ALBERT

Description: 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.

Description: T cell cancers are common in pediatric oncology, and include the clinical entities T cell acute lymphoblastic leukemia (T-ALL) and the Non-Hodgkin Lymphoma, T cell lymphoblastic lymphoma (T-LBL). These diseases carry worse prognoses than their B cell counterparts, and the regimens used in their treatment confer significant short- and long-term morbidities. Unlike many pediatric malignancies, which have characteristic chromosomal translocations, most T cell-derived cancers carry no such cytogenetic hallmark. Because such straightforward oncogenic lesions are typically absent, insights into the genetic underpinnings and molecular pathogenesis of these T cell diseases are also less comprehensive. To address these deficiencies, we have performed a forward genetic screen for T cell malignancies using the vertebrate model organism, zebrafish (Danio rerio). Although others have used transgenic zebrafish to show that known mammalian oncogenes can induce zebrafish leukemias, to date, this powerful animal model has not been used as a discovery tool to elucidate novel genetic mutations underlying de novo T cell malignancies. Zebrafish share ontogeny with other vertebrates, and possess an adaptive immune system strikingly similar to mammals. However, unlike other vertebrate models, zebrafish are amenable to forward genetic approaches, where animals are randomly mutagenized, and then screened using high-throughput strategies to detect rarely-occurring phenotypes. Using a zebrafish line with T lymphocyte-specific expression of green fluorescent protein (GFP), we have conducted a germline mutagenesis screen to identify animals that develop T cell malignancy. Thus far, we have isolated three independent lines with a heritable predisposition to developing T cell cancers. In all three lines, age of onset, patterns of spread, and morphologic appearance resemble human T-ALL/-LBL. In addition, we have compiled histologic and immunohistochemical evidence further characterizing these diseases. Analysis of T cell receptor (TCR) β gene sequences from malignant tissue has verified the monoclonality of malignant cells, based on identical VDJ rearrangements. Malignant cells have also been successfully transplanted serially into sub-lethally irradiated allogeneic recipients, using as few as 2500 donor cells to transfer disease. Furthermore, we have shown the malignancies themselves to be radiation sensitive, like their human correlates. Soon after radiation-induced remissions are achieved, however, high rates of aggressive disease relapse are seen, modeling recurrent human T-ALL/-LBL. In summary, we have identified three independent zebrafish mutant lines, which each recapitulate human T lymphocyte malignancy, and that are all heritably transmissible from one generation to the next. The positional cloning of the underlying genetic mutation of each line will provide exciting candidates for study in these human diseases. Moreover, using these lines as subjects in ongoing investigations, we are examining the genomic, epigenetic, and transcriptional profiles of these malignant cell populations, comparing pre- and post-transplant samples, pre- and post-irradiation isolates, and following other accepted and investigational therapeutic interventions. Finally, these lines are now poised for utilization in additional high-throughput screening approaches, including forward genetic suppressor and enhancer mutagenesis projects, as well as small molecule drug screens. Collectively, these new vertebrate models of T-ALL/-LBL should provide vital experimental platforms for studies of this important class of human cancers.

Description: Acquired and congenital defects in iron metabolism from either deficiency or excess are one of the most common human diseases. Here we present the characterization of the zebrafish frascati mutation, which results in a profound hypochromic anemia and a developmental arrest at the pro-erythroblast stage. Using a positional cloning strategy, we have identified the gene disrupted frascati in mutants as a novel member of the mitochondrial solute transporter family. Members of this family of solute carriers have related tripartite sequence and structure. They function in transporting various metabolites and substrates across the inner mitochondrial membrane. We have verified the identity of the gene in zebrafish by the following criteria: (a) tissue-restricted expression in erythroid progenitors, (b) identification of missense mutations from the frascati five alleles, (c) rescue of anemia by over-expression frascati of cRNA in mutant embryos, and (d) mimicry of anemia using inactivating antisense morpholinos in wildtype embryos. We have also identified the functional ortholog in the mouse which has an analogous tissue and developmental expression pattern. The frascati ortholog in the mouse is highly expressed in fetal liver and adult bone marrow and spleen. The murine frascati transcript and protein are induced during terminal erythroid differentiation in MEL cells treated with either DMSO or HMBA. The over-expression of the mouse frascati cRNA in zebrafish frascati mutant embryos rescued their anemia with equal efficacy as the zebrafish clone. Given the identity of the gene and the requirement for iron in heme biosynthesis in the mitochondria of the developing erythron, we injected exogenous iron-dextran into frascati embryos. The embryos injected with iron-dextran were allowed to develop to 3 days post-fertilization, then stained for hemoglobinized cells with o-dianisidine and genotyped. Using this assay, the anemia caused by frascati the mutation could be partially rescued with exogenous iron supplementation. We therefore propose that the frascati gene functions as the essential transporter for iron importation into the mitochondria for heme biosynthesis and subsequent hemoglobin production in developing erythroid progenitors. Insight into the function of frascati the gene will be directly relevant to our understanding of human disorders of iron deficiency anemia and iron-overload sideroblastic anemia.

Description: 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.

Description: Abstract 273 VPREB1 deletions occur in 1/3 of pediatric ALL (pALL) patients, but have been considered a normal consequence of λ light chain rearrangement due to VPREB1's location among λ chain variable region genes. Recently, VPREB1 deletions were reported in high risk pALL samples with BCR-ABL1-like gene expression (Den Boer 2009) and VPREB1 under-expression was found in a 38-gene signature associated with relapse (Kang 2010). We characterized VPREB1 deletions to determine if they are part of normal λ light chain rearrangement or represent a bona fide copy number alteration (CNA) that predicts worse outcome in pALL. We first used the genome-wide Molecular Inversion Probe (MIP) 330K Cancer Panel (Affymetrix) to identify VPREB1 deletions in a cohort of 52 pALL patients from the University of Utah (n=47 Precursor B-cell (Pre-B), 4 Precursor T-cell (Pre-T), 1 relapse Pre-B ALL). We found focal VPREB1 deletions in 25% (n=12/48) of Pre-B ALL and no (n=0/4) Pre-T ALL patients. We also identified a distinct and often homozygous deletion (14 kb in length) located ≂f 78 kb upstream of VPREB1 that could represent a potential enhancer region; this possible enhancer deletion was seen in 16.7% (n=8/48) of Pre-B and none of the Pre-T ALL patients. 50% of Pre-B ALL samples had at least one deletion of B-cell developmental gene (SPI1, IKZF1, BCL11A, TCF3, EBF1, PAX5, FOXP1, LEF1, VPREB1, BLNK), and of these, half (n=12/24) had deletion only of VPREB1 or its possible enhancer. We next sorted blood leukocytes from 10 healthy volunteers for quantitative PCR (qPCR) analysis on κ V-J junction (deletion=attempted κ rearrangement), λ V-J junction (deletion=attempted λ rearrangement), LOC96610 and PRAME (distal to VPREB1, among λ-light chain variable genes), VPREB1, and VPREB1 enhancer (proximal to VPREB1, among λ-light chain variable genes). As predicted for normal light chain rearrangement, we observed no deletions in monocytes; 100% of κ-sorted B-cells had κ V-J deletions without lambda deletions; and 100% of λ-sorted B-cells had both κ V-J and λ V-J deletions. We also analyzed 10 mature B-cell (Burkitt) lymphoma samples and observed VPREB1 deletions only in lambda-expressing samples with both κ and λ V-J deletions, and deletions always extended contiguously downstream to λ chain V-J region. We re-analyzed 30 Pre-B ALL samples (Utah cohort) and 11 Pre-B ALL cell lines by qPCR. Results validated MIP CNA patterns and differed from normal sorted B-cell and mature B-cell (Burkitt) results, displaying several types of rearrangements: 1) No light chain rearrangements with no VPREB1/enhancer deletion (n=5/30 [17%] clinical samples and 2/11 [18%] Pre-B cell lines), 2) Normal light chain (κ or κ/λ) rearrangement with no VPREB1/enhancer deletion (n=5/30 [17%] samples and 6/11 [55%] cell lines), 3) Normal light chain (κ or κ/λ) rearrangements with contiguous VPREB1/enhancer deletion (n=1/30 [3%] samples and no cell lines), 4) λ-light chain rearrangement with non-contiguous VPREB1/enhancer deletion (n=13/30 [43%] samples and 2/11 [18%] cell lines), and 5) No λ light chain rearrangement and focal VPREB1/enhancer deletion (n=6/30 [20%] samples and 1/11 [9%] cell lines). In the Utah Pre-B ALL cohort (n=47), VPREB1/enhancer deletions were significantly associated (Fisher's Exact Test, 2-tailed) with Hispanic ethnicity (p= 0.013), relapse (p= 0.027), death (p=0.026), and Day 14 M2 Marrow (5-25% blasts; p=0.038). Finally, we examined the publicly available St. Jude Children's Research Hospital ALL dataset (NEJM 351:533) and found VPREB1 under-expression associated with higher LC50 (resistance) for DNR (p=0.04) and VCR (p=0.04), but not PRED or ASP. In summary, we have shown VPREB1 deletions occur outside the context of normal light chain rearrangement. Unlike physiologic λ rearrangements, VPREB1 deletions do not always extend to the λ V-J junction in pALL. We also identified a focal, homozygous deletion just proximal to VPREB1 that may affect a possible enhancer region for this B-cell developmental gene. VPREB1/enhancer deletions were the only B-cell developmental gene lost in 25% of our cohort, perhaps defining a new Pre-B ALL subtype. Finally, deletion of VPREB1 (and its possible enhancer) predict worse clinical outcome and VPREB1 down-regulation correlates with in vitro drug resistance. Further studies of VPREB1's function in pediatric ALL may improve our understanding of leukemogenesis and could further refine clinical risk stratification. Disclosures: No relevant conflicts of interest to declare.