Abstract: Relapse is the most common cause of mortality in patients undergoing allogeneic stem cell transplantation (ASCT) for acute myeloid leukemia (AML). The presence of measurable residual disease (MRD) at the time of transplant is associated with very high relapse rates, and novel strategies to address relapse risk are needed for these patients. Venetoclax has demonstrated single agent activity in AML, and venetoclax combined with azacitidine, decitabine, or low dose cytarabine is FDA approved to treat newly diagnosed patients & gt;75yrs or older and those unfit for induction. Given the ability of venetoclax to target leukemic stem cells combined with its favorable toxicity profile and ease of administration, we have initiated off label post-transplant venetoclax maintenance for AML patients with MRD at the time of transplant. We report outcomes of our experience to date. Venetoclax therapy is planned to be initiated approximately 40-80 days post-ASCT upon count recovery (ANC & gt;1000 109/L and platelets & gt;100 109/L) and recovery from early ASCT toxicity. Dose is initiated at 100 mg daily for one week and titrated up by 100 mg weekly to a final maintenance dose of 400 mg daily. Therapy is planned until one year post-ASCT. Since February 2019, we have administered venetoclax to 23 post-ASCT patients (22 AML (6 with prior diagnosis of MDS) and 1 MDS), median age 65 (range 19-73). Venetoclax therapy was initiated beginning median 67 days (range 36-146) after ASCT. 16 patients underwent transplant following one line of therapy (twelve of whom received venetoclax azacitadine), 5 patients received 2 lines of therapy, and 2 patients received 3 lines of therapy. Three patients were second transplants. Donor sources were cord (n=14), haplo cord (n=5), and matched related donor (n=4). 7 patients underwent myeloablative, 14 reduced-intensity, and 2 non-myeloablative conditioning. All patients had MRD at time of transplant (15 by cytogenetics/FISH, 10 by flow cytometry, and 6 by digital droplet PCR). At time of transplant, 1 patient was aplastic, 6 were in morphologic leukemia free state (MLFS), one was in complete remission with incomplete count recovery (CRi), 15 were in complete remission (CR), and 1 had MDS with ringed sideroblasts with multilineage dysplasia. Patient data is summarized in Table 1. Median follow up among survivors is 219 days (range 92-439). Details of individual patients' courses on venetoclax are summarized in Figure 1. In total, 11 patients were still on venetoclax at the time of this analysis, and 4 had completed the planned 1 year of treatment. 3/23 (11%) had the drug permanently discontinued due to potential adverse effects in the setting of additional post-ASCT complications. Four patients relapsed 191, 288, 325, and 367 days post-transplant and all died, and 3 patients experienced transplant related mortality 146, 164, and 307 days post-ASCT. 6 month overall survival (OS) and relapse free survival (RFS) were both 87%. Venetoclax was temporarily held or dose-reduced in 11/23 (47.8%) patients due to adverse events (AEs). 14/23 (61%) patients experienced at least one AE that could be potentially attributed to venetoclax. Most common AEs were cytopenias (7/23, 30%) and diarrhea (7/23, 30%). 12 patients experienced grade 2-4 acute GVHD (3 grade 3). 10 of these patients developed GVHD symptoms prior to starting venetoclax. 5/7 patients who held venetoclax due to diarrhea had acute GVHD prior to initiation of ventoclax, and whether the diarrhea was GVHD or venetoclax related was unclear. Three patients have developed chronic GVHD (2 mild and 1 severe). Our preliminary data suggests venetoclax is tolerable in the post-ASCT maintenance setting without unexpected side effects. In this small cohort, GVHD rates are comparable to historical controls. Ongoing follow-up will continue to examine the safety and efficacy of this approach. Disclosures Pollyea: Novartis: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Agios: Consultancy; 47: Consultancy, Research Funding; Genentech: Consultancy; Glycomimetics: Other; Celgene/BMS: Consultancy; Syndax: Consultancy; Syros: Consultancy; Abbvie: Consultancy, Research Funding; Daiichi Sankyo: Consultancy. OffLabel Disclosure: off label venetoclax post-transplant maintenance

Abstract: Chronic myeloid leukemia (CML) is a heterogeneous disease, initiated by reciprocal translocation of chromosome 9 and 22, resulting in the generation of a BCR-ABL fusion protein and constitutive activation of the ABL kinase. ABL tyrosine kinase inhibitors (TKIs) have been very successful in suppressing CML disease. However, TKIs may not eliminate leukemia stem cells (LSCs), as evidenced by the frequent re-emergence of the disease upon TKI discontinuation. Moreover, blast phase CML (bpCML) remains a formidable challenge in disease management. Recent clinical evidence suggests that the BCL2 inhibitor venetoclax (Ven) in combination with ABL-targeting tyrosine kinase inhibitors (TKI) can eradicate bpCML LSCs. However, the exact mechanism by which this combination may targets LSCs is not known. In this report, we confirm the efficacy and LSC-targeting capacity of Ven/TKI combination in preclinical models of bpCML and we further identify that inhibition of free fatty acid (FFA) mobilization pathways may provide enhanced efficacy against LSCs. To establish the efficacy of Ven/TKI combination, we treated bpCML samples with Ven+Dasatinib (Das) combination for 24h, this resulted in a significant decrease in the viability of bulk and primitive populations (CD34+, CD38+). Patient-derived xenografts of bpCML samples in NSGS-mice, were treated with Ven/Das as well as single agents. The result showed a significant decrease in leukemia burden in the combination treated group, compared to either drug alone, albeit, some resistant cells survived in the combo treated group. Furthermore, using a syngeneic mouse model of bpCML, co-expressing Bcr-Abl and Nup98-HOXA9 translocations, the mouse leukemic cells treated with Ven/Dasatinib combination demonstrated a significant loss of viability of the bulk as well as phenotypically defined LSCs (Lin-/Sca1+). Treatment of leukemic mice with Ven/Das had a significant survival benefit and remained disease free at 80 days post treatment. We also showed significant survival benefits of Ven/ponatinib in NSGS-mice harboring syngeneic bpCML cells with the T315I gatekeeper mutation. Treatment of normal mice with Ven/Das combo did not affect the colony forming ability of LSK cells from the bone marrow, indicating a leukemia-specific response. Based on these results, we conclude that Ven/TKI combination effects were due to direct targeting of the LSC population. To investigate the potential mechanism of Ven/TKI activity in LSC targeting, we performed gene expression studies using RNA-seq based methods after short term treatment. Our findings indicated that the LSC population from Ven/TKI-treated mice showed enrichment of a gene signature associated with lysosome biology. Pre-treatment of mouse leukemia cells with bafilomycin, an inhibitor of lysosome function, resulted in increased sensitivity to Ven/TKI combo. Intriguingly, we also found significant induction of lysosomal acid lipase (LIPA), an enzyme involved in the generation of free fatty acids for energy needs. Metabolomic analysis of LSCs isolated after short term treatment with Ven/TKI, showed that a number of fatty acids were up-regulated in the Ven/Das treated group compared to control. Knocking down Lipa using CRISPR technology resulted in increased sensitivity to Ven/TKI combination, whereas overexpression of Lipa resulted in decreased sensitivity to the Ven/TKI combination, implicating Lipa upregulation and a resultant increase in free fatty acids as a protective response to Ven/TKI treatment. Furthermore, knocking down CPT1A, an important free fatty acid mitochondrial transporter, resulted in increased sensitivity to Ven/TKI combination both in mouse and primary human leukemic cells, leading to the hypothesis that activation of fatty acid processing through enhanced Lipa activity may represent a compensatory response to venetoclax based therapies in bpCML. In summary, we demonstrate the preclinical efficacy of Ven/TKI combination therapies for targeting of bpCML LSCs. Furthermore, our data suggest that blocking upregulation of free fatty acids through mechanisms such as inhibition of LIPA activity, might synergize with Ven/TKI combinations to eradicate LSCs, allowing for more durable response. Our findings provide a therapeutic rationale for blocking pathways involved in free fatty acids generation, as a potential strategy for increasing remission duration. Disclosures Pollyea: Amgen: Consultancy; Janssen: Consultancy; Genentech: Consultancy; AbbVie: Consultancy, Research Funding; Syndax: Consultancy; Daiichi Sankyo: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Glycomimetics: Other. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding.

Abstract: Recent clinical trials have reported that in combination with hypomethylating agents, the BCL-2 inhibitor venetoclax can induce responses in over 70% of older previously untreated AML patients who are unfit for conventional chemotherapy. These findings led to the recent United States Food and Drug Administration approval of this regimen for this population, and it is now considered to be the standard care. However, a significant minority of patients do not achieve a remission and are refractory. In addition, the majority of patients who do achieve a remission ultimately relapse. It is therefore critical to identify AML patients who are likely to be resistant to venetoclax-based therapy. To initially address this question, we retrospectively reviewed 75 newly diagnosed AML patients who received venetoclax + azacitidine (VEN+AZA) at our institution and analyzed several baseline clinical features to determine the ability of each to predict disease that was refractory to treatment (defined as a lack of complete remission [CR], CR with incomplete recovery of peripheral blood counts [CRi] , partial remission, or morphological leukemia free state [MLFS]). Both univariate and multivariate analyses revealed the presence of FAB-M5 to be associated with disease that was refractory to VEN+AZA (Table 1). Given that FAB-M5 represents AML with monocytic differentiation, these findings indicate a strong correlation between myeloid differentiation status and resistance to venetoclax. Using multicolor flow cytometry, we show bone marrow specimens of typical FAB-M5 patients who were refractory to VEN+AZA presented dominant monocytic disease that has an immunophenotype of CD45-bright/SSC-high/CD117-/CD11b+/CD68+ (Figure A). In contrast, bone marrow specimens of typical FAB-M0/M1/M2 patients who achieved CR with VEN+AZA presented as a single dominant disease population that is CD45-med/SSC-low/CD117+/CD11b-/CD68- (Figure B). In a subset of AML patients, we observed the co-existence of both phenotypically primitive and monocytic populations, which we term "MPM" AML (for Mixed Primitive/Monocytic). We observe that after attaining CR with VEN+AZA treatment and subsequent relapse, MPM-AML showed almost complete loss of the primitive subpopulation, and evolved to a dominant monocytic disease (Figure C). These data indicate that VEN+AZA treatment induces strong selection of the monocytic phenotype. Importantly, when we compared the immunophenotype of six pairs of diagnostic/relapse specimens from AML patients treated with conventional intensive induction chemotherapy, we observed selection of a more primitive phenotype, suggesting the drive toward a monocytic phenotype observed at relapse appears to be a unique consequence of VEN+AZA therapy. To our knowledge, selection of a monocytic phenotype at relapse has never been previously observed in AML, suggesting the relapse after VEN+AZA may represent a new clinical entity. Mechanistically, using RNA-seq we show the global transcriptome of monocytic AMLs are distinct from primitive AMLs, suggesting they represent two broad classes of AML with likely differential responses to therapy. Indeed, we demonstrate that AML with a primitive immunophenotype is dependent on BCL-2 activity as a means to drive oxidative phosphorylation, a critical requirement for survival of leukemia stem cells. Conversely, AML with a more differentiated monocytic phenotype is no longer dependent on BCL-2, but rather switches to MCL-1 as a mediator of oxidative phosphorylation. Using colony-forming and xenograft assays, we show the stem and progenitor potential of monocytic AMLs are selectively more sensitive to MCL-1 inhibition comparing to BCL-2 inhibition. Together, our study suggests a significantly higher refractory/relapse risk for monocytic AML patients treated with VEN+AZA (Figure D). Further, for those AML patients who do respond to initial VEN+AZA treatment, the therapy drives a powerful selective process resulting in emergence of more differentiated monocytic disease in some patients. Based on these findings, we propose that AML exists on a developmental spectrum that is inherently fluid, where with appropriate selective pressure the disease can acquire characteristics of a more differentiated state. Further, our data indicate that optimal AML therapy will require strategies designed to target both primitive and myeloid phenotypes. Disclosures Pollyea: Forty-Seven: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: 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; Diachii Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: 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; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Savona:TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; 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; Sunesis: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Abstract: Responses and survival with venetoclax for “real-world” AML patients were promising but inferior to those treated in a clinical trial. Compared with induction, response rates are as high as would be predicted and venetoclax patients had a lower than expected early death rate.

Abstract: Venetoclax (ven) plus azacitidine (aza) is the standard of care for patients with newly diagnosed acute myeloid leukemia (AML) who are not candidates for intensive chemotherapy (IC). Some patients who are IC candidates instead receive ven/aza. We retrospectively analyzed patients with newly diagnosed AML who received ven/aza (n = 143) or IC (n = 149) to compare outcomes, seek variables that could predict response to 1 therapy or the other, and ascertain whether treatment recommendations could be refined. The response rates were 76.9% for ven/aza and 70.5% for IC. The median overall survival (OS) was 884 days for IC compared with 483 days for ven/aza (P = .0020). A propensity-matched cohort was used to compare outcomes in the setting of equivalent baseline variables, and when matched for age, biological risk, and transplantation, the median OS was 705 days for IC compared with not reached for ven/aza (P = .0667). Variables that favored response to ven/aza over IC included older age, secondary AML, and RUNX1 mutations. AML M5 favored response to IC over ven/aza. In the propensity-matched cohort analyzing OS, older age, adverse risk, and RUNX1 mutations favored ven/aza over IC, whereas intermediate risk favored IC over ven/aza. In conclusion, patients receiving IC have improved OS compared with those receiving ven/aza. However, in a propensity-matched cohort of patients with equivalent baseline factors, there was a trend toward favorable OS for ven/aza. Specific variables, such as RUNX1 mutations, reported here for the first time, can be identified that favor ven/aza or IC, helping to guide treatment decisions for patients who may be eligible candidates for either therapy.

Abstract: Venetoclax+azacitidine is the standard of care for newly-diagnosed patients with acute myeloid leukemia (AML) for whom intensive chemotherapy is inappropriate. Efforts to optimize this regimen are necessary. We designed a clinical trial to investigate two hypotheses: i) higher doses of venetoclax are tolerable and more effective, and ii) azacitidine can be discontinued after deep remissions. Forty-two newly diagnosed AML patients were enrolled in the investigator-initiated High Dose Discontinuation Azacitidine+Venetoclax (HiDDAV) Study (clinicaltrials gov. Identifier: NCT03466294). Patients received one to three “induction” cycles of venetoclax 600 mg daily with azacitidine. Responders received MRD-positive or MRDnegative “maintenance” arms: azacitidine with 400 mg venetoclax or 400 mg venetoclax alone, respectively. The toxicity profile of HiDDAV was similar to 400 mg venetoclax. The overall response rate was 66.7%; the duration of response (DOR), event-free survival (EFS) and overall survival were 12.9, 7.8 and 9.8 months, respectively. The MRD negativity rate was 64.3% by flow cytometry and 25.0% when also measured by droplet digital polymerase chain recation. MRD-negative patients by flow cytometry had improved DOR and EFS; more stringent measures of MRD negativity were not associated with improved OS, DOR or EFS. Using MRD to guide azacitidine discontinuation did not lead to improved DOR, EFS or OS compared to patients who discontinued azacitidine without MRD guidance. Within the context of this study design, venetoclax doses 〉 400 mg with azacitidine were well tolerated but not associated with discernible clinical improvement, and MRD may not assist in recommendations to discontinue azacitidine. Other strategies to optimize, and for some patients, de-intensify, venetoclax+azacitidine regimens are needed.

Abstract: Background: Venetoclax (ven)+azacitidine (aza) is the standard of care for untreated acute myeloid leukemia (AML) patients (pts) ≥75 years or unfit for intensive induction chemotherapy (IC) due to comorbidities. While the standard of care for younger newly diagnosed pts is IC, long term outcomes for all but favorable risk pts are suboptimal. Specifically, pts with adverse risk disease, per the European Leukemia Network (ELN), are more likely to be refractory to IC and have low rates of long term survival. Ven based regimens have a ~70% response rate in the upfront setting; in contrast to IC, those with adverse risk do not have lower response rates. This study explores the feasibility and outcomes of ven+aza for newly diagnosed younger AML pts who are not necessarily unfit for IC, but carry adverse risk features that make them less likely to respond to this therapy. Methods: This is an interim analysis of an ongoing phase 2 study (NCT03573024) of a planned 36 untreated adverse risk AML pts 18-59. Pts have adequate organ function and white blood cell counts & lt;25x109/L (hydrea permitted); "fitness" for IC is not a consideration. In cycle 1, pts receive aza 75mg/m2 on days (d)1-7 and ven, escalated from 100 to 200 to 400 to 600mg on d1-4. Ven continues at 600mg d5-28 and bone marrow biopsies (BMBX) occur on d14 and 28. Pts who achieve morphologic remission without count recovery have up to 14 days off therapy before subsequent cycles. Non responders discontinue or receive up to 1 additional cycle. Post response (defined as a morphologic leukemia free state [MLFS], complete remission [CR] with incomplete count recovery [CRi] or CR), pts who are measurable residual disease (MRD) negative by multiparameter flow cytometry (MFC) and/or droplet digital PCR (ddPCR) may proceed to MRD negative maintenance: ven 400mg d1-28 with aza 75mg/m2 d1-5 of sequential 28-day cycles. Pts who are MRD-positive may receive up to 3 additional cycles of consolidation: ven 600mg/day d1-28 and aza 75mg/m2 d1-7 of a 28-day cycle. Pts who become MRD negative may proceed to MRD negative maintenance. Pts who are MRD positive after induction and consolidation may proceed to MRD positive maintenance: ven 600mg daily d1-28 with aza 75mg/m2 d1-7 of a 28-day cycle. Conversion to MRD negativity results in a transition to MRD-negative maintenance (Fig 1). While these post-remission options are available, pts are encouraged to discontinue the study and proceed to transplant after achieving a morphologic remission. The primary objective is to determine the overall response rate (CR+CRi+MLFS). The endpoint is to show ven+aza is noninferior to historical controls: younger newly diagnosed adverse risk AML pts who receive IC. Frequent analysis of outcomes, using 6 stages, was adopted to ensure the CR rate remains ≥60%. Results: 8 have enrolled between November 2018 and July 2020; median age is 33 (22-52). All have adverse risk disease (Table 1). There were 179 adverse events (AEs); 35 related. The ≥grade 3 events were: fatigue (N=1), leukopenia (N=2), neutropenia (N=2), anemia (N=2), thrombocytopenia (N=1). There were no deaths in the first 60 days. In cycle 1 pts spent a median 10 days (4-19) inpatient and required a median of 2 (0-10) red blood cell transfusions and 3 (0-15) platelet transfusions. Six of 8 (75%) pts responded; all 6 were CRs. 4/6 responses were cytogenetic remissions, 5/6 were MRD negative by MFC and 1/6 was MRD negative by ddPCR. Two pts relapsed, 35 and 84 days after remission; one was successfully salvaged with IC and proceeded to transplant while the other was refractory to IC and died from AML. Two pts were refractory to ven+aza; both were successfully salvaged with IC and have proceeded to transplant. 7/8 proceeded to transplant; 4 required only ven+aza for this outcome and proceeded to transplant a median of 89 days (56-93) after diagnosis. No pts who have proceeded to transplant have relapsed, a median of 340 days (136-444) after transplant. 7/8 remain alive, 345 days (91-586) after enrolling. Conclusions: At the stage 1 interim analysis, the CR rate for ven+aza remains & gt;60% in adverse risk younger patients. With small numbers, up-front treatment with ven+aza allows some younger pts with adverse risk disease a non IC opportunity to achieve a response and proceed to transplant; most of those who relapsed or were refractory were salvaged with IC. Disclosures Pollyea: Syndax: Consultancy; Syros: Consultancy; Abbvie: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Genentech: Consultancy; Amgen: Consultancy; 47: Consultancy, Research Funding; Janssen: Consultancy; Agios: Consultancy; Glycomimetics: Other; Celgene/BMS: Consultancy; Pfizer: Consultancy; Takeda: Consultancy. OffLabel Disclosure: Venetoclax for younger patients fit for induction chemotherapy