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Although the treatment paradigm for chronic lymphocytic leukemia (CLL) is rapidly changing, the disease remains incurable, except with allogeneic bone marrow transplantation, and resistance, relapsed disease, and partial responses persist as significant challenges. Recent studies have uncovered roles for epigenetic modification in the regulation of mechanisms contributing to malignant progression of CLL B cells. However, the extent to which epigenetic modifiers can be targeted for therapeutic benefit in CLL patients remains poorly explored. We report for the first time that expression of epigenetic modifier histone deacetylase 6 (HDAC6) is upregulated in CLL patient samples, cell lines, and euTCL1 transgenic mouse models compared with HDAC6 in normal controls. Genetic silencing of HDAC6 conferred survival benefit in euTCL1 mice. Administration of isoform-specific HDAC6 inhibitor ACY738 in the euTCL1 aging and adoptive transfer models deterred proliferation of CLL B cells, delayed disease onset via disruption of B-cell receptor signaling, and sensitized CLL B cells to apoptosis. Furthermore, coadministration of ACY738 and ibrutinib displayed synergistic cell kill against CLL cell lines and improved overall survival compared with either single agent in vivo. These results demonstrate for the first time the therapeutic efficacy of selective HDAC6 inhibition in preclinical CLL models and suggest a rationale for the clinical development of HDAC6 inhibitors for CLL treatment, either alone or in combination with Bruton tyrosine kinase inhibition.

5-Lipoxygenase (5-LOX) plays key roles in infection and allergic responses. Herein, four 5-LOX–derived lipids comprising 5-hydroxyeicosatetraenoic acid (HETE) attached to phospholipids (PLs), either phosphatidylethanolamine (PE) or phosphatidylcholine (18:0p/5-HETE-PE, 18:1p/5-HETE-PE, 16:0p/5-HETE-PE, and 16:0a/5-HETE-PC), were identified in primary human neutrophils. They formed within 2 minutes in response to serum-opsonized Staphylococcus epidermidis or f-methionine-leucine-phenylalanine, with priming by lipopolysaccharide, granulocyte macrophage colony-stimulating factor, or cytochalasin D. Levels generated were similar to free 5-HETE (0.37 ± 0.14 ng vs 0.55 ± 0.18 ng/106 cells, esterified vs free 5-HETE, respectively). They remained cell associated, localizing to nuclear and extranuclear membrane, and were formed by fast esterification of newly synthesized free 5-HETE. Generation also required Ca2+, phospholipase C, cytosolic and secretory phospholipase A2, 5-LOX activating protein, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. 5-HETE-PLs were detected in murine S epidermidis peritonitis, paralleling neutrophil influx, and in effluent from Gram-positive human bacterial peritonitis. Formation of neutrophil extracellular traps was significantly enhanced by 5-LOX inhibition but attenuated by HETE-PE, whereas 5-HETE-PE enhanced superoxide and interleukin-8 generation. Thus, new molecular species of oxidized PL formed by human neutrophils during bacterial infection are identified and characterized.

Interleukin 6 (IL6) trans-signaling has emerged as a prominent regulator of immune responses during both innate and acquired immunity. Regulation of IL6 trans-signaling is reliant upon the release of soluble IL6 receptor (sIL6R), which binds IL6 to create an agonistic IL6/sIL6R complex capable of activating cell types that would not normally respond to IL6 itself. Here we show that intrinsic and extrinsic apoptotic stimulation by DNA damage, cytokine deprivation, and Fas stimulation promotes shedding of sIL6R. Apoptosis-induced shedding of the IL6R was caspase dependent but PKC independent, with inhibition of ADAM17 preventing IL6R shedding. Such insight is relevant to the control of acute inflammation, where transition from the initial neutrophil infiltration to a more sustained population of mononuclear cells is essential for the resolution of the inflammatory process. This transitional event is governed by IL6 trans-signaling. This study demonstrates that IL6R is shed from apoptotic human neutrophils. In vivo studies in a murine inflammation model showed that neutrophil depletion resulted in reduced local sIL6R levels and a concomitant decrease in mononuclear cells, suggesting that apoptosis-induced IL6R shedding from neutrophils promotes IL6 trans-signaling and regulates the attraction of monocytic cells involved in the clearance of apoptotic neutrophils.

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.

HBG protein, when paired with α-globin, forms fetal hemoglobin (HBF). HBF can substitute for defective adult hemoglobin found in sickle cell disease and β-thalassemia patients to ameliorate disease. We have previously shown that ACY-957, a selective inhibitor of HDAC1/2, induces HBG mRNA and HBF protein in vitro. We have also shown that 5 days of oral dosing with ACY-957 induces HBG mRNA in the blood of non-anemic cynomolgus monkeys. In this work, we used this model to investigate the effects of ACY-957 over an extended dosing period and with several dosing schedules. We have also developed new assays for PD response and HBG protein induction, a predictor of efficacy. We selected a dose of 75 mg/kg based on previous studies. 15 naïve, male monkeys, ~3.5 years in age and ~3.5 kg in weight were randomized into four groups and dosed QD x 5 per week (starting on day 0) for 3 weeks followed by a 10 day washout period (Fig 1A): 'Control' = vehicle days 0, 1, 2, 3, 4; '3 on 4 off' = ACY-957 days 0, 1, 2 and vehicle days 3, 4; '1 on 1 off' = ACY-957 days 0, 2, 4 and vehicle days 1, 3; '5 on 2 off' = ACY-957 days 0, 1, 2, 3, 4. Blood samples were collected pre-dose on days 0 and 3 every week. There were no notable clinical observations during the study. A minor, yet significant, body weight reduction was observed in all groups (Fig 1B). Body weight loss was less in the 3 on 4 off group, and unlike the 1 on 1 off and 5 on 2 off groups, these animals recovered to control levels following the washout period. For all groups, plasma levels of ACY-957 were 2 μM at 24 hr post-dose (Fig 1C). Prior to the next week of dosing, the 1 on 1 off and 5 on 2 off groups had plasma levels of 250 nM, while the 3 on 4 off group had background levels. These findings confirm the target exposure was reached and suggest that ACY-957 does not accumulate with extended dosing. Acetylation of histone H3 lysine 56 (H3K56ac) in nucleated peripheral blood cells was used as a marker of PD response (Fig 1D-E). H3K56ac steadily increased throughout the study in all treatment groups. H3K56ac trended towards baseline levels during the washout period, yet elevated levels persisted in 3 on 4 off and 5 on 2 off groups for up to 144 hours after the last dose. Furthermore, 3 on 4 off dosing yielded a PD response equivalent to 5 on 2 off and superior to 1 on 1 off dosing. These data reveal that the PD response is decoupled from the dosing schedules examined, a finding consistent with the slow off rate of aminobenzamide HDAC inhibitors. Complete blood counts (CBC) were affected in all groups (Fig 2). Neutrophils were suppressed by 70%, with nadir reached after 2 weeks of dosing and a rapid return to control levels during the washout period. Lymphocytes were cyclically suppressed by 50% at day 3 of each week, returning to baseline levels prior to the next week of dosing. Monocytes were elevated 4-fold by the third week of dosing and returned to control levels during the washout period. Platelets dropped by 20% in the 5 on 2 off group only, returning to control levels during the washout period. Red blood cells (RBC), hemoglobin, and hematocrit levels decreased by 15% in the 5 on 2 off group, an effect minimized in the 3 on 4 off and 1 on 1 off groups. HBG mRNA induction was observed in all groups and correlated with PD response over time (Fig 3A). HBG mRNA increased a maximum of 50-fold in the 1 on 1 off group and 200-fold in the 3 on 4 off and 5 on 2 off groups. As a percentage of all β-like globin mRNA, HBG increased from ~0.01% at baseline to ~1% at peak response. HBG protein was also elevated, reaching levels of 20-, 35-, and 90-fold above baseline in the 1 on 1 off, 3 on 4 off, and 5 on 2 off groups, respectively (Fig 3B-C). HBG mRNA trended to control group levels during the washout period, while HBG protein remained elevated, a finding consistent with the transient lifespan of RNA containing reticulocytes and the long lifespan of the protein containing RBC. In conclusion, we have extended our previous results by showing that 3 week dosing of ACY-957 is well-tolerated and leads to an accumulation of HBG protein in monkey. Furthermore, we have established the relationship between PK, PD, hematopoietic toxicity, and potential efficacy for ACY-957. 3 on 4 off dosing elicited a PD response and HBG induction profile comparable to 5 on 2 off dosing, while minimizing body weight loss and hematopoietic toxicity. Our findings suggest that dose level and schedule may be further refined to optimize the therapeutic window of HDAC1/2-selective inhibitors for hemoglobinopathies. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3. Figure 3. Disclosures Chonkar: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Bhol:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Shearstone:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

We have generated a truncated form of the erythropoietin receptor (EPO- R), the extracytoplasmic ligand-binding domain, that is secreted from a transfected Chinese hamster ovary (CHO) cell line. The truncated receptor is readily purified from CHO conditioned media as a 33-Kd glycosylated protein, which is converted to a 25-Kd species upon treatment with protein N-glycan glycosidase F. Cross-linking of radioiodinated EPO to the secreted receptor yielded a complex of 72 Kd. Also, the growth of the EPO-dependent cell line, FDCPE, was inhibited in a dose-responsive manner by the truncated receptor. The complex of the secreted receptor and EPO was isolated by gel filtration and shown to be a one-to-one complex of the receptor and growth factor by quantitative amino terminal sequencing. Finally, analysis of the interaction of the receptor and growth factor by gel filtration indicated an apparent dissociation constant of 1.1 nmol/L for the truncated receptor.

We have generated six high affinity monoclonal antibodies (MoAbs) to the human erythropoietin receptor (hEPO-R) polypeptide. All six MoAbs bind to the extracytoplasmic domain of the hEPO-R, and all immunoprecipitate 35S-labeled hEPO-R from metabolically labeled Ba/F3- hEPO-R cells. Four of the MoAbs neutralize the EPO-dependent growth of Ba/F3-hEPO-R cells, whereas two MoAbs are non-neutralizing. None of the MoAbs inhibit the EPO-dependent growth of Ba/F3 cells expressing the murine EPO-R (mEPO-R), even though the hEPO-R and mEPO-R share 82% amino acid identity. All six of the anti-EPO-R MoAbs bind to the cell surface human EPO-R but none bind to the cell surface murine EPO-R. Of the four neutralizing MoAbs, the one-half maximal inhibition occurs at MoAb concentrations ranging from 1 nmol/L to 50 nmol/L. These MoAbs also compete with radiolabeled EPO for hEPO-R binding. The two non- neutralizing MoAbs fail to inhibit EPO-dependent growth or compete with EPO-binding, even at antibody concentrations as high as 500 nmol/L. The four neutralizing MoAbs, designated group I, compete with each other for an epitope of the hEPO-R polypeptide required for EPO-binding. The two non-neutralizing MoAbs recognize discrete epitopes, and are designated group II and group III MoAbs. In conclusion, this is the first description of MoAbs specific for the hEPO-R. The MoAbs, which recognize three discrete epitopes, may be useful in characterizing the spectrum of cells that display the hEPO-R and in further defining the role of EPO in hematopoiesis.

We have isolated the human homologue of the murine erythropoietin receptor (mEPO-R) from an erythroleukemia line, OCIM1, and from fetal liver. Both the cDNA and protein sequence of the human receptor were 82% homologous to the mEPO-R. Heterologous expression of the human cDNA in COS cells yielded a protein of about 66 Kd. The protein could be specifically immunoprecipitated with either an antibody raised against the amino terminus of mEPO-R or by a monoclonal antibody that bound EPO bound to its receptor. Cross-linking of radioiodinated EPO to COS cells expressing the human EPO-R gave apparent molecular weights of 66 and 100 Kd for the receptor. The murine interleukin-3-dependent pre-B- lymphocyte cell line, Ba/F3, was made EPO-dependent by transfection of the human cDNA into the cells and selecting for growth in EPO- containing media.

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.

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 in models of MM and lymphoma with bortezomib (Santo, Blood, 2012; Amengual, Clin Cancer Res, 2015) or carfilzomib (Mishima, Br J Haematol, 2015; Dasmahapatra, Mol Cancer Ther, 2014). Furthermore, ricolinostat has demonstrated an excellent safety and tolerability profile in phase I trials as an oral liquid formulation (Raje, Haematologica, 2014, Suppl 1). We have now identified ACY-241 as a structurally related and orally available selective inhibitor of HDAC6 that is undergoing clinical evaluation in tablet form. In combination with ricolinostat, the immunomodulatory (IMiD®) class of drugs, including lenalidomide (Len) and pomalidomide (Pom), exhibit striking anti-myeloma properties in a variety of MM models (Quayle, AACR, 2014) and have demonstrated clinical activity in MM patients (Yee, ASH, 2014). In support of our ongoing development of ACY-241, we show here that combination with either Len or Pom leads to synergistic decrease in MM cell viability in vitro. Time course studies demonstrated cell cycle arrest followed by progressive induction of apoptosis after prolonged exposure to Len or Pom. Notably, the addition of ACY-241 to either Len or Pom resulted in synergistic increases in apoptosis of MM cells. At the molecular level, treatment with IMiDs reduced expression of the critical transcription factors MYC and IRF4, which was further reduced by combination treatment with ACY-241. Current studies are exploring the molecular mechanism underlying this effect, which may be a consequence of low level inhibition of HDAC1, 2, and 3 by ACY-241. Prolonged treatment with ACY-241 plus Pom was well tolerated in vivo with no evidence of toxicity, and the combination resulted in a significant extension of survival in a xenograft model of MM. Given the comparable tolerability profiles of ricolinostat and ACY-241 and the similar preclinical activity in combination with IMiDs, a clinical trial (NCT02400242) is currently evaluating ACY-241 in combination with Pom and low-dose dexamethasone in MM patients. Predicated upon the clinical experience with ricolinostat and the non-clinical pharmacokinetics of ACY-241, we designed an expedited first-in-human phase 1a/1b clinical trial of a single cycle of ACY-241 monotherapy followed by ACY-241 in combination with Pom and dexamethasone in MM patients. A merged monotherapy/combination trial design was chosen to grant patients access to combination therapy with an established regimen while enabling insight into the safety, pharmacokinetics, and pharmacodynamics of ACY-241 monotherapy. Patients with relapsed or relapsed-and-refractory MM previously treated with at least two cycles of Len and a proteasome inhibitor were eligible for this trial. The first patient was enrolled in June 2015. This patient tolerated monotherapy well and pharmacokinetics showed maximal plasma levels of ACY-241 in the micromolar range, consistent with predictions. An update on enrollment, pharmacokinetic and pharmacodynamic profiles as well as safety of monotherapy and combination therapy will be provided. Disclosures Niesvizky: Celgene: Consultancy, Speakers Bureau. Richardson:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; 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; 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. Gabrail:Onyx: Honoraria, Speakers Bureau; BI: Honoraria, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Honoraria, Speakers Bureau. Madan:Onyx: Speakers Bureau; Celgene: Speakers Bureau. Quayle:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Almeciga-Pinto:Acetylon Pharmaceuticals, Inc: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Houston:Acetylon Pharmaceuticals, Inc: Employment. Hayes:Acetylon Pharmaceuticals, Inc: Employment. Van Duzer:Acetylon Pharmaceuticals, Inc: Employment. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Trede:Acetylon Pharmaceuticals, Inc: Employment. Raje:Acetylon: Research Funding; Celgene Corporation: Consultancy; BMS: Consultancy; Amgen: Consultancy; Millenium: Consultancy; AstraZeneca: Research Funding; Novartis: Consultancy; Onyx: Consultancy; Eli Lilly: Research Funding; Takeda: Consultancy.

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.

Pan-class HDAC inhibitors are active agents for select subtypes of lymphoma but are often associated with toxicities such as fatigue, diarrhea/constipation, cardiac arrhythmias and myelosuppression. HDAC6 plays a critical role in protein homeostasis by managing misfolded proteins and inducing the unfolded protein response (UPR). Ricolinostat is an isoform selective HDAC6 inhibitor. We evaluated the preclinical effects of ricolinostat and translated the findings into a multicenter Phase 1b study in patients with lymphoma (NCT 02091063). Cytotoxicity was evaluated in 16 lymphoma cell lines (8 DBCL, 4 MCL, 4 TCL) by Cell TiterGlo and Annexin V. Ricolinostat has activity with IC50 values ranging from 0.9-4.7 uM range, which is in the range observed in clinical PK studies. Ricolinostat displayed marked synergy with bortezomib, PI3K inhibitors, disatinib, alisertib, and ibrutinib with synergy coeficients of 0.24, 0.29, 0.64, 0.07, 0.16 respectively (values In an effort to translate these findings, a multi-center Phase Ib open-label, single agent study designed to study the safety of 2 dose schedules of ricolinostat in patients with relapsed or refractory lymphoid malignancies was initiated. Patients were treated with 160 mg of oral ricolinostat continuously on a 28 day cycle. Patients were accrued sequentially to 2 dose cohorts: 3 patients were enrolled on single daily dosing (QD) schedule, and following safety assessment, patients were enrolled to twice daily dosing (BID). Samples were collected for PK and PD analysis. As of July 25th 2015, 11 patients were enrolled on the phase Ib study and were evaluated for safety. The median age was 36 yrs (24-86) and 7 (64%) were male. Sixty-four percent had 5 prior treatments (range: 1-15). Histologies included HL (N=3) and NHL (N=8) including DLBCL=3, MZL=2, FL=1, PTCL=1, PTLD=1. Three patients were enrolled in QD dosing and 8 patients in the BID schedule. There were no DLTs at either of the dosing schedules, and the maximum administrable dose was determined on the BID dosing schedule. The median number of cycles completed was 2 and ranged from 1 through 12+ (currently ongoing). The only grade 3 toxicities reported were anemia (27%), hyponatremia (9%), hypercalcemia (9%) and hypotension (9%), none thought to be attributed to the study drug. Other treatment emergent events included nausea (45%), diarrhea (36%), fatigue (36%) and vomiting (27%) which were all grade 1 and 2. Three patients achieved stable disease, all of whom were treated on the BID schedule (3 of 8, 37.5%), no complete or partial responses have been observed to date. One patient with refractory HL with 14 prior lines of therapy achieved stable disease (SD) and had symptomatic improvement corresponding to an increase of hemoglobin after 9 cycles, as well as resolution of B symptoms. Another patient with PTCL had immediate resolution of B symptoms such as fevers and pruritus after starting QD dosing. This is the first reported experience with an isoform selective HDAC inhibitor to be studied for the treatment of patients with relapsed or refractory lymphoid malignancies. Preclinical studies support the notion that ricolinostat mediates its effects through the UPR and potently synergizes with complementary agents. Ricolinostat is well tolerated with no DLTs. Thirty-seven percent of patients achieved SD on the BID schedule. PK and PD data will be evaluated for acetylation of alpha-tubulin and modulation of the UPR in an effort to confirm preclinical findings. Efficacy will be evaluated in a phase II study. We believe that ricolinostat in rational combinations will lead to effective treatment for patients with relapsed lymphoid malignancies. Disclosures Amengual: Acetylon Pharmaceuticals, INC: Consultancy, Research Funding. Off Label Use: Ricolinostat is being studied for the treatment of lymphoma. Neylon:Seattle Genetics: Speakers Bureau; Celgene: Speakers Bureau; Genentech: Speakers Bureau; Gilead: Speakers Bureau. Deng:TG Therapeutics: Research Funding; Gilead: Research Funding; Amgen/Onyx: Research Funding. Sawas:Seattle Genetics: Research Funding; Gilead Sciences: Honoraria. Shah:Pharmacyclics: Speakers Bureau; NCCN: Consultancy; SWOG: Consultancy; Rosetta Genomics: Research Funding; Acetylon Pharmaceuticals, INC: Membership on an entity's Board of Directors or advisory committees; Plexus Communications: Honoraria; Spectrum: Speakers Bureau; Bayer: Honoraria; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; DeBartolo Institute for Personlaized Medicine: Research Funding; Seattle Genetics: Research Funding; Janssen: Speakers Bureau. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. O'Connor:Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy; Bayer: Consultancy, Honoraria; Acetylon Pharmaceuticals, INC: Consultancy; Mundipharma: Consultancy, Honoraria, Research Funding.

Elevated levels of HbG mRNA, leading to the formation of fetal hemoglobin, is known to ameliorate disease severity in sickle cell and β-thalassemia patients. We have previously shown that the small molecule ACY-957 is a selective inhibitor of HDAC1/2 which induces HbE and HbG in cultured human primary CD34+ cells (Shearstone et al, ASH Annual Meetings 2012-14). In this work, we describe the pharmacokinetics and HbE/HbG induction following once daily oral dosing of ACY-957 in rat and monkey. To determine the duration of ACY-957 exposure required to induce HbE and HbG in vivo, we first tested ACY-957 in drug washout experiments performed in cultured primary erythroid progenitors. The aminobenzamide class of HDAC inhibitors, such as ACY-957, are known to have slow on rates for HDAC1/2 (Kral et al, Biochemistry 2014; Lauffer et al, J Biol Chem 2013). We found that a 6 h pulse of ACY-957 (1 μM) resulted in undetectable increases in histone H3 lysine 9 acetylation (H3K9ac) and that 4 or 8 h pulses of ACY-957 (1 μM) resulted in undetectable HbE and HbG induction. However, continued exposure resulted in a 2.5-fold and 7-fold increase in H3K9ac after 24 and 48 h of incubation, respectively, leading to a time-dependent increase in HbE and HbG. Based on this data, we hypothesized that in vivo studies would require ACY-957 levels of 1 μM for 24 h in order to observe elevated HbE and HbG. Non-fasted Sprague Dawley rats or cynomolgus monkeys received a single oral dose of 20 mg/kg or 12.5 mg/kg ACY-957, respectively, and pharmacokinetic analysis yielded comparable results with T1/2 = 11.8 and 10.9 h, Cmax = 7.8 and 2.4 μM, and Tmax = 5.3 and 4.0 h, in rat and monkey, respectively. At 24 h post dose, ACY-957 plasma levels in rat and monkey were 1.6 and 0.6 μM, respectively. These findings suggested that the targeted drug exposure could be met with a single daily oral dose of ACY-957. Since ACY-957 induced HbE in cultured human primary erythroid progenitors, we attempted to measure HbE induction in rat as a surrogate marker for HbG in primate. Rats were dosed with 0, 10 or 30 mg/kg (n=4 per group) by oral gavage, once daily for 6 days, followed by a 13 day washout period. Peripheral blood was sampled every 3 days for isolation of total RNA. Complete blood counts were performed on day 0, 6 and 18. The low and high dose groups showed ACY-957 plasma levels of 1.3 or 5.2 μM, respectively, at 24 h post final dose. No abnormal clinical signs were found during the in-life phase, although a minor, reversible delay in rat weight gain was observed in the high dose group. White blood cells were suppressed by 33% and 68% at day 6 in low and high dose groups, respectively, but recovered to baseline levels by day 18. ACY-957 administration led to a dose-dependent increase in HbE relative to HbB that was detectable at day 3, peaked at day 6, and returned to baseline levels by day 9. Maximum induction of HbE was 2-fold and 5.6-fold for the low and high dosing groups, respectively, relative to animals receiving vehicle only. Next, monkeys were dosed at 0, 25 or 75 mg/kg (n=3 per group) by oral gavage, once daily for 5 days, followed by a 14 day washout period. Peripheral blood was sampled every 2 to 3 days for isolation of total RNA and analysis of complete blood counts. The low and high dose groups showed ACY-957 plasma levels of 1.9 or 8.0 μM, respectively, at 24 h post final dose. No abnormal clinical signs were found during the in-life phase. White blood cells were suppressed by 25% and 61% at day 5, but recovered to baseline levels by day 9. ACY-957 administration led to a dose-dependent increase in HbE and HbG relative to HbB that was detectable at day 5, peaked at day 7, and returned to baseline levels by day 12. Maximum induction of HbG was 2.2-fold and 7.2-fold for the low and high dosing groups, respectively, relative to animals receiving vehicle only. These results demonstrate that ACY-957 induces HbE in rat and HbG in monkey to a similar extent. ACY-957 appeared well tolerated in both animals, although a reversible suppression of white blood cells was observed. Together, these findings suggest that optimization of dose and schedule could be performed in rats by monitoring HbE as a surrogate for HbG in primates. The optimized regime could then be validated in cynomolgus monkey. Accordingly, we have initiated experiments that explore the effects of several different ACY-957 dose schedules on HbE induction and white blood cell suppression in rats during a 4 week dosing and 2 week recovery period, which will also be presented. Disclosures Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Bhol:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

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.

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

The development of isoform selective HDAC inhibitors has opened the opportunity to better define and target pathways germane to lymphoma. Class IIb HDAC6 binds polyubiquinated, misfolded proteins facilitating their transport to the aggresome for proteosome-independent degradation. The aggresome is a key outlet for the unfolded protein response (UPR), a quality control mechanism that promotes survival during endoplasmic reticulum stress and signals CHOP (C/EBP-homologous protein) mediated apoptosis when homeostasis cannot be reestablished. We investigated the impact of the selective HDAC6 inhibitor ACY-1215 (Acetylon Pharmaceuticals, Inc) in preclinical models of lymphoma, its synergistic potential with BOR, its mechanism of action with respect to inhibiting protein degradation pathways, and potential biomarkers for response. Single agent concentration-effect curves were generated for 8 diffuse large B-cell lymphoma (DLBCL) (4 GCB: OCI-Ly1, OCI-Ly7, Su-DHL4, Su-HDL6; 4 ABC: OCI-Ly10, Su-DHL2, HBL-1, RIVA), 5 mantle cell lymphoma (MCL) (Maver, JVM-1, JEKO, Rec-1, HBL-2 ), and 4 T-cell lymphoma cell lines (HH, H9, CCL-119, Sup-T1). Maximal cytotoxicity was observed at 48-72 hr with IC50 ranging between 240-3500 nM. Activity was greatest in DLBCL, with 72 hr IC50 values as low as 240 nM. ACY-1215 began to show irreversible activity after 6 hr exposure. Single agent exposure at the IC50led to minimal apoptosis as analyzed by annexin V/propidium iodide (PI) staining, and caspase 3 and PARP cleavage, but led to G1 cell cycle arrest. The synergistic potential of ACY-1215 in combination with BOR was measured and evaluated for schedule and dose dependency in 6 cell lines. The greatest synergy was observed with simultaneous exposure of ACY-1215 (750 nM) and BOR (2 nM) at 48 hr with synergy coefficients as low as 0.24. Cell death occurred by apoptosis as evaluated by annexin V/PI, and cleavage of caspase 3 and PARP. Treatment with ACY-1215 led to inhibition of the aggresome as evidenced by increased poly-ubiquitinated proteins and acetylation of α-tubulin. Upregulation of UPR was demonstrated by acetylation of GRP78 and subsequent dissociation of PERK, increased p-eIF2a, spliced XBP-1 and CHOP. These effects were enhanced by treatment with BOR, confirm that accumulation of misfolded proteins activates the UPR response triggering apoptosis, and substantiate blocking two protein degradation systems simultaneously. Minimal acetylation of histone was observed following treatment with ACY-1215, and was considerably less relative to cells treated with pan-HDAC inhibitors confirming selectivity of the drug. These findings were validated in an in vivomodel of DLBCL (OCI-Ly10). Mice were treated with ACY-1215 50 mg/kg days 1-5, 8-12, 15-19 and/or BOR 1.5 mg/kg days 1, 8, 11. Treatment was well tolerated with no toxicity related deaths with the combination. One cycle of the combination led to statistically significant tumor growth delay (p=0.001) and extended overall survival (p Establishing biomarkers for response to isoform selective HDAC6 inhibition may lead to the treatment of patients with specific molecular and phenotypic signatures. Immunoblot levels of basal HDAC6 did not correlate with sensitivity to ACY-1215 in 8 cell lines. Resistance was characterized by combined increase in Bcl2 and loss of BIM. All cell lines expressed more GRP78 than normal peripheral blood mononuclear cells. Fifty-one untreated lymphoma patient samples (10 DLBCL, 11 FL, 10 MZL, 7 MCL, 13 TCL) were analyzed for markers of the UPR by immunohistochemistry. HDAC6 had bright expression in 30/41 lymphoma samples compared to 0/11 reactive lymph nodes. A similar pattern was observed for GRP78 (36/46 vs 0/10), and XBP-1 (26/50 vs 2/12). Selective targeting of HDAC6 with ACY-1215 inhibits aggresome mediated protein degradation inducing the UPR and cell cycle arrest. Dual targeting of protein degradation pathways represents a novel and rational approach for the treatment of lymphoma. Plans to study the clinical efficacy of ACY-1215 in patients with relapsed lymphoma are underway. Disclosures: Amengual: Acetylon Pharmaceuticals, INC: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Off Label Use: ACY-1215 is not approved for the treatment of lymphoma. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. O'Connor:Acetylon Pharmaceuticals, INC: Membership on an entity’s Board of Directors or advisory committees.

Histone deacetylase (HDAC) enzymes represent attractive therapeutic targets in multiple myeloma, but unfortunately non-selective HDAC inhibitors have led to dose-limiting toxicities in patients. ACY-1215 is a first generation, 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). Ongoing Phase Ib clinical trials with ACY-1215 have thus far confirmed an exceptional safety and tolerability profile (Raje, et al, EHA, 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 studies have shown clinical activity of a combination of the non-selective HDAC inhibitor vorinostat with lenalidomide and dexamethasone in myeloma patients (Richter, et al, ASH, 2011). However, many patients experienced significant toxicities with this regimen that significantly limits its clinical utility. In support of our ongoing clinical development program for ACY-1215 in MM, we show here that combining ACY-1215 with either lenalidomide or pomalidomide leads to synergistic decreases in the viability of MM cells in vitro. The relevance of inhibition of HDAC6 to this synergistic 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. Further, 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 ongoing 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 an ongoing Phase Ib clinical trial (NCT01583283) for the treatment of MM. Disclosures: Quayle: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

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.

Background Although non-selective HDAC inhibitors are active in MM, combination therapy is limited by significant adverse effects (AEs) including severe fatigue, gastrointestinal toxicity, and myelosuppression. ACY-1215 is the first-in-class selective oral HDAC6 inhibitor that inhibits the aggresome/autophagy pathway, an alternate pathway to proteasome clearance of misfolded proteins. ACY-1215 has demonstrated potent synergy with bortezomib preclinically in cell and animal models of MM (Santo, Blood, 119(1):2527). Methods ACY-100 is a three part single arm, open label study with cohort dose escalation in a standard 3+3 design as monotherapy (1a) and in combination with bortezomib (1b) followed by a phase 2 extension. Eligible patients (pts) for the phase 1a and 1b portions had relapsed or relapsed and refractory MM, previously received at least two 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 bone marrow reserve, hepatic function and creatinine clearance of >30 mg/mL/min. ACY-1215 was given orally days 1-5 and 8-12 of a 21 day cycle, and bortezomib days 1,4,8,11 and dexamethasone 20 mg days 1,2,4,5,8,9,11,12. Peripheral blood samples were obtained for pharmacokinetic (PK) and pharmacodynamics (PD), assessment of acetylated tubulin (HDAC6 inhibition) and acetylated histones (Class 1 HDAC inhibition). Toxicity was assessed using CTCv4.0 and responses were evaluated by modified EMBT and Uniform Criteria. Results The monotherapy portion of ACY-100 has been previously presented (Raje, Blood, V20(21):4061). Fifteen heavily pretreated pts received ACY-1215 monotherapy at doses of 40 mg to 360 mg. Most AEs were low grade and not related to ACY-1215. Two pts had grade 3 AEs, anemia and neutropenia, considered possibly related to ACY-1215. No dose limiting toxicities (DLTs) were observed. Stable disease (SD) was the best response in 6 patients. Sixteen patients as of June 28, 2013 have received ACY-1215 at doses of 40 mg to 160 mg in combination with bortezomib (1.0 mg/m2 first cohort and 1.3 mg/m2 thereafter) and dexamethasone. Median age was 60, and 16 patients had previously received up to 11 lines of therapy. The first combination cohort was expanded due to a dose limiting toxicity (DLT) of asymptomatic increase in amylase. No other DLTs have been observed. Treatment emergent adverse events were predominantly low grade. Those occurring in >25% of patients were elevated creatinine, thrombocytopenia, anemia, fatigue, elevated ALT, AST and amylase, hypokalemia, cough, decreased appetite, dyspnea, hypoalbuminemia and peripheral neuropathy; most were not considered related to ACY-1215. Grade 3-4 AEs possibly related to ACY-1215 included asymptomatic elevated amylase (2), thrombocytopenia (3), anemia (1), stomach cramps (1) and elevated creatinine (1). Of 16 patients evaluable for response, VGPR (1), PR (2), MR (1) were seen, with 5 pts achieving SD. Responding patients were on study from 2 to 17 cycles. Eleven pts were refractory to bortezomib prior to study entry. Of those the best outcome was MR (1) and SD (4), with the remaining having either progressive disease (5) or not evaluable (1). PK and PD data is available from 16 patients including the 160 mg dose level. PK for ACY-1215 is similar to the same dose levels in phase 1a monotherapy suggesting coadministration of bortezomib does not impact the PK of ACY-1215. Maximal levels were ≥ 1µM at ≥ 80 mg correlating with measurable increases >2x in acetylated tubulin with a minimal increase in acetylated histones. Conclusions ACY-1215 was well-tolerated when administered in combination with bortezomib and encouraging disease responses were observed in this heavily pretreated patient population. Future cohorts in phase 1b will explore twice daily dosing prior to start of phase 2. Disclosures: Raje: Acetylon Pharmaceuticals, Inc: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Amgen: Consultancy. Vogl:Otsuka: Consultancy; Celgene: Consultancy; Millennium: Research Funding; Acetylon Pharmaceuticals, Inc: Research Funding. Hari:Celgene: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Millenium: Consultancy, Research Funding. Jagannath:Celgene: Honoraria; Millenium: Honoraria. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Supko:Acetylon Pharmaceuticals, Inc: Research Funding. Leone:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Wheeler:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity’s Board of Directors or advisory committees; Millenium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.

Proteasome inhibitors (PI) induce significant apoptosis associated with c-Jun NH2-terminal kinase (JNK) and caspase activation in multiple myeloma (MM). When the proteasome is inhibited, aggregated misfolded proteins are ubiquitinated, and polyubiquitinated aggregated proteins are transported to the microtubule organizing center (MTOC). These proteins are bound by the HDAC6-dynein motor complex to form aggresomes. The aggresome is engulfed by autophagosomes before it is finally subjected to degradation by lysosomes. This proteolysis system (autophagy) is considered to be one of the key mechanisms of cell survival. An HDAC6 inhibitor can potentially regulate the autophagosome-proteolysis system by inhibiting aggresome formation. Therefore, combination treatment targeting the proteasomal degradation system with a PI and the autophagosome-proteolysis system with an HDAC6 inhibitor results in accelerated cell death and is the subject of several ongoing clinical trials. Although this mechanism makes rational sense, it has not yet been demonstrated in preclinical or clinical models using clinically approved compounds. Carfilzomib (CFZ) is a selective PI that is approved in the US for the treatment of relapsed and refractory MM. Carfilzomib selectively inhibits the chymotorypsin-like activity of the constitutive proteasome and the immunoproteasome. Here we studied the anti-MM effect of CFZ alone and in combination with the selective HDAC6 inhibitor ACY-1215 with respect to autophagy. Combination treatment with CFZ (50 nM, 1 hr)) with ACY-1215 (2 µM, 48 hr) showed synergistic cytotoxicity in RPMI8226, U266, H929, and MM.1s MM cell lines, including a bortezomib-resistant cell line, ABNL-BR, as demonstrated by MTT assay. Apoptosis activity was evaluated by ELISA and immunoblot analysis, and showed an increase in caspase cleavage and PARP expression in the combination treatment. In U266 and RPMI8226 cells, CFZ (50 nM) increased the autophagosome marker, LC3-II, as well as p62, a binding protein for ubiquitinated proteins within the aggresome and the autophagosome. When ACY-1215 (2 µM) was added to the system, increased LC3-II was observed. However, p62 was decreased, suggesting that aggresome formation was inhibited by ACY-1215. Importantly, in primary CD138+MM cells the combination treatment of CFZ with ACY-1215 showed synergistic cytotoxicity. In primary CD138+ cells, single-agent CFZ (5 nM, 12 hr) increased LC3-II, and p62 levels were reduced by ACY-1215 (2 µM), which was consistent with the findings observed in MM cell lines. Immunofluorescence staining demonstrated that U266 cells treated with CFZ alone showed accumulation of ubiquitinated proteins in the cytoplasm and engulfment of aggresomes by autophagosomes. Although autophagosome formation was also evident in combination treatment as demonstrated by the association between p62 and LC3, association of autophagosome and aggresome was inhibited by the drug combination. This result suggests that ACY-1215 plays a role in disrupting the association between the aggresome and the autophagosome. Electron microscopy confirmed that combination treatment with ACY-1215 inhibits aggresome formation and autophagy induced by CFZ, thereby enhancing apoptosis in U266 cell line. However, electron microscopy in MM1s cells did not show an increase in autophagosome formation following use of single-agent CFZ, suggesting that cell death induced by CFZ and ACY-1215 may be cell specific. Nevertheless, the combination still results in increased cytotoxicity, suggesting stress response may be an additional mechanism. In fact the combination treatment results in an increased p-PERK and p-eIF2 alpha, markers of ER stress. In conclusion our data suggests that combination treatment with ACY-1215 and CFZ has significant synergistic cytotoxic effects against MM cells and may overcome bortezomib-resistance. Moreover, inhibition of aggresome formation and autophagy by ACY-1215 enhances apoptosis in some MM cell lines and in primary CD138+ MM cells. Ongoing animal studies will confirm this data and is the basis for a planned Phase I/II clinical trial in relapsed MM. Disclosures: Arastu-Kapur: Onyx Pharmaceuticals, Inc.: Employment. Kirk:Onyx Pharmaceuticals: Employment. Jones:Acetylon Pharmaceuticals, Inc: Employment. Noopur:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Amgen: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding.

Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Fetal beta-like globin gene (HbG) expression is silenced in adults partly through the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains HDAC1/2 (Sankaran VG, Science, 2008). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). However, the mechanism through which HDAC1/2 inhibition leads to activation of HbG remains largely unknown. In this work, we have utilized our proof of concept molecule, ACY-957, to investigate changes in gene expression and chromatin organization that result from inhibition of HDAC1/2. Gene expression profiling was performed on cells treated with ACY-957 (n=3) or vehicle (n=3) using Affymetrix PrimeView GeneChips. Treatment of early erythroblasts (CD71+, GlyA-) resulted in the up and down regulation of 1294 and 681 transcript probe sets, respectively. In comparison, treatment of late erythroblasts (CD71+, GlyA+) resulted in a total of 255 transcript probe set changes. This finding is consistent with follow-up experiments demonstrating that ACY-957 is unable to induce HbG in cells positive for both CD71 and GlyA. Taken together, these results suggest that erythroblasts become less responsive to HDAC inhibition as they mature. Gene set enrichment analysis using public domain data revealed that genes up- or down-regulated by HDAC1/2 shRNA knockdown are significantly overrepresented in the list of genes induced or repressed by ACY-957, respectively; suggesting pharmacologic inhibition of HDAC1/2 recapitulates genetic ablation. We also identified significant enrichment in other gene sets involving targets linked to HbG regulation, including lysine-specific demethylase 1 (LSD1) (Shi L, Nature Medicine, 2012). GeneChip and quantitative real-time PCR time course experiments show ACY-957 treatment leads to a decrease in Bcl11A (2-fold) and Sox6 (10-fold) mRNA, known repressors of fetal globin synthesis, and an increase in Klf2 (2-fold) and Gata2 (8-fold) mRNA, proposed fetal globin activators. This result is consistent with work by others that show Gata2 is suppressed, in part, by the NuRD complex (Hong W, EMBO Journal, 2005) and that Gata2 binding at the HbG promoter leads to increased levels of HbG expression (Zhu X, PLoS One, 2012). Interestingly, Gata2 induction preceded Sox6 suppression in ACY-957 treated cells and the Sox6 promoter contains 8 canonical WGATAR binding sites and one Gata2-specific binding motif, raising the possibility suppression of Sox6 by ACY-957 is mediated by Gata2 induction. To investigate these possibilities, we have performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) for HDAC1, HDAC2, Gata2, and the HDAC2-specific histone modification H3K56 in ACY-957 and vehicle treated cells. These experiments will be discussed. ChIP-seq data, both by itself and in combination with gene expression data, will provide further insight into the mechanism through which HDAC1/2 regulates HbF synthesis. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe: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.

Induction of HbG is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and fetal hemoglobin (HbF) protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). While a variety of selective HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by variable efficacy and concerns over off-target side-effects observed in clinical trials, potentially due to inhibition of HDAC3. Additionally, it remains to be determined if HDAC1 or HDAC2 is the preferred therapeutic target. In this work we present data that investigates the effects of selective inhibitors of HDAC1, 2, or 3 on cytotoxicity, erythroid differentiation, and HbG induction in cultured human CD34+ bone marrow cells. Acetylon Pharmaceuticals has generated a library of structurally distinct compounds with a range of selectivity for each of HDAC1, 2, or 3 (Class I HDAC) as determined in a biochemical assay platform. From our initial chemical series, we identified ACY-822 as a Class I HDAC inhibitor with IC50 values of 5, 5, and 8 nM against HDAC1, 2, and 3, respectively. In contrast, ACY-1112 is 30-fold selective for HDAC1 and 2, with IC50 values of 38, 34, and 1010 nM against HDAC1, 2, and 3, respectively. Treatment of cells for 4 days with ACY-822 (1 μM) resulted in a 20-fold decrease in cell viability, while ACY-1112 (1 μM) treatment resulted in a minimal reduction in viability (1.2-fold) and a 2-fold increase in the percentage of HbG relative to other beta-like globin transcripts. This result suggests that pharmacological inhibition of HDAC3 is cytotoxic and is consistent with the therapeutic rationale for the design selective inhibitors of HDAC1 and 2. To investigate if HDAC1 or HDAC2 is the preferred therapeutic target, we utilized a second series of structurally distinct compounds. We identified ACY-957 as an HDAC1/2 selective compound biased towards HDAC1 with IC50 values of 4, 15, and 114 nM for HDAC1, 2, and 3, respectively. In contrast ACY-1071 showed balanced HDAC1 and 2 selectivity with IC50 values of 27, 24, and 247 nM for HDAC1, 2, and 3, respectively. Treatment of cells for 6 days with 1 μM of ACY-957 or ACY-1071 resulted in a 3-fold increase in the percentage of HbG relative to other beta-like globin transcripts. However, we found that ACY-957 treatment resulted in an approximately 3-fold decrease in cell viability after 6 days of treatment, while ACY-1071 treatment resulted in a minimal reduction (1.2-fold) in cell viability. Decreased cell viability observed with ACY-957 was associated with a reduction of cells positive for the erythroid differentiation markers CD71 and glycophorinA. This result is consistent with the Mx-Cre mouse model where HDAC1KO; HDAC2het had reduced numbers of erythrocytes, thrombocytes, and total bone marrow cells, while the HDAC1het; HDAC2KO was unaffected (Wilting RH, EMBO Journal, 2010). Our results suggest that compounds with a pharmacological profile of increased selectivity towards HDAC2 inhibition versus HDAC1 may be less cytotoxic and minimize effects on differentiation, while still inducing HbG in human CD34+ bone marrow cells. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe: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.

Background ACY-1215 is the first selective HDAC6 inhibitor in clinical trials and is well-tolerated as monotherapy up to 360 mg/day, the maximum dose examined. Cmax ≥ 1µM was achieved at dose levels >80 mg. Unlike nonselective HDAC inhibitors, which are associated with severe fatigue, vomiting, diarrhea and myelosuppression, DLTs have not been observed with ACY-1215. ACY-1215 synergizes in in vitro with both lenalidomide and dex in MM cell lines providing the rationale to conduct a phase 1b trial of ACY-1215 in combination with these agents. Methods Relapsed and relapsed and refractory pts who have progressed on at least one prior treatment regimen, who have creatinine clearance >50 mg/mL/min, adequate bone marrow and hepatic function, and who gave informed consent were enrolled. In Part A, patients were treated with escalating doses of oral ACY-1215 on days 1-5 and 8-12 of a 28 day cycle with lenalidomide 25 mg d 1-21 and dex 40 mg weekly. In Part B, the schedule includes ACY-1215 on days 15-19. Subsequent cohorts will explore twice daily dosing based on emerging clinical, pharmacokinetic (PK) and pharmacodynamic (PD) data. Peripheral blood samples were obtained for PK and PD analysis. PD assessment measured the fold increase of acetylated tubulin (a marker of HDAC6 inhibition) and acetylated histones (a marker of class 1 HDAC inhibition) in peripheral blood mononuclear cells (PBMC). Results As of July 3, 2013, 15 pts who progressed after 1 to >3 prior therapies have been enrolled; 8 were relapsed and 7 were refractory to the most recent therapy. Patients were treated daily at up to 240 mg ACY-1215. Fourteen pts had received prior lenalidomide of which 6 were previously refractory as defined by having less than a minimal response (MR) to therapy (1) or progressive disease on either full dose or maintenance therapy (5). Pts have completed 0 to 11+ cycles of therapy with 10 pts continuing on therapy. Five pts have discontinued therapy due to progressive disease (PD) (3), travel difficulties (1), or missed doses of lenalidomide (1). The latter pt was replaced. The most common treatment emergent events were fatigue (43%), upper respiratory infection (36%), anemia and peripheral edema (21% each), neutropenia (29%) and muscle spasms (21%). Most were grade 1 and 2 and there was no dose relationship to ACY-1215. There were 9 grade 3-4 events in 6 pts, primarily hematologic, as well as fatigue and asymptomatic laboratory investigations. Only 1 event, grade 3 neutropenia, was considered possibly related to ACY-1215 by the investigator. PK and PD data is available from 12 pts up to 160 mg dose level. PK for ACY-1215 is similar to the analogous dose levels in phase 1a monotherapy suggesting coadministration of lenalidomide does not significantly impact the PK of ACY-1215. Maximal levels were ≥ 1µM at ≥ 80 mg correlating with measurable increases >2x in acetylated tubulin with a minimal increase in acetylated histones. Twelve pts, at doses up to 160 mg ACY-1215, are evaluable for response (after at least two cycles). In addition, 1 pt who discontinued therapy after one cycle had response data available. Nine patients (69%) have ≥ PR, including 1 CR, 4 VGPR, 3 PR, and 1 PRu. Two pts had MR and 2 had SD as the best response. Reponses are durable up to 11+ cycles of therapy. Of the 6 patients who were refractory to lenalidomide, best responses included 1 PR, 1 VGPR, 2 MR and 2 SD. Conclusions ACY-1215 at doses which have biological activity (as determined by PD data in PBMC) can be safely combined with lenalidomide and dex with favorable toxicity to date. Significant responses were observed in pts, and responses have been seen in pts previously refractory to lenalidomide. Future cohorts will explore longer duration of exposure as well as a twice daily dosing schedule for ACY-1215. Disclosures: Vorhees: Acetylon Pharmaceuticals, Inc: Research Funding; GlaxcoSmithKline: Consultancy, Research Funding; Millenium: Research Funding; Celgene: Consultancy, Research Funding; Merck: Research Funding; Janssen: Research Funding; Prolexys: Research Funding; Abbott: Consultancy. Bensinger:Celgene: Consultancy, Honoraria, Research Funding. Supko:Acetylon Pharmaceuticals, Inc: Research Funding. Richardson:Celgene: Membership on an entity’s Board of Directors or advisory committees; Millenium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Patrick:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Wheeler:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Raje:Acetylon Pharmaceuticals, Inc: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Amgen: Consultancy.

Abstract 328 In multiple myeloma (MM), deregulated osteoclast (OC)/osteoblast (OB) cross-talk induces osteolytic bone lesions. The HDAC6 selective inhibitor, rocilinostat (ACY-1215), in combination with bortezomib has shown potent anti myeloma activity in preclinical studies, which provided the rationale for a clinical trial that is currently recruiting relapsed/refractory MM patients (NCT01323751). However, while the beneficial role of bortezomib in tumor-related bone disease has been previously described, the effect of HDAC6 inhibition is not known. Evidence suggests a positive effect on bone turnover as pan HDAC inhibitors accelerate OB maturation and suppress OC maturation in vitro. Here, we evaluated effects of the selective HDAC6 inhibitor rocilinostat (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib in MM bone disease. Rocilinostat (1 μM) alone and in combination with bortezomib (2.5 nM) inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, rocilinostat (1 μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines and in combination with bortezomib inhibited transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS, and NFATC1. Importantly, such an effect was cytokine (RANKL and M-CSF) dependent. Further, rocilinostat, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase (ALP) enzyme activity and alizarin red staining. In addition, we found increased mRNA expression of beta-catenin, osteocalcin, ALP, and RUNX2. Based on this promising in vitro data, we used the xenograft model of disseminated human MM in SCID mice to study the effect of rocilinostat, alone and in combination with bortezomib, on MM bone disease. MM.1S-GFP-Luc cells were injected intravenously, and MM disease progression was followed by bioluminescence imaging. A significant decrease in tumor burden was observed in mice following three weeks of treatment with rocilinostat, alone or in combination with bortezomib. Isolating serum from control and treated mice, we also observed a significant decrease of TRAPc5b levels, a marker of bone resorption, as well as a significant increase in osteocalcin levels, a marker of bone formation, in the serum of the combination treated cohort. Cells isolated from the calvaria from the combination treated group compared to the control group showed a significant increase in the mRNA expression of ALP, RUNX2, and osterix, as well as a significant decrease in the mRNA expression ratio of RANKL/OPG. To elucidate the role of HDAC6 inhibition on bone turnover, we used HDAC6 knockout mice. Cells were isolated from femurs, tibia, and spine of 2 month-old wild type (WT) and HDAC6 knockout (KO) mice and mRNA expression for osteocalcin, ALP, RUNX2 and osterix was assessed by qPCR. We observed a significant increase in osteocalcin mRNA expression without significant changes in the mRNA expression of ALP, RUNX2 and osterix. Bone marrow stromal cells (BMSCs) differentiated from WT and KO mice were co-cultured with MM murine cell lines and, notably, the proliferative advantage conferred by BMSC isolated from HDAC6 KO mice to MM cell lines was significantly decreased compared to WT BMSCs. These data suggest that a microenviroment lacking HDAC6 reduces MM cell proliferation. Moreover, treatment with rocilinostat (1mM) for 24 h inhibited proliferation of MM cells cocultured with WT BMSCs to levels observed in MM cells cultured with KO BMSC lacking endogenous HDAC6. Finally, the effect of co-treatment with rocilinostat (1μM) and bortezomib (2.5 nM) on proliferation of MM cells co-cultured with WT-BMSC was similar to that observed when bortezomib was added to MM cells in cocultures with KO BMSC. In conclusion, the in vitro data and the in vivo results from the xenograft models of human MM in SCID mice, as well as data in the HDAC6 KO mice, indicate a potential beneficial role of HDAC6 inhibition on MM-related bone disease. We are currently performing dynamic and static histomorphometric analysis to confirm this effect on bone remodeling at the tissue level. These effects on bone remodeling are an added benefit for MM patients and will be assessed prospectively in our ongoing clinical trial. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Abstract 4061 Introduction The rate of relapse in multiple myeloma (MM) remains high despite novel therapies such as the proteasome inhibitor bortezomib. Histone deacetylase (HDAC) inhibitors (panobinostat and vorinostat) in combination with bortezomib have demonstrated activity in patients (pts) with relapsed and refractory MM. However, pan-HDAC inhibitors target multiple HDAC enzymes, including HDAC6 and Class 1 enzymes, and are associated with adverse effects such as severe fatigue, nausea, vomiting, diarrhea, and myelosuppression. HDAC6 regulates acetylation of cytoplasmic tubulin, an essential component of aggresome formation in an alternative pathway to the proteasome to remove unfolded or misfolded proteins. Therefore selective inhibition of HDAC6 may lead to efficacy in MM with an improved safety profile. Rocilinostat is a novel selective inhibitor of HDAC6 with 11-fold selectivity over Class 1 HDACs. Preclinical results in models of MM demonstrate synergy between bortezomib and rocilinostat (Blood 119(11):2579-89) and a superior safety profile compared with pan-HDAC inhibitors. We examined the pharmacokinetics, pharmacodynamics, safety, and preliminary activity of rocilinostat, alone and in combination with bortezomib and dexamethasone, in pts with relapsed or relapsed/refractory MM. Methods Patients with relapsed or relapsed/refractory MM, previously exposed to both proteasome inhibitor and immunomodulatory agents, were enrolled in an ongoing, 3-part, phase I/II, single-arm, open-label study. A group sequential dose-escalation design was used to identify optimal dosing of rocilinostat alone (Part 1) and in combination with bortezomib and dexamethasone (Part 2), followed by evaluation of the objective response rate and safety of the combination (Part 3). In Part 1, pts received single oral daily doses of rocilinostat for 5 consecutive days during Weeks 1 (Days 1–5) and 2 (Days 8–12) every 3 weeks, with a starting dose of 40 mg and planned escalation to 80, 160, 240, 360, and 480 mg. Part 2 began in parallel after completion of the third monotherapy cohort, with a starting dose of 40 mg rocilinostat plus bortezomib 1.0 mg/m2twice weekly (Days 1, 4, 8, and 11) and 20 mg dexamethasone given on the day of and day after each bortezomib dose. Pharmacokinetics were assessed on Days 1, 4, 8, 11 and 15 and pharmacodynamics in blood mononuclear cells were measured on Day 1. Patients received up to 6 cycles of therapy, or more if experiencing clinical benefit. Available data are for the first 4 monotherapy cohorts (40, 80, 160, 240 mg) and the initial 3 pts in the first combination cohort (16 pts total). Results Of the 16 pts, 13 were treated with monotherapy and 3 with combination. Median age was 70 yrs (range: 51–79), and 56% of pts were male. The majority of pts were either white (8, 50%) or black (7, 44%). Fourteen pts had relapsed/refractory MM and 2 had relapsed disease; 88% of pts received ≥ 3 prior treatments. Patients received a median of 2 cycles of rocilinostat (range: 1–10). Monotherapy was well tolerated with no dose-limiting toxicities (DLTs) and mostly grade 1–2 adverse events (AEs). The most common AEs with monotherapy were elevated creatinine, diarrhea, fatigue, and upper respiratory tract infection (n=3 each). Grade 3/4 treatment-related AEs on monotherapy occurred in 1 pt (grade 3 anemia). In the first combination cohort, 1 pt had a DLT (elevated amylase). Five monotherapy pts had stable disease. One combination pt with relapsed/refractory disease had a 26% reduction of serum M protein at Cycle 2. Rocilinostat was rapidly absorbed (Table) and displayed approximately dose-linear pharmacokinetics with no accumulation of drug. Maximal levels of acetylated tubulin in peripheral blood were observed 1 hr post dose. The magnitude of the signal increased with dose and exposure (Table), with all cohort 3 pts having at least a doubling of acetylated tubulin levels. Conclusion Preliminary results from this first clinical evaluation of rocilinostat suggest that selective inhibition of HDAC6 with rocilinostat, alone or in combination with bortezomib and dexamethasone, may provide a well-tolerated treatment option for relapsed or relapsed/refractory MM. Updated data will be presented. Disclosures: Raje: Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding. Hari:Celgene: Consultancy, Honoraria. Vogl:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Consultancy, Research Funding; Otsuka: Consultancy; Acetylon: Research Funding. Jagannath:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Milenium Pharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx Pharm: Honoraria, Membership on an entity's Board of Directors or advisory committees. Supko:Acetlon: Research Funding. Wheeler:Acetylon: Employment.

Abstract 1650 The emergence of epigenetic therapies has identified histone deacetylase(HDAC) inhibitors as effective therapeutic agents for the treatment of refractory lymphoma. While pan-class I/II HDAC inhibitors have led to treatment ramifications, recently goals have shifted to the development of discrete HDAC selective inhibitors to further define and better target pathways germane to specific subtypes of lymphoma. We investigated the therapeutic impact of the selective HDAC6 inhibitor rocilinostat (ACY-1215) (Acetylon Pharmaceuticals, Inc) in a panoply of lymphoma cell lines. HDAC6, a class IIb histone deacetylase, binds polyubiquinated, misfolded protein aggregates and facilitates their transport to the aggresome. The aggresome then sequesters these aggregates for degradation. The aggresomal pathway is proteasome-independent and is a key outlet for the unfolded protein response (UPR). The purpose of this study was to determine whether inhibition of HDAC6 with rocilinostat leads to an accumulation of misfolded proteins, activates the UPR stress response and ultimately induces apoptosis. These results have been confirmed in preclinical models of multiple myeloma, and the drug is currently being evaluated in clinical trials for that disease [1]. We evaluated the single agent activity of rocilinostat, its synergistic potential when combined with bortezomib, and the mechanism of action of this combination in lymphoma with respect to inhibiting two separate protein degradation pathways. Single agent dose-effect curves were generated for 8 diffuse large B-cell lymphoma (DLBCL) cell lines (4 germinal-center type: OCI-Ly1, OCI-Ly7, Su-DHL4, Su-HDL6; 4 activated B-cell type: OCI-Ly10, Su-DHL2, HBL-1, RIVA), 4 mantle cell lymphoma (MCL) cell lines (Maver, JVM-1, JEKO, Rec-1), and 2 T-cell lymphoma cell lines (HH and H9). The inhibitory concentration 50% (IC50) values were calculated after exposure for 24, 48, and 72 hours. Maximal cytotoxicity was observed in all cell lines at 48–72 hours with IC50 values ranging between 240–3500 nM. Activity was greatest in the cell lines OCI-Ly10 and OCI-Ly7, with 72-hour IC50 values of 240 nM and 255 nM respectively. Rocinlinostat began to show irreversible activity after 6 hours of exposure. The cytotoxicity of rocilinostat in combination with bortezomib was measured and evaluated for schedule and dose dependency in OCI-Ly10. The greatest synergy was observed with simultaneous exposure and was first established at the concentrations rocilinostat750 nM and bortezomib 3 nM (CI=0.576). Synergy did not depend on the concentration of one drug over the other. Eight additional cells lines were evaluated for synergy with similar results. Cell death occurred by apoptosis as evaluated through FACS analysis for annexin V/propidium iodide, and cleavage of caspase 3 and PARP by immunoblotting. Treatment of OCI-Ly10 and Su-DHL6 cells with rocilinostat led to acetylation of a-tubulin, increased poly-ubiquitinated proteins, PERK, GRP78, p-eIF2a, and spliced XBP-1 as detected by immunoblotting. These effects were enhanced by treatment with bortezomib, and confirm that accumulation of misfolded proteins activates the UPR response triggering apoptosis, and substantiates blocking two protein degradation systems simultaneously. In addition, p65 NF#x2610;#x0025;B subunit was decreased with the combination. Slight acetylation of H3 was observed following treatment with roclinostat, but this was considerably less compared with cells treated with pan-Class I/II HDAC inhibitors and confirms selectivity of the drug. These are the first results to indicate that a selective HDAC inhibitor can have marked activity across a panel of lymphoma cell lines. Future studies will evaluate efficacy and tolerability in in vivo models of lymphoma, and further define mechanism of action. Taken together, these findings raise the prospect that dual targeting of the ubiquitin-proteasome and aggressomal protein degradation pathways can be synergistically effective and provide excellent pre-clinical rationale for expanding the use of rocilinostat in combination with bortezomib for patients with relapsed or refractory lymphoma. Disclosures: Amengual: Acetylon Pharmaceuticals, Inc: Research Funding. Jones:Acetylon Pharmaceuticals, Inc: Employment. O'Connor:Millenium Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics, Inc: Consultancy; Seattle Genetics, Inc: Membership on an entity's Board of Directors or advisory committees; Allos Therapeutics, Inc: Consultancy.

Abstract 3259 Introduction: Fetal hemoglobin (HbF) induction is an established therapeutic strategy in sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). The only drug with proven efficacy in SCD is hydroxyurea, which is cytotoxic, poorly tolerated, and only reduces the frequency and severity of SCD crises in a subset of patients. For bT there are no approved drug treatments and, for the most severe forms of bT, patients require repeated blood transfusions for life and chelation therapy to reduce iron overload. Fetal (γ) globin expression is silenced in adults partly through the action of a complex containing BCL11A and HDAC1/2. Genetic ablation and chemical inhibition of HDAC1 or HDAC2, but not HDAC3, resulted in the induction of γ-globin synthesis in adult bone marrow derived CD34+ erythroid cells (Bradner, Proc Natl Acad Sci 2010). While a variety of non-specific HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by their variable efficacy and concerns over off-target side-effects observed in small clinical trials, potentially due to inhibition of HDAC3. Therefore, development of selective and potent HDAC1/2 inhibitors leading to HbF induction represents a refined and targeted therapeutic approach for the treatment of SCD and bT. Results: Recently, selective inhibitors for HDAC1 and 2 have been described for compounds containing a benzamide zinc chelating group (Witter, Bioorg Med Chem Lett 2008). Acetylon Pharmaceuticals has generated a library of structurally distinct compounds containing a modified benzamide biasing element. We have screened these molecules in an HDAC biochemical assay platform and found IC50 values for HDAC1 and 2 ranging from 5 to 10 nM and 10 to 30 nM, respectively, with approximately 10- to 100-fold selectivity over inhibition of HDAC3. These compounds had good oral bioavailability in rat, with exposure (AUC) exceeding 5000 hr*ng/mL following an oral dose of 5 mg/kg. Furthermore, half-life was found to range between 4 to 8 hours. In cultured human CD34+ bone marrow cells undergoing erythroid differentiation, these compounds induced a dose dependent increase in fetal hemoglobin expression, with a 5-fold induction observed at 1 μM. The γ-globin induction we observe is comparable to MS-275, a non-selective HDAC1/2/3 inhibitor, and decitabine, a DNA methyltransferase inhibitor which has been in clinical trials to induce HbF in SCD. The absolute levels of adult β-globin remained unchanged. These results were confirmed using flow cytometry, where a 2- to 4-fold increase in the number of cells expressing HbF protein was observed. Conclusion: These results suggest that inhibition of HDAC1 and 2 is sufficient to induce fetal globin expression. Our selective HDAC1/2 inhibitors have highly favorable oral pharmacokinetic profiles suitable for further development towards the treatment of SCD and bT. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Bradner:Acetylon Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Mazitschek:Acetylon Pharmaceuticals, Inc. : Consultancy, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment.