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Purpose: New therapies are needed to treat immune checkpoint inhibitor–resistant non–small cell lung cancer (NSCLC) and identify biomarkers to personalize treatment. Epigenetic therapies, including histone deacetylase inhibitors, may synergize with programmed cell death-1 (PD-1) blockade to overcome resistance. We report outcomes in patients with anti-programmed cell death ligand-1 [PD-(L)1]–resistant/refractory NSCLC treated with pembrolizumab plus entinostat in ENCORE 601. Patients and Methods: The expansion cohort of ENCORE 601 included patients with NSCLC who previously experienced disease progression with immune checkpoint inhibitors. The primary endpoint for the phase II expansion cohort is overall response rate (ORR); safety, tolerability, and exploratory endpoints are described. Results: Of 76 treated patients, 71 were evaluable for efficacy. immune-regulated RECIST–assessed ORR was 9.2% [95% confidence interval (CI): 3.8–18.1], which did not meet the prespecified threshold for positivity. Median duration of response was 10.1 months (95% CI: 3.9–not estimable), progression-free survival (PFS) at 6 months was 22%, median PFS was 2.8 months (95% CI: 1.5–4.1), and median overall survival was 11.7 months (95% CI: 7.6–13.4). Benefit was enriched among patients with high levels of circulating classical monocytes at baseline. Baseline tumor PD-L1 expression and IFNγ gene expression were not associated with benefit. Treatment-related grade ≥3 adverse events occurred in 41% of patients. Conclusions: In anti–PD-(L)1–experienced patients with NSCLC, entinostat plus pembrolizumab did not achieve the primary response rate endpoint but provided a clinically meaningful benefit, with objective response in 9% of patients. No new toxicities, including immune-related adverse events, were seen for either drug. Future studies will continue to evaluate the association of monocyte levels and response.

Purpose: Histone deacetylase (HDAC) inhibition improves the efficacy of proteasome inhibition for multiple myeloma but adds substantial toxicity. Preclinical models suggest that the observed synergy is due to the role of HDAC6 in mediating resistance to proteasome inhibition via the aggresome/autophagy pathway of protein degradation. Experimental Design: We conducted a phase I/II trial of the HDAC6-selective inhibitor ricolinostat to define the safety, preliminary efficacy, and recommended phase II dose in combination with standard proteasome inhibitor therapy. Patients with relapsed or refractory multiple myeloma received oral ricolinostat on days 1–5 and 8–12 of each 21-day cycle. Results: Single-agent ricolinostat therapy resulted in neither significant toxicity nor clinical responses. Combination therapy with bortezomib and dexamethasone was well-tolerated during dose escalation but led to dose-limiting diarrhea in an expansion cohort at a ricolinostat dose of 160 mg twice daily. Combination therapy at a ricolinostat dose of 160 mg daily in a second expansion cohort was well tolerated, with less severe hematologic, gastrointestinal, and constitutional toxicities compared with published data on nonselective HDAC inhibitors. The overall response rate in combination with daily ricolinostat at ≥160 mg was 37%. The response rate to combination therapy among bortezomib-refractory patients was 14%. Samples taken during therapy showed dose-dependent increases of acetylated tubulin in peripheral blood lymphocytes. Conclusions: At the recommended phase II dose of ricolinostat of 160 mg daily, the combination with bortezomib and dexamethasone is safe, well-tolerated, and active, suggesting that selective inhibition of HDAC6 is a promising approach to multiple myeloma therapy. Clin Cancer Res; 23(13); 3307–15. ©2017 AACR.

Summary Background Histone deacetylase (HDAC) inhibitors are an important new class of therapeutics for treating multiple myeloma. Ricolinostat (ACY-1215) is the first oral selective HDAC6 inhibitor with reduced class I HDAC activity to be studied clinically. Motivated by findings from preclinical studies showing potent synergistic activity with ricolinostat and lenalidomide, our goal was to assess the safety and preliminary activity of the combination of ricolinostat with lenalidomide and dexamethasone in relapsed or refractory multiple myeloma. Methods In this multicentre phase 1b trial, we recruited patients aged 18 years or older with previously treated relapsed or refractory multiple myeloma from five cancer centres in the USA. Inclusion criteria included a Karnofsky Performance Status score of at least 70, measureable disease, adequate bone marrow reserve, adequate hepatic function, and a creatinine clearance of at least 50 mL per min. Exclusion criteria included previous exposure to HDAC inhibitors; previous allogeneic stem-cell transplantation; previous autologous stem-cell transplantation within 12 weeks of baseline; active systemic infection; malignancy within the last 5 years; known or suspected HIV, hepatitis B, or hepatitis C infection; a QTc Fridericia of more than 480 ms; and substantial cardiovascular, gastrointestinal, psychiatric, or other medical disorders. We gave escalating doses (from 40–240 mg once daily to 160 mg twice daily) of oral ricolinostat according to a standard 3 + 3 design according to three different regimens on days 1–21 with a conventional 28 day schedule of oral lenalidomide (from 15 mg [in one cohort] to 25 mg [in all other cohorts] once daily) and oral dexamethasone (40 mg weekly). Primary outcomes were dose-limiting toxicities, the maximum tolerated dose of ricolinostat in this combination, and the dose and schedule of ricolinostat recommended for further phase 2 investigation. Secondary outcomes were the pharmacokinetics and pharmacodynamics of ricolinostat in this combination and the preliminary anti-tumour activity of this treatment. The trial is closed to accrual and is registered at ClinicalTrials.gov, number NCT01583283. Findings Between July 12, 2012, and Aug 20, 2015, we enrolled 38 patients. We observed two dose-limiting toxicities with ricolinostat 160 mg twice daily: one (2%) grade 3 syncope and one (2%) grade 3 myalgia event in different cohorts. A maximum tolerated dose was not reached. We chose ricolinostat 160 mg once daily on days 1–21 of a 28 day cycle as the recommended dose for future phase 2 studies in combination with lenalidomide 25 mg and dexamethasone 40 mg. The most common adverse events were fatigue (grade 1–2 in 14 [37%] patients; grade 3 in seven [18%]) and diarrhoea (grade 1–2 in 15 [39%] patients; grade 3 in two [5%]). Our pharmacodynamic studies showed that at clinically relevant doses, ricolinostat selectively inhibits HDAC6 while retaining a low and tolerable level of class I HDAC inhibition. The pharmacokinetics of ricolinostat and lenalidomide were not affected by co-administration. In a preliminary assessment of antitumour activity, 21 (55% [95% CI 38–71]) of 38 patients had an overall response. Interpretation The findings from this study provide preliminary evidence that ricolinostat is a safe and well tolerated selective HDAC6 inhibitor, which might partner well with lenalidomide and dexamethasone to enhance their efficacy in relapsed or refractory multiple myeloma. Funding Acetylon Pharmaceuticals.

Background: Immunosuppressive tumor associated macrophages (TAMs) have been shown to correlate with poor prognosis. SNDX-6352 is a humanized, high-affinity monoclonal IgG4 antibody that blocks dual ligand activation of colony stimulating factor- 1 receptor (CSF-1R), which regulates monocyte migration, proliferation and differentiation into TAMs. By inhibiting and reducing TAM numbers, SNDX-6352 may slow tumor growth and enhance anti-tumor immunotherapeutic approaches. Methods: SNDX-6352-0502 was a multi-center Phase 1 study consisting of Phase 1a (monotherapy) and Phase 1b (combination with durvalumab) in patients with pretreated, advanced solid tumors. The primary objective was determining the MTD and RP2D of SNDX-6352 alone and in combination with durvalumab. Dose regimens of 1, 2, 3 and 6 mg/kg bi-weekly (q2wks) and 6 mg/kg once monthly (q4wks) were administered in 1a, and 1, 2, and 3 mg/kg bi-weekly in 1b. Drug pharmacokinetics (PK) and pharmacodynamic (PD) biomarkers were measured. Results: Thirty-two patients with advanced solid tumors were enrolled with a median age of 61. The median number of prior therapies was 5 (range 2-12) and 72% of patients had ECOG performance status of 1. Median exposure in terms of cycles was 2, maximum was 10. Three patients had SD greater than 4 months. Most common (> 10%) treatment-related AEs were fatigue (31%), periorbital edema (31%) aspartate aminotransferase increased (22%), blood creatinine phosphokinase increased (22%) nausea (13%) and decreased appetite (13%). Grade 3/4 related AEs were observed in 12 patients (38%) with ≥ 2 events in CPK (5), amylase (3), AST (3) and lipase (2). One possibly related SAE of pneumonitis at 6 mg/kg q2wks was reported. DLTs reported in two subjects were G3 fatigue at 2 mg/kg and G3 pneumonitis at 6 mg/kg q2wks. Laboratory abnormalities included increases in blood creatinine phosphokinase, ALT, AST, amylase, and lipase. These abnormalities were all asymptomatic and are consistent with the known class effect of CSF-1Ri. There was no evidence of hepatocellular toxicity. Plasma concentrations of SNDX-6352 increased in a dose-proportional manner with drug accumulation observed at > 1 mg/kg. Plasma CSF1 and IL-34 concentrations increased with treatment and remained elevated at all doses > 1 mg/kg. CSF1 receptor occupancy was saturated at 4 hours post-dose in all treatment cohorts. Circulating non-classical monocytes (CD14+CD16hi) were ablated at all dose levels within 24 hrs and remained suppressed at all doses >1 mg/kg. Conclusion: SNDX-6352 demonstrates tolerability at the highest dose level evaluated (6 mg/kg) with robust PD biomarker modulation at doses as low as 1 mg/kg. A RP2D of 6mg/kg q4wks will be explored in future solid tumor studies. Citation Format: Nilo Azad, Drew Rasco, Sunil Sharma, Matthew Taylor, Christine Quaranto, David L. Tamang, Robert Nordness, Michael L. Meyers, Serap Sankoh, Peter Ordentlich, Anthony W. Tolcher. SNDX-6352-0502 - A phase 1, open-label, dose escalation trial to investigate the safety, tolerability, pharmacokinetics and pharmacodynamic activity of SNDX-6352 monotherapy in patients with unresectable, recurrent, locally-advanced, or metastatic solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT149.

Background: We previously reported that ENT + PEMBRO demonstrated promising clinical activity in anti-PD-(L)1 pre-treated NSCLC patients. Identification of circulating and/or tumor-resident biomarkers are essential for optimizing the utility of this combination. Here we report clinical data and the identification of gene expression signatures in tumors associated with response to PEMBRO + ENT in patients previously treated with PD-(L)1. Methods: Gene expression analysis of pre-treatment tumor samples by RNAseq and NanoString’s PanCancer IO360™ assay was conducted in a subset of patients with sufficient RNA yield (n=43 of 76). Comparisons of differential expression were made between responders (R) vs non-responders (NR) and monocyte high (MHi) vs low (MLo) subset; significance was defined by BH adjusted p 1.5 fold difference. Pre-ranked gene set enrichment analysis was performed against all human MSigDB collections and gene sets with positive normalized enrichment score and p-value Results: 76 patients were enrolled, of which 72 were evaluable for efficacy. The confirmed ORR with ENT + PEMBRO was 10% (7 of 72, 95% CI: 4-19%), with a median duration of response of 5.3 mos (4 patients ongoing) and median PFS of 2.8 mos. Results from gene set analysis revealed targets of Myc regulation (HALLMARK_MYC_TARGETS_V1) as the most significant enriched Hallmark collections in R (n=4) vs NR (n=39). Patients were also evaluated based on levels of baseline circulating classical monocytes (CD14+CD16-HLA-DRhi), which associated with response (MHi ORR 21.1%; PFS 5.3 mos vs MLo ORR 6.5%; PFS 2.7 mos). Myc target gene sets were also significantly enriched in MHi (n=11) vs MLo (n= 32) subjects. Oxidative phosphorylation, and E2F signaling were also among the top five for both R and MHi groups. 49.2% of statistically significant differentially expressed pathways were up-regulated in both R and MHi groups suggesting a strong overlap in differential gene expression in tumors from R and MHi patient subsets. Tumor adaptive immune resistance, as measured by the Tumor Inflammation Signature was also associated with both clinical response and longer PFS. Additionally, PD-L1, CTLA4, IFNγ-signaling and Th1 cell signatures correlated with response. Conclusions: Gene expression analysis identified up-regulated Myc signaling in R vs NR as well as in MHi vs MLo. Recent reports have implicated Myc signaling as an anti-PD-(L)1 resistance factor and we and others have shown Myc can be down-regulated by entinostat. Our data may provide a mechanistic basis for the responses seen with ENT + PEMBRO in this anti-PD-(L)1 pre-treated population. Citation Format: Peter Ordentlich, Lei Wang, David Tamang, Amy H. Sullivan, Sally E. Church, Dan Rozelle, Michael L. Meyers, Suresh S. Ramalingam, Matthew D. Hellmann. Identification of gene signatures associated with response in a Phase II trial of entinostat (ENT) plus pembrolizumab (PEMBRO) in non-small cell lung cancer (NSCLC) patients whose disease has progressed on or after anti-PD-(L)1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT041.

Introduction: Entinostat is an oral Class I selective histone deacetylase inhibitor (HDACi), shown in preclinical models to enhance the anti-tumor activity of immune checkpoint blockade through reduction in the number and function of immune suppressive cells in the tumor microenvironment. Encouraging preliminary activity of entinostat (5 mg PO weekly) in combination with pembrolizumab (200 mg IV Q3W) has been previously reported in patients with melanoma and non-small cell lung cancer. This Phase 1 study was conducted to explore the impact on immune correlatives, pharmacokinetics (PK), safety and efficacy of different dose levels and schedules of entinostat combined with pembrolizumab. Methods: Up to 30 patients with advanced solid tumors who previously completed Study SNDX-275-0140 (cardiac safety study of entinostat versus placebo) were planned to be enrolled. Patients were randomized 1:1:1 into three arms: Arm A) 1 mg entinostat Days 1-5 QW; Arm B) 5 mg entinostat Day 1 QW; Arm C) 10 mg entinostat Day 1 Q2W. All patients received 200 mg pembrolizumab Day 1 Q3W, and treatment continued until progression or unacceptable toxicity. Biomarkers were collected pre-dose & Cycle 1 Day 15, and PK samples were collected at Cycles 1, 2 and 3. Results: 26 patients with advanced solid tumors were enrolled to Arm A (n=8), Arm B (n=9) and Arm C (n=9). No notable differences in the safety profile were observed among the 3 arms, and the overall safety profile was consistent with prior experience of entinostat combined with pembrolizumab. The most common treatment-related adverse events (>15%) included fatigue, nausea, neutropenia, dyspepsia, and pneumonitis. All of these events were Grade 1/2 with the exception of neutropenia (19.2% Grade ≥3). Three patients (breast and uterine cancer in Arm B; endometrial in Arm C) had a partial response (PR), and four patients (including 2 PRs and 2 breast cancer patients with stable disease) remain on study treatment beyond 40 weeks. All of these breast cancer patients had experienced progression of disease on hormonal therapy. PK analysis showed dose dependent increases (Arm C > Arm B > Arm A) in exposure to entinostat (AUC(Cycle 1)) with increasing variability at higher dose. Decreased numbers of monocytic myeloid derived suppressor cells (M-MDSCs; CD14+HLA-DRlow/neg) were observed in all arms, with no significant differences among arms. Protein lysine acetylation in multiple immune populations was elevated post-therapy in Arms A and B, but not Arm C, when analyzed at baseline and Cycle 1 Day 15. Conclusions: The combination of entinostat and pembrolizumab continues to show promising activity and acceptable safety in heavily pretreated cancer patients. Consistent with previous reports, entinostat results in reductions in circulating MDSC frequency. Citation Format: Anthony W. Tolcher, Michael L. Meyers, Dmitry Gabrilovich, Fang Wang, Jane Trepel, Min-Jung Lee, Emmett Schmitt, Christine Quaranto, Serap Sankoh, David Tamang, Peter Ordentlich. Safety, efficacy, and immune correlates of alternative doses and schedules of entinostat combined with pembrolizumab in patients with advanced solid tumors - results from SNDX-275-0141 phase I trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT179.

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult β-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene.

Background: The selective HDAC6 inhibitor ACY-241, a tablet, is structurally related to ricolinostat (ACY-1215), the first agent in this class in the clinic.Ricolinostat, an oral liquid, demonstrated clinical efficacy in a Phase 2 combination with pomalidomide (Pom) and dexamethasone (Dex) in patients (pts) with relapsed or relapsed-and-refractory multiple myeloma (RRMM) without toxicities greater than those reported with Pom and Dex alone (Raje et al., EHA 2016, S813). Preclinical data demonstrate synergistic activity of ACY-241 with Pom and lenalidomide (Len) in induction of cell cycle arrest and apoptosis in MM cells as well as significant extension of survival in a mouse xenograft model (Niesvizky et al., Blood 2015, 126: 3040). We present updated data on safety and efficacy of the ACY-241/Pom/Dex combination in pts with relapsed or RRMM (ACE-MM-200, NCT02400242). Aims:Determine the safety, tolerability, and preliminary efficacy of ACY-241 monotherapy and combination with Pom and Dex and the recommended dose for further development. Methods:Based on clinical experience with ricolinostat and non-clinical pharmacokinetics (PK) of ACY-241, we designed a first-in-human phase 1a/1b clinical trial of a single-cycle of ACY-241 monotherapy followed by ACY-241 in combination with Pom (4mg) and low-dose Dex in pts with relapsed or RRMM. The starting dose of ACY-241 was chosen to give similar exposure to the therapeutic dose of ricolinostat (160 mg QD). The trial design was chosen to grant pts access to combination therapy with an active regimen while exploring the safety, PK, and pharmacodynamic profile of ACY-241 alone and in combination with Pom/Dex. The PK of Pom and Dex was also assessed. Pts with relapsed or RRMM previously treated with ≥ 2 cycles of Len and a proteasome inhibitor were eligible. Cohorts of 3 pts had ACY-241 PO QD as monotherapy (180, 360 and 480 mg) on days 1-21 of a 28 day cycle. If no DLT was noted in cycle 1 with ACY-241, pts continued to cycle 2 of combo therapy with ACY-241/Pom/Dex. Pharmacodynamic assessments were acetylated tubulin (HDAC6 marker) and acetylated histones (Class 1 HDAC marker) in peripheral blood mononuclear cells. Results: Since June 2015, 40 pts have enrolled (34 safety-evaluable, 6 had no dosing information in the database). Median age was 62 (34-84) years and median number of prior regimens was 3 (1-7). 90% of pts were refractory to last treatment. 83% were refractory to Len and 50% to both bortezomib and Len. 20% of pts had high risk cytogenetics. No monotherapy DLTs were observed at the highest dose explored (480 mg). Common toxicities in the monotherapy safety population (N=15) were all grade 1/2, except 1 pt with grade 3 anemia at the 480 mg dose level. Toxicities included nausea (4 pts, 27%), anemia (3 pts, 20%), dizziness, fatigue, leukopenia and thrombocytopenia (2 pts each, 13%). Doses of 180 mg and 360 mg were explored in combination; one DLT (grade 4 thrombocytopenia) occurred at 360 mg. Common toxicities in the combination therapy safety population (N=33) included neutropenia (13 pts, 40%), fatigue (9 pts, 27%), anemia, leukopenia (6 pts each, 18%), cough, insomnia, rash (4 pts each, 12%), and hyperglycemia (3 pts, 9%). Grade 3/4 toxicities included neutropenia (10 pts, 30%), leukopenia (3 pts, 9%) and anemia (2 pts, 6%). PK results showed a dose-linear increase in exposure with increasing dose, no accumulation and no drug-drug interaction with Pom and Dex. Selective increase in acetylated tubulin was seen at 180 mg with increasing levels of acetylated tubulin and histones at higher doses. Confirmed efficacy data (median follow-up 3.5 months) for combination treatment (N=22, all refractory to last treatment regimen) shows 1 VGPR, 10 PR, 2 MR and 8 SD and 1 PD. Median PFS and duration of response were not reached at time of the data cut. Given the safety profile, PK exposure (Cmax~6 µM) and PD profile, the 360 mg QD dose level was recommended for further clinical exploration of ACY-241 in combination with Pom/Dex. Summary/Conclusion:ACY-241 is well tolerated in combination with Pom/Dex with dose proportional increase in drug exposure. Early response data to combination treatment parallel those observed with ricolinostat/Pom/Dex and compare favorably to historic controls of Pom/Dex. Cohort expansion at 360 mg ACY-241 with Pom/Dex is ongoing to confirm the dose and schedule for a planned pivotal trial of Pom/Dex +/- ACY-241 and to explore selected biomarkers. Disclosures Richardson: Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nooka:Amgen: Consultancy; Spectrum: Consultancy; Novartis: Consultancy. Raab:Amgen: Consultancy, Research Funding; BMS: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Research Funding. Shain:Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Signal Genetics: Research Funding. Matous:Celgene: Consultancy, Speakers Bureau; Takeda Pharmaceuticals International Co.: Speakers Bureau; Seattle Genetics: Research Funding, Speakers Bureau. Agarwal:Celgene: Speakers Bureau; Onyx: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Consultancy; Millennium: Consultancy; AbbVie: Honoraria, Research Funding. Madan:Amgen: Speakers Bureau; Onyx: Speakers Bureau; Takeda: Speakers Bureau; Celgene: Speakers Bureau. Moreau:Novartis: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria. Mateos:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria. Facon:Acetylon Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Tamang:Acetylon Pharmaceutical Inc.: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Markelewicz:Acetylon Pharmaceutical Inc.: Employment. Wheeler:Acetylon Pharmaceuticals Inc.: Employment. Trede:Acetylon Pharmaceutials Inc: Employment. Raje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding. Terpos:Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Genesis: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Novartis: Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; Celgene: Honoraria. Bensinger:Amgen: Honoraria; Celgene: Honoraria; Acetylon Pharmaceuticals Inc.: Honoraria; Amgen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Bristol-Meyers Squibb: Consultancy; Celgene: Speakers Bureau; Takeda: Speakers Bureau; Amgen: Speakers Bureau; Acetylon Pharmaceuticals Inc: Research Funding; Bristol-Meyers Squibb: Research Funding; Celgene: Research Funding; Karyopharm Therapeutics: Research Funding; Merck: Research Funding; Amgen: Research Funding; Sanofi: Research Funding; Takeda: Consultancy.

INTRODUCTION: HDACs are a family of enzymes that remove acetyl groups from proteins and are involved in key cellular processes. Nonselective HDAC inhibitors have shown promise in treating MM in combination with standard therapies such as proteasome inhibitors (PIs) and immunomodulatory agents, but are limited by adverse effects. The selectivity profile of clinical HDAC inhibitors is critically important as the same drug may exert its effects via alternative mechanisms depending on the partner therapeutic. An example is HDAC6 inhibitors in combination with bortezomib, a proteasome inhibitor (PI). Delivery of protein-bearing vesicles to the aggresome involves HDAC6 through its role in vesicle trafficking, thus supporting a secondary survival pathway. In the case of lenalidomide or pomalidomide, inhibiting class I HDACs can suppress MYC protein levels, which potentially enhances activity in a clinical setting. Ricolinostat (ACY-1215) and ACY-241 that are currently in clinical trials have a selectivity profile tuned toward HDAC6 over HDACs 1, 2, and 3, which is hypothesized to enhance anti-cancer activity with multiple standard of care regimes while minimizing toxicity. METHODS: Peripheral blood from patients that received escalating oral doses of ricolinostat or ACY-241 that range from 40 mg to 360 mg was collected for matched PK and PD assessment. Lymphocytes were examined for tubulin and histone hyper-acetylation by flow cytometry. RESULTS & CONCLUSION: Plasma concentration for both ricolinostat and ACY-241 reach a maximal level (Cmax) 1-2 hr after administration. An exposure plateau was reached at dose levels ≥160 mg of ricolinostat, with Cmax observed to be ∼2 μM. ACY-241 exposures reached higher levels, Cmax of ∼3 μM and ∼7 μM at 180 and 360 mg, respectively, and no evidence of an exposure plateau. Correlating to the PK levels, acetyl-tubulin increases to a maximal level by 1-2 h after dosing compared to predose, and declines to basal level by >4 hr. As plasma levels increased with dose from 40 mg to 160 mg for ricolinostat so do the number of patients that have an increase in acetyl-tubulin such that at dose levels ≥80 mg all patients have a measurable increase in acetylated tubulin. Levels of acetyl-tubulin were similar at dose levels of 180 and 360 mg of ACY-241. Acetyl-histone levels were low- moderate at the 160 and 180 mg dose level for ricolinosat and ACY-241, respectively, and for ACY-241 increased with increasing exposure at doses > 180 mg similar to results observed in vitro by immunoblot analysis of MM cells treated with ACY-241. In conclusion, the PK and PD results suggest that both ricolinostat and ACY-241 have reached a pharmacologically relevant level of HDAC inhibition as determined by acetylated tubulin and histone at the clinical dose levels examined. Citation Format: David L. Tamang, Jeffery Supko, Kailash Bhol, Catherine Wheeler, Simon S. Jones, Min Yang. Tubulin hyper-acetylation in blood lymphocytes: pharmacodynamic (PD) biomarker for the selective histone deacetylase (HDAC) 6 inhibitors ricolinostat and ACY-241 in multiple myeloma (MM) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3958.

INTRODUCTION: Neuroblastoma (NB) is an extra-cranial solid cancer and is among the most common cancers in infants less than 1 year of age, with 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating an urgent unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) treatment. RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified, and chemical inhibition of HDAC1/2 was shown to induce a similar expression pattern. METHODS: In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by RT-PCR, gene expression microarray and retinoic acid receptor (RAR) chromatin immunoprecipitation followed by high-througput sequening (ChIP-Seq). RESULTS: HDAC1/2i and RA together caused increased localization of the RAR to its own RARα and RARβ promoter regions, and increased in RAR mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26, an enzyme responsible for clearing intercellular RA, was reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i enhanced apoptotic pathways and decreased E2F driven cell cycle signaling. We confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage. Consistent with this finding, we observed reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies after combination treatment. Further, combination treatment reduced the E2F-activators CDK4 and CDK6 at the protein level while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress, as are HDAC1, HDAC2, acetylated histone H3K9 ChIP-seq experiments, and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Huang, Olga Golonzhka, Jeffrey R. Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. An orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2477.

Histone deacetylase (HDAC) enzymatic activity has been linked to the transcription of DNA in cancers including multiple myeloma (MM). Therefore, HDAC inhibitors used alone and in combination are being actively studied as novel therapies in MM. In the present study, we investigated the preclinical activity of ACY-1215, an HDAC6-selective inhibitor, alone and in combination with bortezomib in MM. Low doses of ACY-1215 combined with bortezomib triggered synergistic anti-MM activity, resulting in protracted endoplasmic reticulum stress and apoptosis via activation of caspase-3, caspase-8, and caspase-9 and poly (ADP) ribosome polymerase. In vivo, the anti-MM activity of ACY-1215 in combination with bortezomib was confirmed using 2 different xenograft SCID mouse models: human MM injected subcutaneously (the plasmacytoma model) and luciferase-expressing human MM injected intravenously (the disseminated MM model). Tumor growth was significantly delayed and overall survival was significantly prolonged in animals treated with the combination therapy. Pharmacokinetic data showed peak plasma levels of ACY-1215 at 4 hours after treatment coincident with an increase in acetylated α-tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blot analysis. These studies provide preclinical rationale for acetylated α-tubulin use as a pharmacodynamic biomarker in future clinical trials.

Background: Ricolinostat, an oral selective HDAC6 inhibitor, is well tolerated as monotherapy (Raje Blood 2012;120:4061). HDAC6 inhibition impairs the aggresome/autophagy pathway, an alternate pathway to proteasome clearance of misfolded proteins (Santo Blood 2012;119:2578-89) providing the rationale for combining ricolinostat with bortezomib (Btz). Pan-HDAC inhibitors vorinostat and panobinostat are active in multiple myeloma (MM) in combo with Btz, but toxicities (thrombocytopenia, fatigue, and GI events) limit dosing exposure. This trial explores activity of ricolinostat in combo with Btz and dexamethasone (Dex) in patients with relapsed or relapsed-and-refractory MM. Methods: Phase 1b was a 3+3 design that explored ricolinostat combined with Btz (1.0-1.3 mg/m2 IV or SQ on days 1, 4, 8, 11) and Dex (20 mg PO on days 1, 2, 4, 5, 8, 9, 11, 12). Patients previously received at least 2 lines of therapy including a proteasome inhibitor and an immunomodulatory agent, had either progressed after or were ineligible for autologous stem cell transplant, and had adequate BM reserve, hepatic function and CrCl ≥30 mL/min. Patients with prior HDAC inhibitor therapy were excluded. Peripheral blood samples were obtained for PK/PD assessment of acetylated tubulin and histones. Results: 50 patients were enrolled to 8 combination dose cohorts to date. Median age was 65, median number of prior regimens was 5 (2-12) and 33 patients were refractory to Btz. The first combination cohort was expanded due to a DLT of asymptomatic increase in amylase. No other DLTs have been observed in the cohort escalations. Common toxicities were predominately low grade (grade 1/2) and included diarrhea (46%), anemia (38%), thrombocytopenia (22%), increased creatinine (18%), fatigue (16%), and nausea (16%). Important grade 3/4 related toxicity included thrombocytopenia (9 patients, 18%) and anemia (6 patients, 12%). Cohorts were expanded sequentially at two dose levels: 160 mg BID and 160 mg QD. Of the 22 patients receiving ricolinostat 160 mg BID common toxicities were diarrhea (68%, grade 1/2), thrombocytopenia (50%, grade 3/4), anemia (45%, grade 2/3), fatigue (36%, grade 1/2), and nausea (36%, grade 1/2). There was a marked increase in the incidence of SAEs related to diarrhea leading to dehydration and hospitalization (27%). Therefore a second cohort expansion at 160 mg QD was initiated. Of the 15 patients receiving ricolinostat at 160 or 240 mg QD common toxicities were diarrhea (33%, grade 1/2), thrombocytopenia (20%, grade 4), anemia (20%, grade 3), increased creatinine (20%, grade 3), hyponatremia (20%, grade 1), and increased ALT (20% grade 3), and peripheral neuropathy (20%, grade 1/2). PK for ricolinostat is similar to that observed in Phase 1a monotherapy, indicating that co-administration of Btz does not significantly impact ricolinostat exposure. Maximal blood levels of ricolinostat were ≥0.5 µM at ≥80 mg correlating with a >2x increase in the HDAC6 PD marker acetylated tubulin. 48 patients were evaluable for response with median follow-up of 3 (1-18) months; ORR (≥PR) was 39% and clinical benefit rate (≥MR) was 44% with 81% SD or better. Of the 22 patients receiving ricolinostat 160 mg BID with median follow-up of 3 (1-18) months, ORR (≥PR) was 32% and clinical benefit rate (≥MR) was 41% with 62% SD or better. Of the 12 patients receiving ricolinostat at 160 or 240 mg QD with median follow-up of 4 (1-8) months, ORR (≥PR) was 33% and clinical benefit rate (≥MR) was 33% with 67% SD or better. Of the 33 Btz refractory patients, 42% had SD or better, and 12% responded including 1 VGPR. Conclusion: Overall, ricolinostat in combination with Btz and Dex was well tolerated. Diarrhea and hematologic toxicity, mostly low grade, was less prevalent with QD than BID dosing and led to fewer hospitalizations and early treatment discontinuation. Response rates were similar between both QD and BID dosing. 160 mg QD is the recommended dose in combination with Btz and Dex. Disclosures Vogl: Constellation Pharmaceuticals: Research Funding; GSK: Research Funding; Millennium Pharmaceuticals: Research Funding; Calithera Biosciences: Research Funding; Celgene Corporation: Consultancy; Acetylon Pharmaceuticals, Inc.: Research Funding. Raje:Acetylon: Research Funding; Amgen: Consultancy; Eli Lilly: Research Funding; Onyx: Consultancy; Celgene Corporation: Consultancy; AstraZeneca: Research Funding; BMS: Consultancy; Takeda: Consultancy; Millenium: Consultancy; Novartis: Consultancy. Jagannath:Celgene: Honoraria; MERCK: Honoraria; Janssen: Honoraria; Novartis Pharmaceuticals Corporation: Honoraria; BMS: Honoraria. Richardson:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Hari:Sanofi: Consultancy; Takeda: Consultancy; Spectrum: Consultancy; Novartis: Consultancy; Janssen: Consultancy; BMS: Consultancy; Celgene: Consultancy. Orlowski:Millennium Pharmaceuticals: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Acetylon: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Spectrum Pharmaceuticals: Research Funding; Forma Therapeutics: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Wheeler:Acetylon Pharmaceuticals, Inc: Employment. Markelewicz:Acetylon Pharmaceuticals, Inc: Employment. Lonial:Millennium: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

Background: Ricolinostat, an oral selective HDAC6 inhibitor, is well tolerated as monotherapy (Raje Blood 2012;120:4061) and demonstrates potent synergistic activity with lenalidomide (Len) and pomalidomide (Pom) in preclinical models (Quayle Blood 2013;122:1952). In an ongoing trial of ricolinostat in combination with Len and dexamethasone (Dex) in patients with relapsed or refractory multiple myeloma (MM), no dose limiting toxicities (DLTs) were observed at up to doses of 160 mg BID, with excellent activity and tolerability (Yee Blood 2014;124:4772). Pan-HDAC inhibitors vorinostat and panobinostat are active in MM in combination with bortezomib (Btz) and Len, but toxicities (thrombocytopenia, fatigue, and GI events) limit dosing exposure. This trial explores activity of ricolinostat in combination with Pom and Dex in a patient population comparable to that in MM-003 (San Miguel Lancet Oncol 2013;14:1055-66). At the time of achieving the primary endpoint of PFS in MM-003, the ORR was 16.6% at 18.1 weeks median follow-up (EMA Pom Celgene Assessment Report EMA/CHMP/427059/2013), and was 31% at 10 months in the same clinical trial (San Miguel Lancet Oncol 2013;14:1055-66). Methods: Phase 1b was a 3+3 design which explored ricolinostat 160 mg QD or BID combined with Pom (4 mg) for 21 days of a 28 day cycle with Dex (40 mg) on days 1, 8, 15 and 22. Patients had measurable disease, adequate BM reserve and hepatic function with CrCl ≥45 mL/min. Refractory was defined as PD on or within 60 days of last therapy. Patients with non-secretory MM, prior Pom or HDAC inhibitor therapy were excluded. Peripheral blood samples were obtained for PK/PD assessment of acetylated tubulin and histones. Patients with serum FLC only disease were excluded from Phase 2. The dose level of 160 mg dose was chosen based on prior experience in which PK plateau was reached at 160 mg of ricolinostat. A sample size of 66 was determined to be adequate to detect an ORR of 44% against a historical rate of 29%. Results: 7 patients were treated in phase 1b: 3 at 160 mg QD and 4 at 160 mg BID. No DLTs were observed; however, all 4 patients had grade 2 diarrhea in the 160 mg BID cohort and grade 3 fatigue and grade 3 or 4 neutropenia attributed to Pom were reported in patients in phase 1b. 1 patient remained on study for 12 cycles before PD; the other 6 Phase 1b patients were withdrawn for PD at 2-4 cycles. Following safety review committee (SRC) review, phase 2 opened at a dose of 160 mg BID, with a predetermined SRC review to occur after 6 patients had completed 1 cycle of therapy. 3 of the 6 patients had clinically relevant diarrhea requiring ricolinostat dose reduction to 160 mg QD in cycle 2 leading to SRC recommendation of QD dosing of ricolinostat. As of April 28, 2015 39 phase 2 patients were evaluable for safety and 28 evaluable for response. Median age was 68 and median number of prior regimens was 4 (2-9) in 3 (2-6) line of therapy. 37 patients were refractory to Len as defined in the entry criteria. Common toxicities were predominantly low grade and included fatigue (40%), diarrhea (38%), neutropenia (36%), anemia (31%), thrombocytopenia (26%), and constipation (21%). Important grade 3/4 related toxicities included neutropenia (5 patients, 13%) and diarrhea (3 patients, 8%). PK of ricolinostat was similar to that observed in combination with Btz and Len. There was no evidence of ricolinostat accumulation or drug-drug interaction with Pom. Median follow-up of 12 (4-32) weeks; ORR (≥PR) was 29% with 3 VGPR and clinical benefit rate (≥MR) was 50% with 68% SD or better for the 28 response evaluable patients. Updated safety and efficacy data will be presented as enrollment continues and data matures including for the 49 patients enrolled in phase 2 to date. Conclusion: These early data with a median follow-up of 12 weeks (compared to historical control of 16.6% ORR at 18.1 weeks for Pom/Dex alone in MM-003) in a rigorously defined patient population suggest that selective HDAC6 inhibition is associated with encouraging tolerability and promising clinical activity. Accrual is continuing in US, Canada, and Europe. Overall, ricolinostat is an active and safe oral agent which combines favorably with Pom and Dex in relapsed-and-refractory MM. Disclosures Raje: Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Millenium: Consultancy; Onyx: Consultancy; Acetylon: Research Funding; Celgene Corporation: Consultancy; BMS: Consultancy. Bensinger:Sanofi: Research Funding; Acetylon Pharmaceuticals, Inc: Research Funding; BMS: Research Funding; Novartis: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Research Funding; Onyx: Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Lonial:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Jagannath:BMS: Honoraria; Novartis Pharmaceuticals Corporation: Honoraria; Janssen: Honoraria; MERCK: Honoraria; Celgene: Honoraria. Valent:Takeda/Millennium: Speakers Bureau; Celgene: Speakers Bureau. Harb:Idera Pharmaceuticals: Research Funding; Astex Pharmaceuticals, Inc.: Research Funding. Sandhu:Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Bahlis:Johnson & Johnson: Research Funding; Johnson & Johnson: Speakers Bureau; Johnson & Johnson: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Reece:Merck: Research Funding; Lundbeck: Honoraria; Amgen: Honoraria; Onyx: Consultancy; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Millennium Takeda: Research Funding; Otsuka: Research Funding. Terpos:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Takeda: Honoraria; Celgene: Honoraria, Other: Travel expenses; Novartis: Honoraria; Amgen: Honoraria, Other: Travel expenses, Research Funding. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Markelewicz:Acetylon Pharmaceuticals, Inc: Employment. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

Background: Ricolinostat, an oral selective HDAC6 inhibitor, is well tolerated as monotherapy (Raje Blood 2012;120:4061) and demonstrates potent synergistic activity with lenalidomide (Len) and pomalidomide (Pom) in preclinical models (Quayle Blood 2013;122:1952). Immunomodulatory drugs (thalidomide, Len, and Pom) trigger anti-tumor activities in multiple myeloma (MM) in part by targeting cereblon, a component of an E3 ubiquitin ligase complex, leading to decreased levels of IZF1/3, c-Myc and IRF4, critical factors in MM proliferation. Preclinical models using ricolinostat in combination with Len was shown to both avoid cereblon downregulation caused by non-selective HDAC inhibitors, and enhance Len mediated downregulation of c-Myc, IRF4 and IZF1/3 to induce synergistic cytotoxicity in MM (Hideshima Blood Cancer J 2015;5:e312). Pan-HDAC inhibitors vorinostat and panobinostat are active in MM in combination with bortezomib (Btz) and Len, but toxicities (thrombocytopenia, fatigue, and GI events) limit dosing exposure. This trial explores activity of ricolinostat in combination with Len and dexamethasone (Dex) in patients with relapsed and relapsed-and refractory MM. Methods: Phase 1b was a 3+3 design which studied ricolinostat combined with Len (15-25 mg QD on Days 1-21 of a 28-day cycle) and Dex (40 mg weekly). In Regimen A, patients were treated with escalating doses of ricolinostat on Days 1-5 and 8-12 of a 28-day cycle with Len and Dex. In Regimen B, ricolinostat was also given on Days 15-19. Doses up to 240 mg QD and 160 mg BID were explored. In Regimen C, patients were treated with 160 mg ricolinostat BID or QD for 21 of 28 days with Len and Dex. Patients had measurable disease, adequate BM reserve, hepatic function, and CrCl ≥50 mL/min. Per the IMWG, refractory was defined as disease that is nonresponsive while on primary or salvage therapy, or progresses on or within 60 days of last therapy. Patients with non-secretory MM or prior HDAC inhibitor therapy were excluded. Peripheral blood samples were obtained for PK/PD assessment of acetylated tubulin and histones. Results: 33 patients have been enrolled to date. Median age was 63 with a median of 3 (2-9) lines of prior therapy. 22 patients were previously treated with Len, and 12 of these patients were refractory to Len as defined in the entry criteria. No MTD was observed at up to 160 mg BID. Common toxicities were predominately low grade and included fatigue (55%), neutropenia (36%), diarrhea (33%), upper respiratory tract infections (30%), and anemia (27%). Important grade 3/4 related toxicities included neutropenia (6 patients, 18%), fatigue (2 patients, 6%), diarrhea (2 patients, 6%), and vomiting (2 patients, 6%). 2 DLTs have been observed: grade 3 syncope in Regimen B at 160 mg BID on Days 1-5, 8-12 and 15-19 and grade 3 muscle cramps in Regimen C at 160 mg BID Days 1-21. The last cohort is being expanded at 160 mg QD Days 1-21 based on prior cohort expansions and other trials of ricolinostat in combination with Btz and Pom where 160 mg ricolinostat QD is better tolerated than BID dosing (Vogl Blood 2014;124:4764). PK for ricolinostat is similar to that observed in Phase 1a monotherapy, indicating that co-administration of Len does not significantly impact ricolinostat exposure. Maximal blood levels of ricolinostat were ≥0.5 µM at ≥80 mg correlating with a >2x increase acetylated tubulin (HDAC6 PD marker) with minimal increase in acetylated histones (class 1 HDAC PD marker). 31 patients were evaluable for response with ORR (≥PR) was 55% with 1 sCR and 7 VGPR and clinical benefit rate (≥MR) was 61% with 71% SD or better. The median follow-up of all 31 patients compared to the 17 responders was 6 (1-33) months and 12 (1-33) months, respectively. 19 patients were previously naïve or responsive (≥MR) to Len with median follow-up of 11 (1-33) months had ORR (≥PR) of 68% and clinical benefit rate (≥MR) of 74%. 12 patients refractory to Len with median follow-up of 5 (3-10) months had ORR (≥PR) of 33% and clinical benefit rate (≥MR) of 42% with 67% SD or better. Conclusion: Ricolinostat is an active and safe oral agent in combination with Len and Dex in relapsed and relapsed-and-refractory MM. No MTD has been identified. A dose of 160 mg QD on Days 1-21 of a 28-day cycle is proposed for phase 2 based on excellent tolerability. The median duration on study for responders was a year, with some patients continuing up to 33 months. Disclosures Bensinger: Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Research Funding; BMS: Research Funding; Novartis: Research Funding; Sanofi: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Acetylon Pharmaceuticals, Inc: Research Funding. Voorhees:Millennium Pharmaceuticals: Consultancy, Research Funding; Onyx Pharmaceuticals: Research Funding; Celgene: Research Funding; Oncopeptides: Research Funding; Acetylon Pharmaceuticals, Inc.: Research Funding; Janssen: Research Funding; GSK: Research Funding; Oncopeptides: Consultancy; Array BioPharma: Consultancy; Novartis: Consultancy; Celgene: Consultancy; A Takeda Oncology Company: Consultancy, Research Funding; GSK: Consultancy. Berdeja:BMS: Research Funding; Acetylon: Research Funding; Array: Research Funding; Janssen: Research Funding; Curis: Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; MEI: Research Funding; Takeda: Research Funding; Onyx: Research Funding. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Markelewicz:Acetylon Pharmaceuticals, Inc: Employment. Raje:AstraZeneca: Research Funding; Acetylon: Research Funding; Onyx: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Millenium: Consultancy; Eli Lilly: Research Funding; Amgen: Consultancy; BMS: Consultancy.

Neuroblastoma (NB) is an extra-cranial solid cancer arising from the neural crest and is among the most common cancers in infants less than 1 year of age (Park, JR et al., 2008). Approximately one child per 100,000 is diagnosed with NB, resulting in 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating a clear unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) (Yang, RK et. al., 2010; Cheung, NK et. al., 2012). RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified (Hahn, CK et. al., 2008; Frumm, SM et. al., 2013), and inhibition of HDAC1/2 was shown to induce a similar expression pattern. In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by microarray and retinoic acid receptor (RAR) ChIP-seq. HDAC1/2i and RA together caused increased localization of the RAR to its own RARa and RARß promoter regions, and an increase in mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26a1/b1, enzymes responsible for clearing intercellular RA, were reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i was enhancing apoptotic pathways and decreasing E2F driven cell cycle signaling. In further experiments, we confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage, which is consistent with reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies. Further, the E2F-activators, CDK4 and CDK6, were reduced at the protein level in the combination setting while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Haung, Olga Golonzhka, Jeff Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. A novel, orally bioavailable and selective histone deacetylase (HDAC) 1 & 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A136.

Neuroblastoma is an extra-cranial solid cancer arising from the neural crest and is among the most common cancers in infants less than 1 year of age (Park, JR et al., 2008). Approximately one child per 100,000 is diagnosed with neuroblastoma, resulting in 650 new cases each year in the United States. Half of the children with neuroblastoma have high risk disease and 20% - 50% of those children will fail to respond adequately to current therapies, illustrating a clear unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (Yang, RK et. al., 2010; Cheung, NK et. al., 2012). Retinoic acid is a pro-differentiation agent that acts on neuroblastoma cells to slow growth and promote cell death. A gene expression pattern associated with retinoic acid induced neuroblastoma differentiation was recently identified (Hahn, CK et. al., 2008; Frumm, SM et. al., 2013), and it was further shown that inhibition of HDAC1/2 was able to induce a similar expression pattern. In this work, we demonstrate that next generation selective and orally bioavailable HDAC1/2 inhibitors can induce gene expression changes in neuroblastoma cells consistent with differentiation. The action of HDAC1/2 inhibitors potently enhances the retinoic acid differentiation effect at sub-optimal concentrations of retinoic acid or HDAC inhibitor, as well as with intermittent (pulse) HDAC1/2 inhibition. Retinoic acid alone and in combination with HDAC1/2 inhibitors is able to slow cell proliferation in long term growth assays and alter morphology in a manner consistent with differentiation. The observed enhancement of differentiation by selective HDAC1/2 inhibitors occurs at concentrations below that required for cell death as evidenced by viability assays and caspase 3/7 activation. Acute toxicity is induced by elevated concentrations of HDAC1/2 inhibitors, and synergy is observed in combination with retinoic acid. Ongoing studies exploring global gene expression changes, ChIP-seq examining retinoic acid receptor and HDAC1/2 chromatin binding, and activity of the selective HDAC1/2 inhibitor in combination with retinoic acid in animal models of neuroblastoma will be discussed. Taken together, these findings support a role for selective HDAC1/2 inhibitors in combination with retinoic acid for the treatment of patients with high risk neuroblastoma. Citation Format: David L. Tamang, Emily Lurier, Olga Golonzhka, Steven N. Quayle, Simon S. Jones, Min Yang. Novel and selective histone deacetylase (HDAC) 1 & 2 inhibitors enhance differentiation of neuroblastoma cells in combination with retinoic acid. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2196. doi:10.1158/1538-7445.AM2015-2196

AML is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. There is a great need for novel approaches to treat AML due to limited improvements in the treatment of patients over the past several decades since the development of cytarabine and daunorubicin combination therapy. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and the DNA methyltransferase inhibitor azacitidine is approved for the treatment of myelodysplastic (MDS) which frequently progresses to AML. Numerous clinical studies are ongoing to investigate the benefit of combining azacitidine with other investigational agents in AML. HDAC inhibitors are emerging as promising agents for the treatment of AML. Selective HDAC inhibitors are predicted to reduce the combination drug toxicity and other side effects observed with non-selective HDAC inhibitors, while also realizing beneficial therapeutic effects. One example is ricolinostat (ACY-1215), a first-in-class orally available HDAC inhibitor that is 11-fold selective for HDAC6, synergizes with proteasome inhibitors (Santo, et al, Blood, 2012) and immunomodulatory agents (Quayle, et al, ASH, 2013) in preclinical models of B-cell malignancies, which has thus far demonstrated an improved safety and tolerability profile in Phase I trials in multiple myeloma (Raje, Haematological, 2014, Suppl 1). In this work, we evaluated the efficacy of azacitidine in combination with selective inhibitors of either HDAC1/2, HDAC3 or HDAC6 on AML cells. Comparison of the panel of selective HDAC inhibitors demonstrated that HDAC1/2 inhibition is sufficient to reduce cell viability, induce differentiation, arrest cell cycle progression and initiate apoptosis in multiple AML cell lines. Selective inhibition of HDAC3 induced differentiation yet had no effect on cell cycle progression or apoptosis, while selective inhibition of HDAC6 had no effect on differentiation, cell cycle arrest or apoptosis in these cells. Furthermore, combining HDAC1/2 inhibition with azacitidine led to significantly enhanced induction of differentiation and apoptosis, and decreases in AML cell viability compared to either agent alone. Ongoing studies exploring the molecular mechanism underlying the activity of selective HDAC1/2 inhibitors, as well as the activity of combination treatment with azacitidine in primary AML cells and animal models of AML, will be discussed. Together, these findings provide support for the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML and potentially MDS patients. Citation Format: Chengyin Min, Steven N. Quayle, David Tamang, Simon S. Jones, Min Yang. Novel and selective inhibitors of histone deacetylases (HDAC) 1 and 2 significantly enhance the activity of the DNA methyltransferase inhibitor azacitidine in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5372. doi:10.1158/1538-7445.AM2015-5372

Histone deacetylase (HDAC) enzymes are attractive therapeutic targets in oncology, but non-selective HDAC inhibitors have led to dose-limiting toxicities in patients, particularly in combination with other therapeutic agents. Ricolinostat (ACY-1215), a first-in-class orally available HDAC inhibitor that is 11-fold selective for HDAC6, synergizes in vitro and in vivo with bortezomib in models of MM (Santo, Blood, 2012) and with carfilzomib in models of lymphoma (Dasmahapatra, Mol Cancer Ther, 2014). Furthermore, ricolinostat has demonstrated an excellent safety and tolerability profile in Phase I trials (Raje, Haematologica, 2014, Suppl 1). In addition to ongoing clinical studies with ricolinostat, we have identified ACY-241 as a next generation, orally available, selective inhibitor of HDAC6 for clinical evaluation. The IMiD® immunomodulatory class of drugs, including lenalidomide (len) and pomalidomide (pom), exhibit striking anti-myeloma properties in a variety of MM models, and have demonstrated significant clinical activity in MM patients. Beyond MM, len is also approved for the treatment of MCL patients. In support of our ongoing clinical development programs for ricolinostat and ACY-241, we show here that combinations with either len or pom leads to synergistic decreases in the viability of MM and MCL cells in vitro. Time course studies demonstrated accumulation of cell cycle arrest in cells after prolonged exposure to len or pom, as well as progressive induction of apoptosis in these cells. Notably, though, the addition of ricolinostat or ACY-241 to either len or pom resulted in synergistic increases in the percentage of MM and MCL cells undergoing apoptosis. At the molecular level, treatment with IMiD® immunomodulatory compounds reduced expression of the critical transcription factors MYC and IRF4, which were reduced even further by treatment with ricolinostat or ACY-241 in combination with either len or pom. The molecular mechanism underlying this effect is currently being explored, though retention of low level inhibition of HDAC1, 2, and 3 by these compounds may contribute to the enhanced effects on gene expression reported here in combination with len or pom. Finally, prolonged treatment with ACY-241 plus pomalidomide was well tolerated in vivo with no overt evidence of toxicity, and combination treatment resulted in a significant extension of survival in a xenograft model of MM. Given the comparable tolerability profiles of ricolinostat and ACY-241, and their preclinical activity in combination with IMiD® immunomodulatory drugs, these findings support clinical evaluation of these agents in MM and MCL patients. Citation Format: Steven N. Quayle, Ingrid Almeciga-Pinto, David Tamang, Min Yang, Simon S. Jones. Selective HDAC inhibition by ricolinostat (ACY-1215) or ACY-241 synergizes with IMiD® immunomodulatory drugs in Multiple Myeloma (MM) and Mantle Cell Lymphoma (MCL) cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2015-5380

Background Ricolinostat is the first oral, selective HDAC6 inhibitor in clinical trials and was well tolerated as monotherapy up to 360 mg/day, the maximum dose examined (Raje, ASH 2012). Ricolinostat synergizes in vitro with both lenalidomide (len) and pomalidomide in MM cell lines and down-regulates MYC and IRF4 protein expression (Quayle, ASH 2013). Methods Relapsed and relapsed refractory patients (pts) who have progressed on at least one prior treatment regimen, with creatinine clearance >50 mg/mL/min, adequate bone marrow and hepatic function were enrolled. In Part A, pts were treated with escalating doses of ricolinostat on days 1-5 and 8-12 of a 28 day cycle with len 25 mg on days 1-21 and dexamethasone (dex) 40 mg weekly. In Part B, ricolinostat was also given on days 15-19. Doses up to 240 mg QD and 160 mg BID were explored, and a final cohort treated with 160 mg BID for 21 of 28 days is currently enrolling. Peripheral blood samples were obtained for pharmacokinetic (PK) and pharmacodynamic analysis. Results As of July 1, 2014, 25 pts have been enrolled; 12 were relapsed and 13 were refractory to the most recent therapy. Seven pts had more than three prior therapies. Twenty pts (80%) had received prior len with 11 previously refractory defined as having less than a minimal response (MR) to therapy or progressive disease on either full dose or maintenance len. Pts have completed 0 to 24+ cycles of therapy with 14 pts continuing on therapy. Six pts discontinued therapy due to progressive disease (PD,1), travel difficulties (1), adverse events (AE) of secondary malignancy (1), alternate therapy chosen after completion of 6 cycles (2), or missed doses of len (1). The latter pt was replaced. The most common treatment emergent events (>15%) were fatigue, upper respiratory infection, anemia, neutropenia, diarrhea, nausea, thrombocytopenia, muscle spasms, dizziness and rash as well as asymptomatic laboratory abnormalities. Most were grade 1 and 2 with no clear relationship to ricolinostat dose. Seven grade 3 and 4 events in five patients were attributed to ricolinostat: neutropenia, thrombocytopenia, anemia, fatigue and syncope. Syncope, at 160 mg BID, was the only DLT; the pt continued on study with a dose reduction and there were no other DLTs in the expanded cohort. PK for ricolinostat is similar to that observed in phase 1a monotherapy, suggesting that co-administration of len does not significantly impact ricolinostat exposure. Maximal blood levels of ricolinostat were ≥0.5 µM at ≥80 mg correlating with a >2x increase in acetylated tubulin (a marker of HDAC6 inhibition) with minimal increase in acetylated histones (a marker of class 1 HDAC inhibition). Twenty-four pts at doses up to 160 mg ricolinostat BID Part B are evaluable for response (≥2 cycles). The overall response rate (ORR) was 63%: 15 pts have ≥partial responses (PR), including 1 stringent complete response (sCR), 5 very good partial responses (VGPR), 9 PR. Four pts had minimal response (MR) and 5 had stable disease (SD) as the best response. Responses are durable up to 23+ cycles of therapy. Of the 11 pts refractory to len, best responses included 1 VGPR, 3 PR, 3 MR and 2 SD. Median time on therapy for the len refractory pts was 4 months (range 3-13). Conclusions Ricolinostat at doses of 160 mg BID, which have biological activity as determined by PD marker in blood cells, can be combined with len and dex on a 21 day, schedule 5 or 7 days/week and was well tolerated. Durable and significant responses were observed, including responses in pts previously refractory to len. The last escalation cohort 160 BID 21/28 days is currently enrolling to determine the recommended phase 2 dose. Disclosures Voorhees: Celgene Corporation: Consultancy, Research Funding; GSK: Consultancy, Research Funding; Acetylon Pharmaceuticals: Research Funding; Prolexys Pharmaceuticals: Research Funding; Oncopeptides: Research Funding; Millennium/Novartis: Research Funding. Bensinger:Celgene Corporation: Consultancy, Research Funding. Supko:Acetylon Pharmaceuticals: Research Funding. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals Inc.: Employment. Patrick:Acetylon Pharmaceuticals: Employment. Wheeler:Acetylon Pharmaceuticals: Employment. Raje:novartis, Amgen, Celgene, Millenium, Onyx: Consultancy; Eli Lilly, Acetylon: Research Funding.

Background Ricolinostat is the first selective oral HDAC6 inhibitor studied clinically in the treatment of MM. Preclinical and phase 1a clinical data support the hypothesis that the safety profile of a selective HDAC inhibitor will facilitate combination therapy with other active agents. No dose limiting toxicities (DLT) were noted in 15 patients (pts) in a phase 1a dose escalation study of ricolinostat at doses up to 360 mg/day where the best response was stable disease (SD). (Raje Blood 2012;120:4061). HDAC6 inhibition impairs the aggresome/autophagy pathway, an alternate pathway to proteasome clearance of misfolded proteins (Santo L Blood 2012;119(11):2578-89) providing the rationale for combining ricolinostat with bortezomib (Btz). Methods We report the first part of a phase 1b open label dose escalation study of ricolinostat in combination with Btz and dexamethasone (Dex) using a standard 3+3 design. Eligible pts had relapsed or relapsed and refractory MM, previously received at least two lines of therapy including a proteasome inhibitor and an IMiD® immunomodulatory agent, had either progressed after or were ineligible for autologous stem cell transplant, and had adequate bone marrow reserve, hepatic function and creatinine clearance of >30 mg/mL/min. Ricolinostat was given orally days 1-5 and 8-12 of a 21 day cycle, with Btz on days 1,4,8,11 and Dex on days 1,2,4,5,8,9,11,12. Peripheral blood samples were obtained for pharmacokinetic (PK) and pharmacodynamic assessments of acetylated tubulin (HDAC6 inhibition) and acetylated histones (Class 1 HDAC inhibition). Definitions of response, relapsed and refractory were by IMWG criteria (Rajkumar SV Blood:2011;117(18):4691-5) and included response less than minimal response (MR) as refractory. Seventeen pts were treated in an expansion cohort at the potential recommended phase 2 dose and schedule. Results Forty-two pts were enrolled to 8 combination dose cohorts. The first cohort dosed ricolinostat 40 mg QD with Btz 1.0 mg/m2; subsequent cohorts used Btz 1.3 mg/m2 with ricolinostat doses of 40, 80 , 160, and 240 mg QD, and 160 mg BID. Median age was 65, and 79% were refractory to the most recent therapy. 34 pts had previously received >4 prior regimens. The first combination cohort was expanded due to a DLT of asymptomatic increase in amylase. No other DLTs have been observed in the cohort escalations. Treatment emergent adverse events (AEs) were predominantly grade 1-2. The most common AEs were diarrhea, increased creatinine, fatigue, anemia, nausea and thrombocytopenia and only diarrhea increased with ricolinostat dose. Grade 3-4 AEs possibly related to ricolinostat included thrombocytopenia (24%), anemia, diarrhea, and asymptomatic laboratory abnormalities (1%). Fatigue, dehydration, orthostatic hypotension, nausea, vomiting, stomach cramps and pulmonary embolism were seen in ≤1% of pts. Of 25 pts evaluable for response, overall response rate (ORR) was 44%: 2 pts had a very good partial response (VGPR), 9 had a partial response (PR), and 2 had an MR, with 9 pts achieving SD and 3 with progressive disease (PD). Responding pts remained on study for a median of 4 cycles (range 2 to 18). Pts who were not evaluable were those who were too early in treatment (3), or who came off study before cycle 2 (14). Of these 10 had early PD and 3 died due to disease or unrelated AE. 15 pts refractory to Btz prior to study entry were evaluable for response. Of those, response rate (PR or better) was 27% with best outcome VGPR (1), PR (3), SD (8) and PD (3). Pts with SD were on study a median of 3 (2-10) mos. PK and pharmacodynamic data from the first 16 pts were similar to the same dose levels in phase 1a monotherapy suggesting coadministration of Btz does not impact the PK of ricolinostat. Maximal levels were ≥1µM at ≥80 mg correlating with measurable increases >2x in acetylated tubulin with a minimal increase in acetylated histones. Updated PK and pharmacodynamic data will be provided. Conclusions Ricolinostat in combination with Btz and Dex was well tolerated at doses up to 240 mg QD and 160 mg BID in the dose escalation cohorts. Diarrhea was the only dose-related AE. Results of the expansion cohort as well as cytogenetic risk categories will be presented. Responses were observed even in Btz-refractory pts. Disclosures Vogl: Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Raje:Eli Lilly, Acetylon: Research Funding; novartis, Amgen, Celgene, Millenium, Onyx: Consultancy. Jones:Acetylon Pharmaceuticals Inc.: Employment. Supko:Acetylon Pharmaceuticals: Research Funding. Leone:Acetylon Pharmaceuticals: Employment. Wheeler:Acetylon Pharmaceuticals: Employment. Orlowski:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Array BioPharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; JW Pharmaceutical: Research Funding; Spectrum Pharmaceuticals: Research Funding; Millennium: The Takeda Oncology Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Onyx: Consultancy; Sanofi: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jagannath:Celgene Corporation: Consultancy; Millennium: Consultancy; Sanofi: Consultancy.

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Histone deacetylase (HDAC) enzymes represent attractive therapeutic targets in MM, but non-selective HDAC inhibitors have led to dose-limiting toxicities in patients, particularly in combination with other therapeutic agents. ACY-1215 is a first-in-class, orally available HDAC inhibitor that is 11-fold selective for HDAC6, and synergizes in vitro and in vivo with bortezomib in preclinical models of MM without inducing unfavorable toxicities (Blood, 20[210]: 4061). In support of its potential tolerability benefits, ongoing Phase Ib clinical trials with ACY-1215 have thus far demonstrated an excellent safety and tolerability profile (Yee, et al, ASH, 2013). The IMiD class of drugs, including lenalidomide and pomalidomide, exhibit striking anti-myeloma properties in a variety of MM models, and have demonstrated significant clinical activity in MM patients. Prior clinical studies have demonstrated activity of the non-selective HDAC inhibitor vorinostat in combination with lenalidomide and dexamethasone in MM patients (Richter, et al, ASH, 2011). However, many patients experienced considerable toxicities with this regimen that significantly limited clinical utility. In support of our ongoing clinical development program for ACY-1215 in MM, we show here that combining ACY-1215 with either IMiD leads to synergistic decreases in the viability of MM cells in vitro. The relevance of inhibition of HDAC6 to this effect was validated by demonstrating synergistic interactions of either IMiD molecule with ACY-775, which is more than 300-fold selective for HDAC6 over class I HDAC's. Time course studies demonstrated accumulation of cell cycle arrest in cells after prolonged exposure to either IMiD, as well as progressive induction of apoptosis in these cells. Notably, though, the addition of ACY-1215 to either IMiD resulted in synergistic increases in the percentage of MM cells undergoing apoptosis. At the molecular level, MM cells are known to be dependent on expression of the MYC and IRF4 transcription factors. Both ACY-1215 and IMiDs as single agents reduced expression of the critical genes MYC and IRF4, which were reduced even further by combination treatment. The molecular mechanism underlying this effect is currently being explored. Finally, the combination of ACY-1215, lenalidomide, and dexamethasone was well tolerated in vivo with no overt evidence of toxicity, and combination efficacy studies with this combination are now being explored in models of MM. By demonstrating that a selective inhibitor of HDAC6 synergizes with IMiD's while maintaining an improved safety profile, these results provided a rational basis for the clinical development of the orally available combination of ACY-1215 and lenalidomide plus dexamethasone in the ongoing Phase Ib clinical trial ([NCT01583283][1]) for the treatment of MM. Citation Format: Steven N. Quayle, David Tamang, Min Yang, Simon S. Jones. ACY-1215, a selective inhibitor of HDAC6, synergizes with immunomodulatory drugs (IMiDs) to induce apoptosis of multiple myeloma (MM) cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5456. doi:10.1158/1538-7445.AM2014-5456 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01583283&atom=%2Fcanres%2F74%2F19_Supplement%2F5456.atom

Histone Deacetylase (HDAC) inhibitors have demonstrated significant clinical benefit as single agents in cutaneous and peripheral T cell lymphomas, and have received FDA approval for these indications. Numerous clinical studies are also ongoing to investigate the potential benefit of combining HDAC inhibitors with other standard of care and investigational agents in various subtypes of lymphoma. ACY-1215 is a first-in-class, orally available selective inhibitor of HDAC6 (approximately 11-fold selective over class I HDAC’s) that is currently in two Phase Ib clinical trials ([NCT01323751][1] and [NCT01583283][2]) in combination with dexamethasone and either bortezomib or lenalidomide in multiple myeloma (MM) patients. Beyond MM, preclinical studies in NHL cell lines also demonstrated increased benefit from treatment with ACY-1215 in combination with the proteasome inhibitors bortezomib or carfilzomib (Amengual, et al , ASH, 2012; Dasmahapatra, et al , ASH, 2012). In addition to standard of care therapies, a number of novel targeted agents have recently demonstrated potential clinical benefit in subtypes of NHL, including agents targeting Bruton’s tyrosine kinase (eg. ibrutinib) and the phosphatidyl inositol-3’ kinase family (eg. GS-1101, IPI-145, and GDC-0941). We describe here the therapeutic potential of ACY-1215 in a collection of NHL cell lines both as a single agent and in combination with these novel targeted agents. NHL cell lines derived from diffuse large B cell lymphomas (DLBCL; both germinal center and activated B cell type) and mantle cell lymphomas (MCL) exhibit significantly decreased viability in response to treatment with selective inhibitors of HDAC6, including both ACY-1215 and ACY-775 (approximately 300-fold selective for HDAC6 over HDAC1/2, and 1500-fold selective over HDAC3). Furthermore, combination treatment of NHL lines with ACY-1215 and either ibrutinib, GDC-0941, or GS-1101, in a dose-matrix format resulted in synergistic decreases in cell viability. The relevance of HDAC6 inhibition to this synergistic response was confirmed through the observation of similarly decreased viability when treating cells in combination with the highly selective HDAC6 inhibitor ACY-775. Detailed molecular mechanism of action studies are ongoing and will be presented. Additional studies are also underway to assess the tolerability and efficacy of combinations of these targeted agents with ACY-1215 in xenograft models of lymphoma. Overall, these data support the continued investigation of the activity of ACY-1215 in lymphoma both as a single agent and in combination with the next generation of targeted therapies for B cell lymphomas. Disclosures: Quayle: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Tamang: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01323751&atom=%2Fbloodjournal%2F122%2F21%2F3071.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01583283&atom=%2Fbloodjournal%2F122%2F21%2F3071.atom

Introduction The combination of HDAC inhibitors and proteasome inhibitors has demonstrated preclinical benefit in several settings, including multiple myeloma and lymphoma, and is being explored in clinical trials testing various HDAC inhibitors in combination with proteasome inhibitors. ACY-1215 is an investigational, orally available HDAC6-selective inhibitor that has demonstrated preclinical combination benefit with bortezomib in vitro and in vivo (Santos et al, Blood 2012; 119: 2579). These preclinical studies also support the hypothesis that the improved selectivity of ACY-1215 for HDAC6 over class I HDACs (HDAC1,2,3) may provide an improved tolerability profile compared to pan-HDAC inhibitors, while still providing the anti-myeloma effect of other HDACi/proteasome inhibitor combinations. ACY-1215 is currently in a Phase I/II trial in multiple myeloma with bortezomib (VELCADE) and dexamethasone to test this hypothesis (NCT01323751). Ixazomib citrate (MLN9708) is an investigational oral proteasome inhibitor in Phase III clinical trials in multiple myeloma (NCT01850524, NCT01564537). To examine the potential efficacy of the all-oral combination of ixazomib citrate and ACY-1215, we evaluated the combination of these agents in cell lines and xenograft models of multiple myeloma. Results In vitro viability experiments in 2 multiple myeloma cell lines (RPMI-8226 and MM.1S) using a dose matrix format demonstrated a combination benefit of ACY-1215 and ixazomib over a range of concentrations, very similar to the previously reported benefit of ACY-1215 plus bortezomib. Likewise, the combination benefit of the selective HDAC6 inhibitor ACY-1215 with ixazomib was similar to the combination effect observed with the pan-HDAC inhibitor SAHA (vorinostat). Together, these in vitro studies support the hypothesis that the combination of ACY-1215 and ixazomib provides similar levels of benefit as do combinations including other HDACi/proteasome inhibitors. Furthermore, experiments in MM.1S xenograft-bearing mice demonstrated an in vivo combination benefit of ACY-1215 and ixazomib. An all-oral regimen was well tolerated when ACY-1215 was dosed at 100 mg/kg PO twice daily for 5 days per week in combination with ixazomib dosed at 5 mg/kg PO twice weekly, and the combination regimen demonstrated additive antitumor activity (Figure 1). The in vivo combination benefit of ACY-1215 and ixazomib was further demonstrated in MM.1S xenograft-bearing mice using alternate routes of administration (IV dosing of ixazomib and IP dosing of ACY-1215). The combination of ACY-1215 dosed at 30 mg/kg IP once daily for 5 days per week with ixazomib dosed IV at 1.5 mg/kg twice-weekly was also well tolerated and had striking antitumor activity. This combination regimen in fact caused regression of the MM.1S xenograft tumors below the starting volumes, and this level of activity was maintained throughout the entire 17 day dosing period (Figure 2). In an accompanying pharmacodynamic (PD) study of the PO and IP doses of ACY-1215, we confirmed selective HDAC6 inhibition in MM.1S xenograft tumors as evidenced by elevated acetylation levels of the HDAC6 substrate tubulin, with little if any change in the levels of acetylated histone H3, a class I HDAC substrate. In vivo experiments in a second xenograft model, RPMI-8226, also demonstrated a combination benefit of ACY-1215 (30 mg/kg IP for 5 days per week) with ixazomib (0.75 mg/kg IV twice-weekly). Conclusion The combination benefit of ACY-1215 and ixazomib observed here in preclinical experiments utilizing in vitro and in vivo models of multiple myeloma provides rationale for clinical evaluation of this first all-oral combination of a proteasome inhibitor with an HDAC inhibitor. Disclosures: Berger: Takeda Pharmaceutical Company Ltd: Employment. Bannerman:Takeda Pharmaceutical Company Ltd: Employment. Quayle:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yu:Takeda Pharmaceutical Company Ltd: Employment. Garcia:Takeda Pharmaceutical Company Ltd: Employment. Ciavarri:Takeda Pharmaceutical Company Ltd: Employment. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

ACY-1215 is a novel and selective inhibitor of HDAC6 currently in Phase 1b clinical trials in combination with bortezomib (Velcade) or lenalidomide (Revlimid) in relapsed/refractory MM. HDAC6 has been to be involved in misfolded protein clearance, stress response and cytoskeleton functions. This activity is in part due to the regulation of the acetylation status of α-tublin, a critical component of microtubules and a specific substrate for the deacetylase activity of HDAC6. ACY-1215 in combination with inhibitors of the major protein clearance pathway, the proteasome, has shown synergistic effects leading to enhanced MM cell death. However, except for acetylated α-tubulin (lysine 40), there are few other HDAC6 specific biomarkers to illustrate additional cellular functions of targeting HDAC6 in the observed combination activity. In the present work, we undertook the identification of specific acetylated lysine (AcK) containing peptides caused by HDAC6 inhibition in MM cells using a quantitative mass spectrometry method, Stable Isotope Labeling by Amino acids in Cell culture (SILAC). The MM cell line MM.1S was used to identify HDAC6 specific AcKs when exposed to the HDAC6 inhibitors ACY-1215, ACY-775 or Tubastatin A at 2 µM, a biologically active concentration on MM cells. ACY-775 and Tubastatin A are both HDAC6 inhibitors with much greater selectivity of HDAC6 over class I HDACs (500-1000x) than ACY-1215 (11x) and were used as reference compounds to identify HDAC6 specific AcK biomarkers. A total of 2,558 unique AcKs were detected for all three treatments. Specifically, for each treatment group, ACY-1215, ACY-775 and Tubastatin A, 1,367, 1,859 and 1,123 AcKs were detected, respectively. Of these 427, 186 and 322 AcKs were significantly changed from DMSO treated cells (>1.3 fold) for ACY-1215, ACY-775 and Tubastatin A, respectively. In addition the ACY-775 treated MM cell lysate was further fractionated into three subcellular fractions, cytoplasm, soluble nuclear and insoluble nuclear, for AcK quantification. In the total unique AcKs detected 1,868, 1,093, 1,338 and 716 were identified from cytoplasm, soluble nuclear and insoluble nuclear, respectively, and less than 10% (173) of these showed either elevated (86) or reduced (87) levels of AcKs. Surprisingly, since HDAC6 has been recognized as a predominately a cytoplasmic deacetylase, most changes were found in the nuclear fraction (soluble and insoluble) than those in cytoplasm. We also identified changes in absolute levels of peptides in ACY-775 treated MM cells. Of the 4,007 quantified peptides, 109 peptides were increased and 78 were decreased following treatment compared to controls. These peptides identified several proteins involved in acetylation, methylation, ubiquitination, DNA repair, stress response, apoptosis and transcription. Orthogonal confirmation of some of the acetylated and un-acetylated peptides identified is underway using alternative (acetyl-) peptide approaches for example immunoblot studies. In conclusion the AcK peptides identified in MM cells by the SILAC approach utilizing selective inhibitors of HDAC6, confirmed the critical function of HDAC6 in protein folding, ubiquitination, degradation, cytoskeleton structure and apoptosis, and also suggested other new functional targets for HDAC6 inhibition. These AcK peptide biomarkers will expand our knowledge on the role of HDAC6 inhibition particularly in combination with other MM therapeutic agents and assist in the development of predictive biomarkers of ACY-1215 activity in MM patients. Disclosures: Yang: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

Introduction HDACs are a family of enzymes that remove acetyl groups from proteins and are involved in many key cellular processes, including apoptosis, cell cycle arrest and angiogenesis. Nonselective HDAC inhibitors has shown promise in treating MM in combination with standard therapies such as proteasome inhibitors (PIs) [1]. However, nonselective HDAC inhibitors have been of limited utility due to adverse effects. HDAC6 is a Class IIb enzyme found in the cytoplasm that is intimately involved in cytoskeletal remodeling and vesicle transport associated with degradation of misfolded protein through the regulation of the acetylation status of α-tubulin. Recent work [2, 3] suggests that selectively inhibiting HDAC6 in MM is an important therapeutic modality as part of a combination therapy with PIs and immunomodulatory agents (IMiDs) and may minimize toxicity due to Class I HDAC inhibition. ACY-1215 is a novel HDAC6-selective small molecule inhibitor that has been shown to be effective in reducing tumor burden in preclinical animal models of MM, and the anti-tumor activities correlate with increased levels of acetyl-tubulin, a HDAC6 specific substrate, in blood and tumor cells. ACY-1215 has now advanced to phase I clinical trials as a combination agent for the treatment of relapsed and refractory MM. A clinical PD biomarker for HDAC6 inhibition is critical to demonstrate that therapeutic plasma levels of ACY-1215 also increase the HDAC6 specific biomarker, acetylated tubulin, versus the Class I HDAC biomarker, acetylated histones. In addition HDAC6 inhibition is expected to be well-tolerated and dose limiting toxicity may not be reached implying continued dose escalation in phase 1 may not be warranted if there is a suitable increase in the PD HDAC6 biomarker. We describe the development of a clinical PD biomarker of HDAC6 inhibition, acetylated α-tubulin, to determine when blood levels of ACY-1215 are reached that give a specific increase in the biomarker versus acetylated histones. Methods Assay sensitivity, dynamic range and reproducibility have been established using peripheral whole blood from healthy donors and MM patients incubated with ACY-1215 ex vivo. In the clinic, peripheral blood is collected at up to six different PK matched time points after drug administration.Lymphocytes were assessed for tubulin and histone hyper-acetylation by flow cytometry using specific antibodies that recognize acetyl groups in the context of the specific protein targets. ACY-1215 plasma concentration was also determined in the peripheral blood by an appropriate bioanalytical method. More than 50 MM patients have been enrolled in two separate clinical trials and received escalating oral ACY-1215 doses from 40 mg to 240 mg. The ACY-100 study is composed of two parts, ACY-1215 monotherapy and combination therapy with both bortezomib and dexamethasone. A second study, ACE-MM-101, examines ACY-1215 in combination with lenalidomide and dexamethasone. Results & Conclusion Patients in ACY-100 and ACE-MM-101 clinical trials have shown elevated tubulin hyper-acetylation in peripheral blood lymphocytes as ACY-1215 dose increases from 40 mg to 240 mg. ACY-1215 plasma concentration reaches a maximal level (Cmax) 1 hr after administration. An apparent exposure plateau was reached at dose levels >160 mg where Cmax reached 1.2 - 1.8 µM. Correlating to the PK levels, the PD biomarker acetyl-tubulin increases to a maximal level 1 hr after dosing compared to predose, and declines to basal level by >4 hr with an apparent lag period compared to plasma levels of ACY-1215. Acetyl-histone levels were not significantly changed at these doses indicating a predominant HDAC6 selective effect. As plasma levels increased with dose from 40 mg to 160 mg so do the number of patients that have a measurable increase in acetyl-tublin such that at dose levels ≥80 mg all patients have a measurable increase in acetylated tubulin. In conclusion the clinical PK and PD results, comparing to prior preclinical studies [4], suggest that ACY-1215 has reached a pharmacologically relevant level of HDAC6 inhibition at clinical doses ≥80 mg. These results will aid in the determination of the recommend phase 2 dose of ACY-1215 in combination with PIs and IMiDs. Disclosures: Tamang: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Supko:Acetylon: Research Funding.

ACY-1215 is a novel and selective oral inhibitor of HDAC6 currently in Phase I clinical trials in combination with bortezomib or lenalidomide in relapsed/refractory multiple myeloma. Inhibitors of HDAC6 have demonstrated anti-cancer proliferative activity in various cancer types, including Her2+ breast cancer. Blocking HDAC6 activity has been shown to cause Her2+ breast cancer cell growth inhibition through various mechanisms, including destabilization of Her2 mRNA and protein. In the present work, the impact of HDAC6 inhibition has been investigated on Her2+ breast cancer cell growth and cell migration using HDAC6 inhibitor alone or in combination with either a Her2 inhibitor (lapatinib) or a PI3K inhibitor (GDC-0941, IPI-145 and CAL-101). Anti-tumor activity of ACY-1215 was investigated using a panel of nearly 100 human tumor cell lines derived from the major cancer types and Her2+ breast cancer cells were among the most sensitive cells to ACY-1215. Combinations of ACY-1215 with either the Her2 inhibitor lapatinib or PI3K inhibitor GDC-0941 demonstrated synergistic cytotoxicity against these cancer cells. To illustrate the effects are due to HDAC6 inhibition, we used a highly HDAC6 selective inhibitor ACY-775, which has greater than 1,500 fold of selectivity against HDAC6 over Class I HDACs (HDAC1, 2 and 3), in our experiments. ACY-775 is able to sensitize Her2+ breast cancer cells MDA-MB-453 and BT-474 to lapatinib, and PI3K inhibitors (IPI-145, GDC-0941 and CAL-101). ACY-775 also blocks cancer cell migration in the presence of absence of epidermal growth factor (EGF). In agreement with the proposed mechanism of HDAC6 mediated Her2+ breast cancer cell death, ACY-1215 treatment correlated with decrease of Her2 protein, p-EGFR and p-AKT in a Her2+ breast cancer cell line MDA-MB-453. By taking advantage of our highly selective HDAC6 inhibitor ACY-775, we have shown growth and migration suppressive activities by HDAC6 inhibition in Her2+ breast cancer cells. These findings suggest selection of metastatic Her2+ breast cancer for further in vitro and in vivo validation and mechanism of action studies with ACY-1215 in combination with novel and approved standard of care therapeutics for breast cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B280. Citation Format: Min Yang, David Tamang, Simon Jones. Histone deacetylase (HDAC) 6 inhibition potentiates the cytotoxicity of lapatinib and PI3K inhibitors on Her2+ breast cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B280.

Abstract 2908 Bone disease in multiple myeloma (MM) is due to the disruption of the delicate balance between osteoblast (OB)-mediated bone formation and osteoclast (OC)-mediated bone resorption. Agents that target both tumor cells and restore normal bone homeostasis can improve long-term disease control and prolong MM patient survival. It has been demonstrated that in vitro pan HDAC inhibitors accelerate OB maturation and suppress OC maturation, while bortezomib triggers OB activation and inhibits osteoclastogenesis. However it has recently been shown that vorinostat (SAHA), a non-selective HDAC inhibitor, causes bone loss in vivo by inhibiting immature OB. Here, we evaluated effects of a selective HDAC6 inhibitor ACY-1215 (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib, on MM cell growth and related bone disease. ACY-1215 in combination with bortezomib has synergistic cytotoxicity due to simultaneous inhibition of the proteasome and aggresome pathways. We confirm the in vivo anti-MM activity of ACY-1215 in combination with bortezomib in two different xenograft mouse models: human MM injected subcutaneously; and luciferase-expressing human MM injected intravenously (disseminated MM model). Tumor growth was significantly delayed and overall host survival significantly prolonged in animals treated with combined therapy (34 vs 22 days, n=7, p Since there is decreased OB function and new bone formation in MM, we next assessed the effect of ACY-1215 on OB differentiation. ACY-1215, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase enzyme activity and alizarin red staining. In addition, we show increased mRNA expression of b-catenin, osteocalcin, Runx2 and Sp7 (OB differentiation markers) in immature OB triggered by ACY-1215. Finally, ACY-1215 was not toxic to PHA stimulated PBMCs, suggesting a favorable side effect profile and therapeutic index. Our studies therefore demonstrate that ACY-1215, alone and in combination with bortezomib, can inhibit osteoclastogenesis enhance osteoblastogenesis, and inhibit MM cell growth. Based upon these studies, ongoing clinical trials are examining the efficacy of ACY-1215 in relapsed MM and associated bone disease. Disclosures: Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Bradner:Acetylon Pharmaceuticals, Inc.: Consultancy. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals, Inc.: founder; Merck: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

Abstract 2912 HDAC enzymes are being studied as novel therapeutic targets in several cancers including multiple myeloma (MM). In particular, the combination of proteasome inhibitors (e.g. bortezomib (BZ)) with Histone Deacetylase (HDAC) inhibitors have shown very promising results in pre-clinical MM models. HDAC6, a Class II HDAC, has been linked to the activity of aggresomes that degrade unfolded and misfolded ubiquitinated proteins. Targeting both proteasomal and aggresomal protein degradation systems with proteasome inhibitors and HDAC inhibitors, respectively, induces accumulation of polyubiquitinated proteins, followed by activation of apoptotic cascades. Here we investigated the preclinical activity of an HDAC6 selective inhibitor ACY-1215 in MM, either alone or in combination with BZ. In vitro enzyme assays showed that ACY-1215 has potent inhibitory activity against HDAC6 (IC50 0.0054 mM) compared to the other HDACs, including Class I HDACs. Maximal cytotoxicity of ACY-1215 against MM cell lines was observed at 48h, with IC50 values ranging from 2–8 μM. To investigate the specific inhibitory effect of ACY-1215 on HDAC6 activity, we evaluated its effect on acetylation of a-tubulin. ACY-1215 induces potent acetylation of a-tubulin at low doses and triggers acetylation of lysine on histone H3 and histone H4 only at much higher doses, confirming its selective inhibitory effect on HDAC6 activity. Importantly, this selective inhibition was also observed in patient MM cells, where ACY-1215 increased acetylated a-tubulin after 4 h of treatment. We next combined low doses of ACY-1215 with BZ and showed synergistic anti MM activity, resulting in apoptosis via caspase-3, -8, -9 and poly (ADP) ribosome polymerase activation. Moreover, the combination of ACY-1215 plus BZ increased the accumulation of polyubiquitinated proteins compared to either agent alone. To investigate the effect of ACY-1215 on aggresome formation, MM.1S cells treated with ACY-1215 1 μM and/or BZ 2.5 nM for 12 h were stained with immunofluorescent anti-ubiquitin antibody. BZ-treated cells showed perinuclear structures consistent with aggresome formation, which was disrupted when BZ and ACY-1215 were combined. This result supports the synergistic anti MM activity of ACY-1215 with BZ. We also evaluated the in vivo anti-MM effect of combination therapy using two different xenograft models in SCID mice: plasmacytoma model and disseminated MM model. ACY-1215 in combination with BZ triggered more significant anti-MM activity than either agent alone in suppressing tumor growth and prolonging host survival in both models, without significant adverse effects. To optimize the design of future clinical trials, we conducted pharmacokinetic and pharmacodynamic studies in our plasmacytoma model. ACY-1215 peak plasma levels were observed at 4 h, which were unaffected by the addition of BZ. To further characterize the activity of ACY-1215 against HDAC6 in vivo, we evaluated the acetylation of α-tubulin in mouse blood cells by flow cytometry. The maximum levels of blood cell α-tubulin acetylation were observed at 4 h, providing an important biomarker for future clinical trials. Importantly, levels of acetylated α-tubulin were also detected in tumor tissue from treated mice in a similar time frame to peak blood levels, suggesting that ACY-1215 is readily absorbed by tumor tissue. Moreover, ACY-1215 did not accumulate in tumor tissue, as shown by the decline of acetylated α-tubulin in blood cells and tumor tissue by 24 h post-dose, which parallels the elimination of ACY-1215 from blood. We further confirmed the HDAC6 selectivity of ACY-1215 in our in vivo models by investigating the effect of the drug combination on histone acetylation in tumor tissue. WB analysis and IHC did not show a significant increase in acetylated histone H3 (lys 18), while demonstrating a robust acetylation of α-tubulin, the primary marker of HDAC6 inhibition by ACY-1215 at the cellular level. The results from our in vitro and in vivo studies therefore show significant and synergistic anti-MM activity of ACY-1215 in combination with BZ and provided the rationale for the ongoing phase I/II clinical trial in patients with relapsed or relapsed/refractory MM. Moreover, our pharmacodynamic helped inform the design of correlative studies, which will establish whether acetylated α-tubulin can be used as predictive biomarker of HDAC6 inhibition and disease response. Disclosures: Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Ogier:Acetylon Pharmaceuticals, Inc.: Employment. Bradner:Acetylon: Consultancy. Anderson:celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Acetylon Pharmaceuticals Inc: founder. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

e18569 Background: HDACs are being studied as novel therapeutic targets in MM. Here, we investigated the preclinical activity of an HDAC6 selective inhibitor ACY1215 in MM, alone and in combination...

IL-4 down-regulates perforin and granzymes and depresses rapid (4 hr) cytotoxic T lymphocyte (CTL) killing. Curiously, IL-4 can increase anti-tumor immunity in animal models. We wanted to know how IL-4 polarized CTLs kill and found that they have potent delayed cytotoxicity. We cultured BALB/c mouse splenocytes with conA and 500 units/ml mouse IL-4 or IL-2 for 3 days and then re-cultured the cells for 3 days with cytokines without conA. The IL-4 CTLs were polarized as indicated by expression of the IL-4 inducible lipase PLRP2. By flow cytometry, IL-4 CTLs expressed Grz B at lower levels than IL-2 CTLs. We assayed cytotoxicity using anti-CD3-redirected death of P815 tumor cells. Death was measured at 48 hrs by loss of EGFP+ P815 cells using flow cytometry with cell count beads to account for dead cell disintegration. Cytotoxicity by the IL-4 CTLs was 80% at effector lymphocyte to P815 (E:T) ratios of 1:10. IL-4 CTLs had perforin-independent mechanisms, with similar activity for WT and Prf1-/- CTLs. We monitored T cells for death receptor ligands. Quantitative RT-PCR indicated that IL-4 cells maintained comparable levels of mRNA for LTβ, LIGHT, TRAIL and FasL, but had lower levels of TNFα and TNFβ, than IL-2 CTLs. We conclude that IL-4 polarized CTLs have potent cytotoxicity which could be mediated by death receptor ligands and/or by novel IL-4 inducible potential cytotoxins. Supported in part by NIH grants R01CA38942, T32CA09563, P20 RR-016464 & P20-RR015581.