baseline ECGs: –No dose-related increase in QTcF time –No QTcF >500 ms or increase in QTcF from baseline >60ms Sezer O et al. IMW 2009, Abstract 337 - data update at ASH09

39. Preliminary efficacy of panobinostat combined with bortezomib ± dexamethasone in relapsed MM (B2207) 11 responses to date: 3 CRs, 1 VGPR and 7 PRs, across cohorts 1–3 5 of these responders were refractory (a, b) to their last bortezomib- based therapy Three of 22 treated patients were not evaluable for efficacy as they discontinued treatment in cycle 1 Cohort 1 panobinostat 10 mg bortezomib 1.0 mg/m 2 Cohort 2 panobinostat 20 mg bortezomib 1.0 mg/m 2 Cohort 3 panobinostat 20 mg bortezomib 1.3 mg/m 2 All responders b a b a b a: Non-responder to prior bortezomib (i.e. best response SD (IMWG 2006) b: Disease progression on prior bortezomib- based therapy Sezer O et al. IMW 2009, Abstract 337 - data update at ASH09 Number of patients

40. Phase Ib study: Panobinostat combined with lenalidomide and dexamethasone in relapsed MM (B2206) Study design Multicentre, international open-label phase Ib dose-escalation study Patients Adults with active MM (IMWG criteria) whose disease has relapsed after at least 1 previous line of therapy primary refractory MM, grade >2 peripheral neuropathy, or cardiac diseases/factors associated with QT prolongation were excluded Treatment Patients were treated on 28-day cycles until PD or unacceptable toxicity –Escalating doses of oral panobinostat (cohorts 1–3: 5, 10, 20 mg) TIW –Lenalidomide 25 mg given orally on days 1–21 –Dexamethasone 40 mg given orally on days 1–4, 9–12 and 17–20 for cycles 1–4, and on days 1–4 for cycle 5 onwards Spencer A et al. ASCO 2009 Abstract #8542 (data updated in the poster)

41. Safety of panobinostat combined with lenalidomide + dexamethasone in relapsed MM (B2206) Safety MTD for panobinostat not reached at 20 mg TIW –Study is now recruiting patients at the 25 mg dose level Assessment of DLTs (cycle 1): –Cohort 1 (5 mg panobinostat): 7/8 evaluable, no DLT –Cohort 2: (10 mg panobinostat): 6/8 evaluable, 1 DLT (grade 1 QTcF prolongation) –Cohort 3: (20 mg panobinostat): 6/11 evaluable, 1 DLT (grade 4 neutropenia lasting for >5 days) Most frequent grade 3/4 AEs: neutropenia (5/23 pts), thrombocytopenia (5/23 pts), fatigue (4/23 pts), hyponatraemia (3/23 pts) –High-dose dexamethasone may be responsible for many AEs After 1375 post-baseline ECGs: no QTcF >500 ms, no QTcF change >60 ms from baseline Spencer A et al. ASCO 2009 Abstract #8542 (data updated in the poster)

42. Responses with panobinostat combined with lenalidomide + dexamethasone in relapsed MM (B2206) 20 patients evaluable for efficacy (cohorts 1–3 combined) –12/20 responded: 1 sCR, 1 CR, 5 VGPR, 4 PR, 1 MR –1 responder was refractory to their last bortezomib-based regimen –4 responders were refractory to their last thalidomide-based regimen Spencer A et al. ASCO 2009 Abstract #8542 (data updated in the poster) NE, not evaluable; sCR, stringent CR Number of patients

43. Weber et al, ASH 2008

46. Siegel et al,

47. Harrison et al, ASH 2008

49. Pivotal Phase III Study: D2308 A multicentre, randomized, double- blind, placebo-controlled phase III study of panobinostat in combination with bortezomib and dexamethasone in patients with relapsed multiple myeloma

50. D2308 study design Bortezomib 1.3 mg/m 2 BIW, 2 weeks on, 1 week off + dexamethasone on same days as and 1 day after each bortezomib dose Bortezomib 1.3 mg/m 2 QW, 2 weeks on, 1 week off (x2) + dexamethasone on same days as and 1 day after each bortezomib dose Bortezomib 1.3 mg/m 2 BIW, 2 weeks on, 1 week off + dexamethasone on same days as and 1 day after each bortezomib dose + Placebo TIW 2 weeks on, 1 week off Panobinostat 20 mg TIW 2 weeks on, 1 week off n = 672 Relapsed or Relapsed & Refractory MM a (≥1 up to 3 lines of prior therapy) Treatment with panobinostat or placebo in combination with bortezomib +dexamethasone (phase 1, 24 weeks) Patients with ‘no change‘ of disease status or response continue into treatment phase 2 (a further 24 weeks). Continuation of combination therapy up to a total of 48 wks, or until PD, withdrawal of consent, or unacceptable toxicity. Screening 3 weeks Panobinostat 20 mg TIW 2 weeks on, 1 week off (x 2) + Placebo, TIW 2 weeks on, 1 week off (x2) Bortezomib 1.3 mg/m 2 QW, 2 weeks on, 1 week off (x2) + dexamethasone on same days as and 1 day after each bortezomib dose R Treatment phase 1 8 cycles of 21 days each (weeks 1–24) Treatment phase 2 4 cycles of 42 days each (weeks 25–48) Follow-up after therapy: 1 year for PD, up to 4 years for OS a Not refractory to bortezomib

51. Worlwide recruitment 672 patients across 200 clinical sites North America (n=90) Latin America (n=168) Europe (> 200 pts) Japan (n=30) Other countries (n=168) Timelines First patient, first visit: 21 Dec 2009 Last patient, first visit: 21 June 2011 Last patient, last visit: 21 June 2012 18-month enrolment period