ALBERT

Description: The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is critical for normal hematopoietic stem cell development. EZH2 mediates transcriptional repression through histone tri-methylation (H3K27me3). The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis is becoming increasingly recognized. Its role in hematological malignancies however, is complex. Both gain-of-function and loss-of-function mutations have been respectively reported in lymphoma and leukemia, suggesting that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. Impaired self-renewal via EZH2 inhibition has been observed and offers a potentially attractive therapeutic approach in acute myeloid leukemia. Indeed, overexpression of EZH2 has been reported in patients with AML, particularly in those with complex karyotypes. In the present study, we show that deletion of EZH2 compromises the growth potential of AML cells by promoting their differentiation. To understand the role of EZH2 in vitro, we first examined the cell growth and colony-forming ability of EZH2 knockdown vs WT HL-60 cells. We found that proliferation of HL-60 cells was severely compromised following deletion of EZH2. Additionally, EZH2 deletion resulted in retarded cell-cycle entry and resulted in increased apoptotic cell death Similarly, the number of total colonies generated by EZH2 deleted cells in the secondary and tertiary re-plating assays was considerably less than that of controls. EZH2 deleted cells tended to form dispersed colonies that were mainly composed of differentiated myeloid cells, whereas control cells mostly formed compact colonies composed of myeloblasts. The proportion of dispersed colonies in the EZH2deleted cell culture increased with serial replatings. Deletion of EZH2 affects the growth and replating capacity of AML cell in vitro. When EZH2 deleted HL-60 cells were treated with the retinoid all-trans-retinoic acid (ATRA), we observed a marked induction of differentiation (as measured by the myeloid maturation marker CD11b) compared to the effects of ATRA on differentiation in wild type (WT) cells. Similarly, impaired clonogenic survival was more pronounced following ATRA treatment in EZH2 deleted vs WT HL-60 cells (see figure). We then profiled a number of small molecule inhibitors of EZH2 alone (EPZ005687, EPZ-6438, GSK126, El1, DZNeP, UNC1999 and GSK343) and in combination with ATRA, confirming these phenotypic changes. To elucidate the mechanism for how EZH2 regulates the balance of self-renewal vs differentiation in AML, we examined the genome-wide distribution of H3K27me3 by ChIP-seq analysis. First, western blot analysis revealed a marked decrease in the levels of H3K27me3 in EZH2 deleted AML cells. Next, we examined the presence of H3K27me3 marks in leukemia cells purified by ChIP-seq analysis. We focused on the region from 5.0 kb upstream to 3.0 kb downstream of transcription start sites (TSSs) of reference sequence (RefSeq) genes (http://www.ncbi.nlm.nih.gov/RefSeq/) because H3K27me3 marks are usually enriched near TSSs or across the body of genes. As expected, the deletion of EZH2 caused a drastic reduction in these H3K27me3 marks. Targeting EZH2 presents and interesting dichotomy as a novel drug target since inhibition of this protein could potentially be beneficial or detrimental depending on the context of the disease. In the case of AML, EZH2 mutations likely impede differentiation and block retinoic acid led differentiation programs. Updated studies outlining the interaction between the retinoic acid signaling pathway and EZH2 will be presented. These studies justify clinical investigation of EZH2 inhibitors combined with ATRA for patients with AML. Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Figure 1. Knockdown of EZH2 (C) promotes differentiation of AML cells (A), impairs clonogenic survival and synergistically enhances the anti-leukemic effects of the retinoid all-trans-retinoic acid (ATRA) (B). Disclosures No relevant conflicts of interest to declare.

Description: Background Protein arginine methyltransferase 5 (PRMT5) is the primary enzyme responsible for symmetric arginine dimethylation of multiple proteins that impact cell proliferation. Its substrates include proteins involved in mRNA splicing, signal transduction, gene transcription, and DNA repair. PRMT5 overexpression occurs in many cancers and correlates with poor prognosis. GSK3326595 is a potent, specific, and reversible inhibitor of PRMT5 that inhibits proliferation and induces cell death in a broad range of solid and hematologic tumor cell lines. It also exhibits potent anti-tumor activity in vivo in animal models, including in preclinical models of myeloid malignancies. One mechanism of action of GSK3326595 is via inhibition of cellular mRNA splicing and upregulation of tumor suppressor function. Mutations in splicing factors are frequent in myeloid malignancies (including approximately 40% of patients with myelodysplastic syndrome [MDS], and over 60% of patients with chronic myelomonocytic leukemia [CMML]), and further inhibition of mRNA splicing via GSK3326595 may lead to a synthetic lethal phenotype specifically in splicing mutant disease. Study 208809 is the first trial of a PRMT5 inhibitor in participants with myeloid malignancies. Methods Study 208809 is a Phase I/II study to evaluate the safety, tolerability, and clinical activity of GSK3326595 monotherapy in participants with relapsed and refractory MDS, CMML, and hypoproliferative acute myeloid leukemia (AML) that has evolved from an antecedent MDS. Part 1 will identify a tolerated dose and establish preliminary evidence of efficacy in this population. At the end of Part 1, if pre-specified criteria are met, then the study will be expanded with three additional Parts that will be opened in parallel. Part 2A is a Phase II randomized comparison of monotherapy GSK3326595 versus investigator's choice of best available care in participants with relapsed and refractory MDS, CMML, and hypoproliferative AML. Part 2B is a single-arm investigation of safety and efficacy of GSK3326595 plus 5-azacitidine in participants with newly diagnosed high-risk MDS. Part 2C is a single-arm investigation of the safety and efficacy of monotherapy GSK3326595 in participants with relapsed or refractory AML whose tumors harbor mutations in components of the pre-mRNA splicing machinery. All participants enrolled in this study have a diagnosis of MDS, CMML, or AML, with enrollment into each cohort as defined above. Participants are adults with adequate organ function as defined in the protocol. Prior allogeneic transplant is permitted. There are no required biomarkers for enrollment to Parts 1, 2A, and 2B, though central confirmation of pre-mRNA splicing factor mutations will be performed to stratify participants for overall analysis. Enrollment to Part 2C is limited to participants with splicing factor mutations. It is estimated that a maximum of 302 participants will be enrolled in the study, divided as follows: Approximately 41 participants in Part 1, approximately 192 participants in Part 2A, approximately 41 participants in Part 2B, and approximately 28 participants in Part 2C. In Part 1, the primary endpoint is clinical benefit rate, as defined as the percentage of participants achieving a complete remission, complete marrow remission, partial remission (PR), stable disease lasting at least 8 weeks, or hematologic improvement, as per standard criteria. In Part 2A, the primary endpoint is overall survival. In Part 2B and Part 2C, the primary endpoint is overall response rate (ORR), defined as the percentage of participants achieving a PR or better. Samples are collected to evaluate symmetric dimethylated arginine (SDMA), the enzymatic product of PRMT5. This has been demonstrated to be a pharmacodynamic marker of PRMT5 inhibition in plasma and tumor tissue. In addition, participants will be stratified based on the presence or absence of spliceosome mutations and analyzed separately to evaluate the effect of these mutations on clinical activity. As of 1 August 2019, recruitment is ongoing across six centers in the United States and Canada; ten participants have been enrolled, all into Part 1. ClinicalTrials.gov identifier: NCT03614728 Study is funded by GlaxoSmithKline Disclosures Watts: Takeda: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bradley:AbbVie: Other: Advisory Board. Brunner:Novartis: Research Funding; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Minden:Trillium Therapetuics: Other: licensing agreement. Papadantonakis:Agios: Consultancy, Honoraria. Abedin:Actinium Pharmaceuticals: Research Funding; Pfizer Inc: Research Funding; Helsinn Healthcare: Research Funding; Agios: Honoraria; Jazz Pharmaceuticals: Honoraria. Baines:GlaxoSmithKline: Employment, Equity Ownership. Barbash:GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties, Research Funding. Gorman:GlaxoSmithKline: Employment, Equity Ownership. Kremer:GlaxoSmithKline: Employment, Equity Ownership. Borthakur:Cantargia AB: Research Funding; Eisai: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Xbiotech USA: Research Funding; Novartis: Research Funding; Oncoceutics: Research Funding; Oncoceutics, Inc.: Research Funding; PTC Therapeutics: Consultancy; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agensys: Research Funding; AstraZeneca: Research Funding; Bayer Healthcare AG: Research Funding; BMS: Research Funding; Eli Lilly and Co.: Research Funding; NKarta: Consultancy; Cyclacel: Research Funding; GSK: Research Funding; Janssen: Research Funding; Incyte: Research Funding; AbbVie: Research Funding; Merck: Research Funding; Arvinas: Research Funding; Polaris: Research Funding; Strategia Therapeutics: Research Funding.

Description: Background: Isocitrate dehydrogenase 1 mutations (IDH1m) occur in 7-14% of AML patients (pts) and approximately 3-4% of MDS pts. Olutasidenib is a highly potent, selective small molecule inhibitor of IDH1m with the therapeutic potential to restore normal cellular differentiation. Azacitidine (AZA) has shown synergistic effects with IDHm inhibitors on releasing differentiation block in IDHm leukemia models in vitro. Methods: The Phase 1 study (NCT02719574) assessed the safety, PK/PD, and clinical activity of olutasidenib in patients with IDH1m AML or MDS. Eligibility criteria included: IDH1m AML or MDS [relapsed/refractory (R/R) or treatment naïve (TN) pts not eligible for or refusing standard therapy], adequate liver and renal function. There were no restrictions for concomitant non-anticancer medications. IDH1m variant allele frequency (VAF) and co-mutations were measured at baseline and during treatment. Available safety data are presented for all pts (AML/MDS); efficacy data are presented for AML pts only. Results: As of April 12, 2019, 32 pts had been treated with single agent (SA) olutasidenib and 46 pts with olutasidenib in combination (COMBO) with AZA; median time on treatment was 4.2 mo for SA (range: 27 mo); disease control (SD 〉13 mo) was observed in pts without an IWG-defined response. Of the 59 AML pts (23 SA; 36 COMBO) who were transfusion-dependent at baseline, 26 (11 [48%] SA; 15 [42%] COMBO) and 21 (9 [39%] SA; 12 [33%] COMBO) became transfusion-independent (seen in all response categories) during 28 and 56 days on treatment, respectively. For R/R AML pts, median survival was 8.7 mo for SA and 12.1 mo for COMBO; for TN AML pts, median survival was 8.8 mo for SA (n=4) and not reached for COMBO. For R/R and TN AML patients with available pre- and on-treatment samples, IDH1m clearance or significant reduction (VAF

Description: Background: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in ~3% of individuals with myelodysplastic syndrome (MDS) and have been associated with increased transformation to acute myeloid leukemia (AML). Ivosidenib (AG-120) is an oral, potent, targeted inhibitor of the mutant isocitrate dehydrogenase 1 enzyme (mIDH1) and is approved in the US for the treatment of newly diagnosed AML with a susceptible IDH1 mutation in patients ≥75 years of age or who have comorbidities that preclude the use of intensive induction chemotherapy, and in adult patients with relapsed or refractory (R/R) AML. The first-in-human, phase 1 dose escalation and expansion study of ivosidenib (NCT02074839) enrolled adults with mIDH1 advanced hematologic malignancies, including R/R MDS, and the study is ongoing. In the initial phase of the study (DiNardo et al. N Engl J Med 2018), the 12 patients with R/R MDS received 500 mg ivosidenib once daily and were characterized as follows: 75% were male, median age was 72.5 years (range 52-78), and 42% were ≥75 years of age; median number of prior therapies was 1 (range 1-3). Adverse events (AEs) of any grade, irrespective of causality, occurring in ≥20% of the 12 patients were diarrhea, fatigue, back pain, rash (n=4 each, 33.3%), anemia, urinary tract infection, decreased appetite, hypokalemia, arthralgia, dyspnea, pruritus, and hypotension (n=3 each, 25.0%). No AEs led to permanent discontinuation of treatment. Response was assessed according to International Working Group 2006 criteria for MDS. According to investigators, five of 12 patients achieved complete remission (CR) (41.7%; 95% CI 15.2%, 72.3%); median duration of CR was not estimable for these patients (95% CI 2.8 months, not estimable). Nine of 12 patients were transfusion independent for at least 56 days during study treatment. Mutation clearance was observed in one of the 5 CR patients. Here we report the design of a new sub-study of this trial, which is being undertaken to further assess the safety, tolerability, and clinical activity of treatment with ivosidenib in patients with R/R MDS. Methods: This sub-study is evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of ivosidenib. Adults with R/R MDS with an IDH1 mutation will be enrolled in the MDS sub-study. These individuals must have R/R disease after treatment with standard agents indicated for MDS. Eligible patients must have a platelet count of ≥20,000/μL, and adequate hepatic function (total bilirubin ≤1.5 × upper limit of normal [ULN]; aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase ≤3.0 × ULN) and renal function (serum creatinine ≤2.0 × ULN or creatinine clearance 〉40 mL/min). Additional key inclusion criteria are bone marrow blasts 〉5% and/or transfusion dependence. Ivosidenib is to be administered at a dose of 500 mg once daily orally on Days 1 to 28 of 28-day cycles. The addition of the MDS sub-study to this phase 1 clinical study in patients with hematological malignancies will provide additional insights into the use of ivosidenib for the treatment of mIDH1 R/R MDS. Disclosures Foran: Agios: Honoraria, Research Funding. DiNardo:notable labs: Membership on an entity's Board of Directors or advisory committees; medimmune: Honoraria; daiichi sankyo: Honoraria; abbvie: Consultancy, Honoraria; agios: Consultancy, Honoraria; jazz: Honoraria; celgene: Consultancy, Honoraria; syros: Honoraria. Watts:Takeda: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Stein:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. De Botton:Daiichi Sankyo: Consultancy; Astellas: Consultancy; Bayer: Consultancy; AbbVie: Consultancy; Syros: Consultancy; Forma: Consultancy, Research Funding; Janssen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Servier: Consultancy; Pierre Fabre: Consultancy; Celgene: Consultancy, Speakers Bureau; Agios: Consultancy, Research Funding. Fathi:Amphivena, Kite, Jazz, NewLink Genetics,: Honoraria; Agios, Astellas, Celgene, Daiichi Sankyo, Novartis, Takeda, Amphivena, Kite, Forty Seven,Trovagene, NewLink genetics, Jazz, Abbvie, and PTC Therapeutics: Consultancy. Stein:Stemline: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Speakers Bureau. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Patel:France Foundation: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dava Oncology: Honoraria. Tallman:UpToDate: Patents & Royalties; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biosight: Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees. Choe:Agios: Employment, Equity Ownership; Agios: Employment, Equity Ownership. Wang:Agios: Employment, Equity Ownership. Zhang:Agios: Employment, Equity Ownership; Agios: Employment, Equity Ownership. Fan:Agios: Employment, Equity Ownership. Yen:Agios: Employment, Equity Ownership. Oluyadi:Agios: Employment, Equity Ownership. Winkler:Agios: Employment. Hickman:Agios: Employment, Equity Ownership. Agresta:Agios: Employment, Equity Ownership. Liu:Agios: Employment, Equity Ownership. Wu:Agios: Employment, Equity Ownership. Attar:Aprea Therapeutics: Employment; Agios: Employment, Equity Ownership. Kantarjian:Astex: Research Funding; Takeda: Honoraria; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immunogen: Research Funding; AbbVie: Honoraria, Research Funding; Jazz Pharma: Research Funding; Agios: Honoraria, Research Funding; Ariad: Research Funding; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Novartis: Research Funding; Pfizer: Honoraria, Research Funding. OffLabel Disclosure: Ivosidenib (AG-120) is an IDH1 inhibitor indicated for the treatment of AML with a susceptible IDH1 mutation as detected by an FDA-approved test in: 1) adult patients with newly-diagnosed AML who are more than 75 years old or who have comorbidities that preclude use of intensive induction chemotherapy and 2) adult patients with relapsed or refractory AML. It is being evaluated in clinical trials for mutant IDH1 advanced hematologic malignancies.

Description: Background: Some patients receiving a tyrosine kinase inhibitor (TKI) for chronic phase (CP) Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) experience drug intolerance and require switching to an alternative TKI. Bosutinib, a TKI approved for newly diagnosed CP Ph+ CML and Ph+ CML resistant/intolerant to prior therapy, has a distinct adverse event (AE) profile vs other TKIs used to treat Ph+ CML. Methods: The ongoing phase 4 BYOND study (NCT02228382) is further evaluating efficacy and safety of bosutinib for CML resistant/intolerant to prior TKIs. Patients with Ph+ CP CML (n=156) previously treated with imatinib, dasatinib, and/or nilotinib received bosutinib (starting dose 500 mg once daily). Cross-intolerance (AEs leading to permanent discontinuation of both prior TKI and bosutinib), recurrent AEs (grades 1/2 or 3/4), and bosutinib dose modifications due to recurrent AEs were assessed across AEs and AE clusters. This analysis was based on ≥1 year after the last enrolled patient (median treatment duration 23.7 months [range 0.2-42.2]). Results: Of 141, 95, and 79 patients who received prior imatinib, dasatinib, or nilotinib, respectively, 63 (45%), 70 (74%), and 60 (76%) were intolerant and discontinued treatment due to an AE as the primary reason. 1 (2%) imatinib-intolerant and 5 (7%) dasatinib-intolerant patients had cross-intolerance with bosutinib; no cross-intolerance with bosutinib was reported for the 60 nilotinib-intolerant patients (Table). 15 patients discontinued 〉1 TKI due to the same AE. Of these, only 1 with prior imatinib and dasatinib intolerance due to anemia (below), was cross-intolerant to bosutinib. No deaths occurred due to cross-intolerance to bosutinib. Imatinib-intolerant: The most common cause of imatinib intolerance was musculoskeletal pain in 19 patients. Of these, 9 patients experienced grade 1/2 recurrence and 1 had a grade 3/4 AE. There was no cross-intolerance with bosutinib due to musculoskeletal pain. 6 patients were intolerant due to edema, which recurred as grade 1/2 in 3 bosutinib-treated patients. In 2 patients with intolerance due to diarrhea, both had grade 1/2 diarrhea with bosutinib. 2 patients were imatinib-intolerant due to neutropenia or thrombocytopenia (n=1 each) and experienced grade 3/4 recurrence with bosutinib. 1 patient was cross-intolerant to bosutinib due to anemia. Dasatinib-intolerant: The most common reason for prior dasatinib intolerance was pleural effusion. Of 36 patients, 10 (28%) had recurrence of grade 1/2 AE and 4 (11%) had grade 3/4 AE with bosutinib that caused dose delay and reduction in 6 (17%) and 4 (11%) patients, respectively. 1 patient had cross-intolerance with bosutinib due to pleural effusion. Of 8 patients dasatinib-intolerant due to dyspnea, 1 had cross-intolerance with bosutinib (this patient also developed pleural effusion with dasatinib). Of 2 patients with dasatinib intolerance due to pulmonary hypertension, 1 had grade 1/2 and 1 had grade 3/4 recurrence with bosutinib, leading to cross-intolerance in 1 patient. 2 other dasatinib-intolerant patients experienced cross-intolerance due to anemia and nausea (n=1 each). Grade 3/4 recurrence of thrombocytopenia was experienced in 1 of 3 patients with prior dasatinib-intolerance. 1 patient with dasatinib intolerance due to diarrhea had grade 3/4 diarrhea with bosutinib that was managed with dose delay/reduction. Nilotinib-intolerant: 6 patients discontinued nilotinib due to peripheral ischemia, of whom 1 experienced grade 3/4 recurrence with bosutinib. None of the 4 patients with nilotinib intolerance due to acute coronary syndrome had recurrence with bosutinib. Of 3 patients with prior intolerance due to pancreatitis, 1 had grade 3/4 recurrence with bosutinib. 2 patients had recurrent diarrhea with bosutinib, both grade 1/2. There were no bosutinib dose reductions, delays, or recurrence of AEs in patients with prior nilotinib intolerance due to rash, hematologic AEs, hepatotoxicity, or metabolic disorders. Conclusions: Incidence of cross-intolerance, dose delay, or dose reduction with bosutinib in patients intolerant to prior TKIs was low. Despite recurrence of certain same grade 1/2 or grade 3/4 AEs that caused prior TKI intolerance, these were manageable and patients were able to remain on bosutinib. These findings support the use of bosutinib in patients with CP Ph+ CML intolerant to prior TKI treatment. Disclosures Gjertsen: Haukeland University Hospital / University of Bergen: Employment; ERA PerMed: Research Funding; BerGenBio: Consultancy; Astellas: Consultancy; BerGenBio AS: Membership on an entity's Board of Directors or advisory committees; KinN Therapeutics AS: Equity Ownership; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy; ACTII AS: Equity Ownership; Seattle Genetics: Consultancy; Research Council of Norway: Research Funding; EU Horizon 2020: Research Funding; The Norwegian Cancer Society: Research Funding; Helse Vest Health Trust: Research Funding. Hochhaus:Pfizer: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding; Novartis: Research Funding. Rosti:BMS: Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Watts:Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees. Ortí:Bristol-Myers Squibb: Consultancy, Other: Travel Expenses, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy, Other: Travel Expenses, Speakers Bureau. le Coutre:Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Leip:Pfizer: Employment, Equity Ownership. Viqueira:Pfizer Inc: Employment, Equity Ownership. Cortes:Biopath Holdings: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Sun Pharma: Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; BiolineRx: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding. Giles:Novartis: Consultancy; Epigene Therapeutics Inc: Consultancy, Other: leadership, stock/other ownership ; Actuate Therapeutics Inc: Employment. Gambacorti-Passerini:Pfizer: Honoraria, Research Funding; Bristol-Meyers Squibb: Consultancy.

Description: After failure of DNA methyltransferase inhibition (DNMTi) there is no standard of care therapy for high-risk myelodysplastic syndromes (MDS), and median survival for higher risk disease is less than 6 months (Prebet et al, JCO 2011; Jabbour et al, Cancer 2015). Pevonedistat, a first in human small molecule inhibitor of the NEDD8 activating enzyme (NAE), downregulates Cullin ring ligases (CRL) which interferes with the shuttling and degradation of proteins in the proteasome and leads to accumulation of CRL substrates. Combining pevonedistat (Pev) with azacitidine (AZA) resulted in synergistic cell killing in in vitro and xenograft models of acute myeloid leukemia (AML) (Smith et al, Blood 2011), elicited favorable response rates in treatment naïve elderly or unfit AML patients (Swords et al Blood 2018), and is currently under study in treatment-naïve MDS. The study presented herein (NCT03238248) investigates the utility of adding pevonedistat to azacitidine (PevAz) after DNMTi failure in MDS and MDS/MPN overlap syndromes. Methods: In this on-going single-arm phase II study, MDS and MDS/MPN patients were eligible if they were refractory to DNMTi treatment, progressing after at least 2 cycles of therapy; had failed to achieve a complete remission (CR) after at least 4 cycles of DNMTi therapy; or had relapsed after an initial response to DNMTi therapy. Enrolled subjects received AZA 75mg/m2 sc/iv daily on days 1-5 and Pev 20mg/m2 iv on days 1, 3 and 5 of each 28-day cycle. Survival is the primary endpoint and is assessed at regularly scheduled study visits and every 3 months after ending protocol-directed therapy. Hematologic and bone marrow response rates are secondary endpoints. Responses to treatment are determined by the MDS International Working Group (IWG) response criteria (Cheson et al, Blood 2006) or for MDS/MPN, by the modified MDS/MPN IWG response criteria (Savona et al, Blood 2015). Results: As of the data cutoff on 15 MAR 2019, 23 subjects (21 with MDS, 2 with MDS/MPN) had enrolled and initiated treatment. Subjects had previously been treated with AZA (n=11/23), decitabine (n=11/23), and ASTX727 (n=4/23); some subjects had been treated with more than one DNMTi prior to enrollment. Median number of cycles of any prior DNMTi therapy was 7 (range 2-35). 65% of subjects were female. Median age at enrollment was 67 years (range 51 - 85). 65% had Intermediate-2 or High risk disease by IPSS at time of enrollment. Median number of PevAz cycles completed prior to the data cutoff was 4 (range 1-19). One subject had not reached the first response assessment at the time of the data cutoff and data was unavailable for one subject. The overall response rate including complete and partial remission, hematologic improvement and clinical benefit (CB) was 42.9% (9/21), and CR rate (including 1 CR + 4 marrow CR) was 23.8% (5/21) with a median duration of response (DOR) of 8.7m (range 2.8m-15.7m). An additional 38.1% (8/21) had stable disease as best response (Table 1). The most common Grade 〉2 adverse events (any attribution) include thrombocytopenia (39%), anemia (35%), leukopenia (26%), neutropenia (22%), infections (17%), and febrile neutropenia (13%). Six subjects experienced Grade ≤ 2 elevations in AST/ALT and 4 had Grade ≤ 2 elevation in bilirubin, whereas only one subject experienced Grade 〉 2 LFT abnormality (increase in ALT). There was one death on study due to intracerebral hemorrhage related to a previously undiagnosed metastatic carcinoma. PevAz treatment was discontinued in other subjects due to disease progression (n=7), adverse event (n=1), lack of response (n=1), or to pursue allogeneic stem cell transplant after achieving a satisfactory response to PevAz (n=3). Ten subjects were continuing PevAz therapy on study as of the data cutoff. Summary: PevAz was well-tolerated in MDS and MDS/MPN patients who had previously failed DNMTi, with the most common adverse events of cytopenias, which are a common feature of these diseases. 5/21 subjects achieved CR/mCR with meaningful DOR, and the ORR of 42.9% exceeded expectations for MDS patients with previous failure of DNMTi therapy; both MDS/MPN patients responded with CR and CB. For these patients whose treatment options are limited and prognosis very poor, these preliminary data are especially encouraging and warrant further investigation. This therapy combination is being tested in a phase 3 study in treatment naïve high risk MDS, CMML and low-blast AML. Disclosures Watts: Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Research Funding. Strickland:Astellas Pharma: Consultancy; Sunesis Pharmaceuticals: Research Funding; AbbVie: Consultancy; Jazz: Consultancy; Kite: Consultancy; Pfizer: Consultancy. Byrne:Karyopharm: Research Funding. Bradley:AbbVie: Other: Advisory Board. Savona:Sunesis: Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties.

Description: Background: Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell disorders with risk of progression to AML. Isocitrate dehydrogenase 1 mutations (IDH1m) occur in approximately 3-4% of MDS patients (pts). Olutasidenib is a highly potent, selective small molecule inhibitor of IDH1m that has shown clinical activity in AML (De Botton, 2019). Methods: The ongoing Phase 1/2 study (NCT02719574) has evaluated the safety, PK/PD, and clinical activity of olutasidenib alone or in combination with azacitidine (AZA) or cytarabine in IDH1m AML/MDS pts. Safety for all pts and efficacy for evaluable pts with MDS in Phase 1 and 2 are reported here. IDH1m variant allele frequency (VAF) was measured at baseline and on treatment by central droplet digital PCR (ddPCR) assay (peripheral blood). Duration of FT-2102 exposure was calculated using Kaplan-Meier methodology; patients on ongoing treatment were censored at the data cutoff date. Results: As of the 19-Jun-2019 data cutoff date, 20 pts with IDH1m MDS had received olutasidenib continuously either as a single agent (SA; n=6) or in combination (COMBO; n=14) with AZA (75 mg/m2 × 7 days q 4 weeks). The study population (3 intermediate risk, 12 high risk, 4 very high risk MDS and 1 missing at data cutoff) included 7 newly diagnosed pts and 13 relapsed/refractory pts who had received a median of 1 prior regimen (maximum 4). Eleven pts received prior hypomethylating agents for a median 7.8 months and 2 pts were missing prior treatment records. The median time on treatment for all MDS pts treated with olutasidenib +/- AZA was 8 months (range:

Description: Introduction - The Myc proteins are transcription factors that have essential roles in cell growth and proliferation by both positively and negatively regulating gene expression. Mutation, amplification, or activation of the MYC oncogene family is one of the most frequent events associated with cancer. In acute myeloid leukemia (AML), c-Myc is commonly activated and plays an important role in the initiation and maintenance of the disease. In particular, c-Myc is upregulated by activating mutations of the Flt3 receptor tyrosine kinase, one of the most prevalent types of mutations in AML, and also by the AML-associated fusion proteins AML1-ETO, PML/RARα, and PLZF/RARα. It has also been shown that c-Myc is negatively regulated by C/EBPα, a transcription factor essential for granulocytic differentiation, and that c-Myc expression is elevated in myeloid leukemias in which C/EBPα is mutated. Additionally, c-Myc is stabilized in AML with mutations leading to aberrant cytoplasmic localization of nucleophosmin (NPM), the most frequent genetic alteration in AML without karyotypic aberrations. Importantly, the MYC gene itself, located at 8q24, has been found to be one of the most commonly amplified regions in AML. Lastly, the importance of c-Myc in myeloid leukemogenesis has been further demonstrated by the induction of myeloid leukemias in mouse models overexpressing c-Myc in bone marrow progenitors. Methods - In the current study, we disrupt MYC transcription in AML blasts, by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains, specifically BRD4) of putative coactivator proteins implicated in transcriptional initiation and elongation. Results - Following extensive chemical optimization and biological characterization, we identified two advanced pre-clinical candidates, EP11313 and EP11336. These compounds have excellent affinity for BRD4 and achieve high in-vivo exposure in animal models. Both agents possess drug-like physical characteristics, and their pharmacologic properties compared favorably to the BRD4 inhibitor IBET-762 (GSK525762A). Respectively for IBET-762, EP11313 and EP11336 we calculated BRD4 IC50 (23, 7, 5 nM), BRD4 KD (55, 2, 6 nM), molecular weight (424, 415, 445 Da), solubility (194, 20, 85 μM), cLogP (1.8, 3.7, 3.0), PSA (81, 78, 98 Ǻ2), MDR-MDCK Efflux ratio BA/AB (27.9, 1.3, 1.9) and MDR-MDCK Papp (1.1, 7.3, 10.2). In vitro, both molecules have nanomolar anti-leukemic activity across a panel c-myc dysregulated AML cell lines (OCI-AML2, OCI-AML3, KG-1a, HL-60, MV-411 and NB-4). Growth inhibition was variable and context dependent (GI50 range: 265 nM - 1618 nM). Following a brief incubation in-vitro, both EP11313 and EP11336 lead to activation of caspase 3 and initiation of apoptotic cell death. Over-expression of c-myc is known to impair myeloid differentiation in response to the retinoid all-trans-retinoic acid (ATRA) in AML, considering this and the research interests of our group in broadening the anti-leukemic effects of ATRA, we showed that ATRA combined with bromodomain inhibition led to greater cell kill in vitro than was observed for ATRA, or either bromodomain inhibitor tested alone (see figure). Conclusions - Both EP11313 and EP11336 are currently in late pre-clinical development, and are being optimized for first in man studies in AML and other tumor types. Updated information on the in-vivo activity of these compounds and mechanism of action in AML will be presented. Figure 1. The BET bromodomain inhibitors EP11313 and EP11336, impair the viability of AML blasts in vitro and have synergistic activity when combined with the retinoid all-trans -retinoic acid (AtRA). MV-411 cells were incubated with the indicated concentrations of EP11313, EP11336 and AtRA for 48 hours. Cell viability was measured using a bioluminescence assay (CellTiter-Glo¨, left) and induction of caspase 3, was measured by FACS (right). Growth inhibition (GI) in OCI-AML3 cells was calculated using linear extrapolation (top). Figure 1. The BET bromodomain inhibitors EP11313 and EP11336, impair the viability of AML blasts in vitro and have synergistic activity when combined with the retinoid all-trans -retinoic acid (AtRA). MV-411 cells were incubated with the indicated concentrations of EP11313, EP11336 and AtRA for 48 hours. Cell viability was measured using a bioluminescence assay (CellTiter-Glo¨, left) and induction of caspase 3, was measured by FACS (right). Growth inhibition (GI) in OCI-AML3 cells was calculated using linear extrapolation (top). Disclosures Johnstone: Epigenetix: Employment. Albert:Epigenetix: Employment. Collard:Epigenetix: Employment.

Description: Introduction: There is a paucity of data on the distribution of acute leukemia (AL) across ethnic groups. Historically, B-cell acute lymphoblastic leukemia (B-ALL) and acutepromyelocytic leukemia (APL) are over-represented in Hispanic patients, while acute myeloid leukemia (AML) is less frequent. We analyzed the ethnic distribution of AL in Florida based on incidence rates and nativity differences using Florida Cancer Data System (FCDS). In our study, B-ALL and APL were more common in Hispanics vs. non-Hispanics (IRR of 1.627 and 1.302, respectively); however, contrary to prior reports, AML was also more common (IRR 1.533) (Swords et al, BCJ 2016, in press). No nativity differences were observed. Given this novel finding of increased AML incidence in Hispanics in South Florida, we examined AML patients at Sylvester Comprehensive Cancer Center (SCCC) to look for ethnic differences in mutational frequency. Methods: We obtained molecular genetic data on AML patients treated from 2012-2015 across multiple centers.Genomic DNA from bone marrow or peripheral blood was sequenced for ASXL1, DNMT3A, FLT3 TKD, IDH1, IDH2, KIT, NPM1, PHF6, TET2 on the IlluminaMiSeq platform using a lower limit of detection of 5% and minimum coverage of 500X. Alignment and variant calling were performed usingNextGENe® software as previously described byGenoptix, Inc. MLL PTD, FLT3 ITD and CEBPA mutations were detected by PCR amplification followed by fragment analysis.For patients treated at SCCC, we obtained IRB approval to conduct retrospective chart reviews for clinical and pathologic data. Categorical data was analyzed by Fisher exact test or Chi-square test as appropriate. Continuous data was analyzed by independent t-test and Wilcoxon-Mann Whitney test for parametric and non-parametric data, respectively. Statistical analysis was performed using Stata (version 13.0).Circos plots were created usingCircos. Results: We examined the 11-gene mutational profile of 927 unique AML patients. The frequency of these mutations was consistent with previous reports (Park et al, NEJM 2012). DNMT3A, NPM1, and FLT3 were the most common abnormalities (Figure 1A). 75 of these 927 patients were treated at our center. Of these, median age was 60.2 years; 53.3% were female; 80% were white, 13.3% black,6.7% other. 68% (n=51) self-identified as non-Hispanic and 32% (n=24) as Hispanic (Table 1). Median WBC was 3.95 K/µL (1.4-100.7); 37% had antecedent MDS or MDS-related changes; 8% had favorable-, 69% intermediate-, and 23% poor-risk cytogenetics. There were no differences in WBC, MDS, or cytogenetic risk by ethnicity. In the 75 patient SCCC cohort, TET2 (n=14, 18.7%), CEBPA (n=14, 21.9%), and DNMT3A (n=11, 14.7%) were the most common mutations. 16 patients had an IDH mutation (10 IDH1 and 6 IDH2). We found no statistically significant difference in mutation frequency in Hispanic vs. non-Hispanic patients (Figure 1B); however, there was a non-significant increase in MLL-PTD mutations in Hispanic patients (15.8% vs. 9.3%, p=0.665) and IDH1 mutations in non-Hispanics (11.8% vs. 0%, p=0.168). Conclusions: We present one of the largest examinations to date (n=927 patients) of mutational frequency in AML. Our findings corroborate prior literature; however, we did note the rare co-occurrence of TET2 and IDH mutations-showing the 2 are not always mutually exclusive. We examined demographic data on a subset of these patients treated at SCCC, where we have a large Hispanic population, and assessed for ethnic differences in mutational frequency. We found a suggestion of increased MLL PTD mutations, a poor-risk abnormality, in Hispanic patients, and increased IDH1 mutations in non-Hispanics, but found no significant differences. This may be due to our relatively small sample size. We are now examining the full 927 patient cohort for demographic data and updated results will be presented. If certain AML mutations cluster by ethnicity, this might explain differing incidence rates and outcomes in Hispanics, and examination of predisposing heritable or environmental factors should be pursued. Table 1. Distribution of mutations in an 11-gene AML molecular profile by ethnicity in 75 patients with AML. Figure 1.Circosplots characterizing the distribution of concomitant mutations in 927 patients with AML (A); and the incidence of mutations by ethnicity in 75 patients with AML (B). Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Vaupel: Genoptix, a Novartis Company: Employment. Hall:Genoptix, a Novartis Company: Employment.

Description: Introduction - With rapidly advancing sequencing technology, the extent of genetic diversity in AML has never been more apparent. A Òone size fits allÓ approach can no longer be justified. Sequencing studies have also uncovered several actionable targets, yet no targeted therapies are FDA approved for use in the US. AML therefore, has significant potential for personalized therapeutics. Several challenges exist, however. The masses of data generated by high-throughput technologies are challenging to manage, visualize, and convert to knowledge required to improve outcomes. A cross-disciplinary systems biology effort is required, to visualize inter-connected events within leukemic blasts that ultimately contribute to the disease phenotype and inform on rational selection of therapeutic approaches. In the current study, we outline a complimentary functional and genomic screening approach to identify clinical drug candidates for re-purposing in patients with relapsed refractory AML. Methods - In this proof-of principle study, we optimized an ex vivo high-throughput drug screening platform measuring AML cell survival after exposure to over 200 U.S. FDA approved oncology drugs (including conventional chemotherapeutics, proteasome inhibitors, anti-metabolites, transcriptional inhibitors and targeted kinase inhibitors). This multiplex assay is designed for individual AML patients and tests agents over a 10,000-fold concentration range. We screened patient derived blasts against normal bone marrow mononuclear cells to identify the most effective leukemia selective agents. To compliment this functional ex-vivo screen, we employed a genomics approach using predictive simulation software to generate patient specific avatars which map individual dysregulated and interconnecting signaling pathways. The avatar technology will then identify candidate agents at critical impact points within these pathways. Results - Through phenotype screening of primary cells collected from a highly refractory AML patient (patient # PD001), our ex vivo assay identified a list of drugs based on their ability to effectively and selectively reduce the viability of the patients leukemic blasts in culture. Candidate agents are listed according to selective drug sensitivity scores (sDSS), calculated based on the comparative ability of each drug to reduce viability of primary cells vs normal bone marrow mononuclear cells. The highest sDSS indicates the most selective and effective drugs for each individual patient (Figure 1, patient sample PD001). The patient specific avatar generated for patient PD001 identified a series of dysregulated pathways converging on cell proliferation and viability. Both functional and genomic approaches identified the tyrosine kinase inhibitor ponatinib, as a potentially relevant clinical candidate. Potentially effective combination approaches were also predicted (e.g. ponatinib with rosuvastatin, ponatinib with decitabine). Since our ex vivo assay identified bortezomib (Velcade) as a clinical candidate and since we successfully negotiated off-label use of this agent, we selected Velcade for a therapeutic trial in patient PD001. After three doses of Velcade at 1.5 mg/m2 (Days 1,4 and 8), serial blood counts revealed a dramatic fall in total white count and circulating blasts (96% to 20%) (Figure 2). This is noteworthy since single agent Velcade is generally not capable of producing clinically meaningful responses for patients with refractory AML. Conclusions - Our study justifies continued development of this novel, iterative functional/genomics approach to personalized therapeutics in AML. Our model identifies candidate drugs that can be readily re-purposed for immediate clinical use, whilst at the same time providing insights into underlying mechanism of action, informing on rationally designed combination strategies and biomarker candidates. Figure 1. Ex vivo drug screening results from refractory patient_PD001 identifies the proteasome inhibitor bortezomib (Velcade) as one of the top selective and effective drugs. The higher the sDSS, the more effective and selective the drug is. Figure 1. Ex vivo drug screening results from refractory patient_PD001 identifies the proteasome inhibitor bortezomib (Velcade) as one of the top selective and effective drugs. The higher the sDSS, the more effective and selective the drug is. Figure 2. Clinical follow-up of AML patient-PD001 shows chemosensitivity to bortezomib (Velcade). Arrows arrows indicate dosing. Velcade decreased the percentage of leukemic blasts from 96% to 20%. Figure 2. Clinical follow-up of AML patient-PD001 shows chemosensitivity to bortezomib (Velcade). Arrows arrows indicate dosing. Velcade decreased the percentage of leukemic blasts from 96% to 20%. Disclosures Vega: Seatle Genetics: Honoraria; NIH: Research Funding.