pVAX14 DNA, a Non-Specific DNA Vaccine, Improves Survival In An Acute Promyelocytic Leukemia (APL) Mouse Model Treated With All-Trans Retinoic Acid (ATRA) and Arsenic Trioxide (ATO) and Targets Leukemia Initiating Cells (LICs)

Abstract Background The combination of ATRA and ATO is very effective in treating APL, both in mouse models (Lallemand JEM 1999) and in patients (Lo Coco NEJM 2013). Having previously shown that the combination of ATRA and a specific DNA vaccine (pCDNA3PML-RARAFrC) rescues APL mice from relapse and enhances specific immune responses (Padua Nat Med 2003; Robin Blood 2006; Furugaki Blood 2010; Pokorna Mol Cell Probes 2013), we applied this approach using a novel non-specific DNA, pVAX14, as add-on therapy to ATRA +/- ATO. Methods APL transplanted mice (APL cells injected in syngeneic 6-week old mice, Brown PNAS 1997) were treated with various combinations of ATRA +/- ATO and DNA (pVAX1 (control DNA), pCDNA3PML-RARAFrC (specific DNA vaccine) or pVAX14. pVAX14 is a novel construct containing GC-rich sequences and coding for unique peptides, 3 of which we have shown to be immunogenic. Mice were monitored for survival, MRD (by RQ-PCR of PML-RARA) on days 19, 37, 57 and 67 in peripheral blood (PB), and as a measure of LICs by secondary transplantation of bone marrow (BM) or spleen cells from long term (more than 120 days) survivors (LTS) into syngeneic lethally irradiated mice. Immune responses were assessed on CD4 Memory T-cells (Tmem), IFNg production (ELISPOT) and anti-RARa (ELISA) antibody production. Functional studies including challenge experiments of LTS with leukemic cells assessed the protective effects of immunotherapy. Results 1) Survival . In a first set of experiments, ATRA+pVAX14 significantly increased survival compared to placebo, ATRA alone or ATRA+pVAX1 (control DNA) (p<0.001, p<0.03, p<0.05 respectively (Fig. 1); survival was similar with ATRA+pVAX14 and ATRA+pCDNA3PML-RARAFrC, with about 40% LTS. In a second set of experiments where ATO was introduced, ATO+pVAX14 was not superior to ATO alone, but ATRA+ATO+pVAX14 combination was significantly better than ATRA+ATO (p<0.05), providing 85% LTS (Fig. 2). MRD for PML-RARA in ATRA+ATO+pVAX14 mice was significantly lower than in ATRA mice (p<0.001) but the difference with ATRA+ATO mice was not significant up to day 67. Moreover, when we injected BM or spleen cells from both ATRA+ATO and ATRA+ATO+pVAX14 LTS none of the recipients develop APL (follow-up period of 140 days), indicating the clearance of LICs. 2) Immune response monitoring showed that effector Tmem were significantly higher in ATRA+ATO+pVAX14 > ATRA+ATO > ATRA (p<0.05 and p<0.001 respectively). LTS of ATRA+ATO+pVAX14 had increased IFNg producing lymph node cells when stimulated with APL cells or PML-RAR peptide compared to ATRA+ATO (p<0.02). Serum anti-RARa antibodies were significantly increased in all combinations compared to controls with ATRA+ATO+pVAX14 > ATRA+pVAX14 ≈ ATRA+ATO+pVAX1 (control DNA) ≈ ATRA+ATO > ATRA+pVAX1 (Control DNA) > ATRA (p<0.05, p<0.03, p<0.0006 respectively). When LTS mice of ATRA+ATO+pVAX14 and ATRA+ATO groups were challenged with APL spleen cells, survival was significantly higher in both groups (median survival 105 vs 80 days, p=NS) than in controls (syngeneic mice injected with APL cells) (p<0.001 and p<0.01 respectively) Conclusion The combination of the non-specific vaccine (pVAX14) with ATRA+ATO further improved survival over ATRA+ATO alone and elicited specific humoral and T-cell mediated responses targeting LICs. This vaccine is also effective in a mouse model of high risk MDS (Submitted to ASH 2103). SP, LG and CLP contributed equally to this work. Disclosures: Patel: CEFIPRA: Research Funding. Fenaux:Celgene: Honoraria, Research Funding. Chomienne:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. , I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees. Padua:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. I, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. I Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees.

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ABT-737 Targets Leukemic Stem Cells In Mouse Models of Mutant NRASD12/hBCL-2- Mediated Acute Myeloid Leukemia Progression with Increased Survival

Blood ◽

10.1182/blood.v116.21.3308.3308 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3308-3308

Author(s):

Rose Ann Padua ◽

Stephanie Beurlet ◽

Patricia Krief ◽

Nader Omidvar ◽

Carole Le Pogam ◽

...

Keyword(s):

Stem Cells ◽

Disease Progression ◽

Board Of Directors ◽

Research Funding ◽

Leukemic Stem Cells ◽

Spleen Cells ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Bh3 Mimetic

Abstract Abstract 3308 Background: Animal models enable us to understand disease progression and provide us with reagents to test various therapeutic strategies. We have previously developed a mouse model of myelodysplasia/acute myelogenous leukemia (MDS/AML) progression using mutant NRASD12 and overexpression of human hBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). Expanded leukemic stem cells (LSC) were identified as Lin-/Sca1+/KIT+ (LSK) populations, with increased myeloid colony growth and were transplantable. Increased hBCL-2 and RAS-GTP complex were observed in both MDS/AML diseases. The MDS-like disease had increased apoptosis, whilst the AML-like mice had liver apoptosis patterns similar to wild type. The single NRASD12 line also had increased apoptosis. In this present study using a BCL-2 homology domain 3 (BH3) mimetic ABT-737 (Abbott), we have evaluated the effects of targeting BCL-2 in our preclinical models. Methods & Results: Treatment with the inhibitor shows a reduction of LSK cells, reduced progenitor numbers in colony assays and clearance of the liver infiltrations in both MDS and AML models. Gene expression profiling of the MDS mice shows regulation of 399 genes upon treatment including 58 genes expressed by the single mutant RAS mice and not expressed in the untreated AML mice. 78 genes were shared between single NRASD12 and diseased mice and not the treated mice. These studies potentially identify the contribution of NRASD12 genes to disease progression. By confocal microscopy we observed that in the MDS mice the majority of the RAS and BCL-2 co-localized to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localized in the mitochondria, where BCL-2 is normally found (Omidvar et al 2007). After treatment with the inhibitor the AML co-localization of RAS and BCL-2 shifted to the plasma membrane where single NRASD12 is normally localized. Furthermore, increased RAS-GTP levels was detected in both Sca1+ and Mac1+ enriched spleen cells and interestingly an increase in BCL-2 expression was observed in peripheral blood and in spleen cells after treatment; this increase in BCL-2 was associated with a decrease in the phosphorylation of serine 70 and an increase in phosphorylation of threonine 56 of BCL-2. ABT-737 treatment led to increased phosphorylated ERK resembling RAS and reduced MEK and AKT phosphorylation, changes detected by western blots and the nanoimmunoassay (NIA, NanoPro, Cell Biosciences) that might account for the increased apoptosis, measured by TUNEL and In vivo imaging by single-photon emission computed tomography (SPECT) using Tc-99m-labelled AnnexinV (SPECT). In contrast, although treated MDS mice had increased apoptosis they did not have an increase in overall expression of BCL-2 or in RAS-GTP levels. Treatment of both MDS and AML models with this inhibitor significantly extended lifespan from diagnosis with mean survival of 28 days untreated vs 80 days treated (p=0.0003) and mean survival from birth of 39 untreated vs 85 days treated (p<0.0001) respectively Conclusions: Genomics, proteomics and imaging have been employed in the MDS/AML models to characterize disease progression and follow response to treatment to the BH3 mimetic ABT-737 in order to gain molecular insights in the evaluation of the efficacy. ABT-737 appears to target LSCs, induce apoptosis, regulating RAS and BCL-2 signalling pathways, which translated into significantly increased survival. Disclosures: Padua: Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Auboeuf:GenoSplice technology: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. de la Grange:GenoSplice technology: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Fenaux:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Tu:Cell Biosciences Inc;: Employment. Yang:Cell Biosciences Inc;: Employment. Weissman:Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Felsher:Cell Bioscience:. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Expression of PD-L1 in Leukemic Progenitor Cells Defines NPM1 Mutated AML As a Potential Subgroup for PD1/PD-L1 Directed Immunotherapy

Blood ◽

10.1182/blood-2018-99-111458 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2734-2734

Author(s):

Jochen Greiner ◽

Vanessa Schneider ◽

Hubert Schrezenmeier ◽

Markus Wiesneth ◽

Lars Bullinger ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Immune Responses ◽

Progenitor Cells ◽

Board Of Directors ◽

Research Funding ◽

Significant Inhibition ◽

Leukemic Cells ◽

Advisory Committees ◽

Oncology Research

Abstract Clinical and preclinical data suggest that acute myeloid leukemia (AML) with mutated nucleophosmin 1(NPM1mut) may constitute an immunogenic leukemia subtype. NPM1mut AML generally correlates with a better prognosis, however the underlying mechanisms still need to be clarified. Checkpoint inhibition targeting Programmed cell death protein 1 (PD-1)/Programmed cell death 1 ligand 1 (PD-L1) has been proven to be an effective novel immunotherapeutic approach in cancer treatment including the treatment of hematological malignancies. Expression of CD34/CD38/CD274 was evaluated in 20 NPM1mut versus 20 wild-type (NPM1wt) AML patient samples via flow cytometry analyses to assess PD-L1 (CD274) expression in leukemic cells, including leukemic progenitor and stem cells (LSC). We also investigated the influence of the anti-PD-1 antibody Nivolumab® on the antigen-specific immune responses in ELISpot assays. Additionally, we assessed the effect of Nivolumab in colony forming unit (CFU) immunoassays. Many AML cases showed relevant expression of PD-L1. Bulk cells of NPM1mut AML showed a significantly higher PD-L1 expression in comparison to NPM1wtAML patients (median of 1.5%, range 0.0-8.5%, versus 0.3%, range 0.1-1.1%). Importantly, PD-L1 expression was detected at a higher level in leukemic progenitor cells (CD34+CD38-) of NPM1mut than of NPM1wtAML (median of 3.3%, range 0.0-17.2%, versus 0.3%, range 0.0-3.0%). In general, the LSC fraction showed a higher PD-L1 expression than the non-LSC fraction. CFU immunoassays showed a significant inhibition of CFU when adding T cells stimulated against various LAA. In all patient samples, effectors activated against at least one LAA were successful to decrease the colony number significantly. Immune effects increased adding Nivolumab to the CTL for several days before starting CFU immunoassays. In summary, we detected higher PD-L1 expression in NPM1mut patients, especially in the leukemic progenitor compartment. This observation further supports the hypothesis that NPM1-directed immune responses might play an important role in tumor cell rejection, which tumor cells try to escape via expression of PD-L1. Immunogenicity of neoantigens derived from NPM1mut with higher PD-L1 expression constitute promising target structures for individualized immunotherapeutic approaches. Disclosures Schrezenmeier: Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Bullinger:Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria.

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Bortezomib-Associated Peripheral Neuropathy: Relationship Between Clinical Neurophysiologic Evidence in Previously Untreated Multiple Myeloma Patients and Preclinical Characterization in a Mouse Model.

Blood ◽

10.1182/blood.v114.22.3860.3860 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3860-3860 ◽

Cited By ~ 1

Author(s):

Paul G. Richardson ◽

Jordi Bruna ◽

Anthony A. Amato ◽

Esther Udina ◽

Constantine S. Mitsiades ◽

...

Keyword(s):

Mouse Model ◽

Board Of Directors ◽

Research Funding ◽

Sensory Nerve ◽

Preclinical Studies ◽

Employment Equity ◽

Advisory Committees ◽

Small Fiber ◽

Neuroprotective Strategies ◽

Equity Ownership

Abstract Abstract 3860 Poster Board III-796 Bortezomib (Velcade®) therapy for multiple myeloma (MM) results in high overall and complete response rates, but can also lead to peripheral neuropathy (PN). Bortezomib-associated PN has been shown to be a cumulative, dose-related, primarily sensory neuropathy that is reversible to baseline in the majority of cases (Richardson et al, Br J Haematol 2009). Further research is needed to determine the mechanisms by which PN arises and to determine potential neuroprotective strategies. A preclinical model that reflects the neurophysiologic changes seen in patients developing PN during bortezomib treatment would prove highly valuable. Here we assess the relationship between clinical neurophysiologic findings in untreated MM patients who received single-agent bortezomib and developed PN in a phase 2 study (Richardson et al, J Clin Oncol 2009) and preclinical neurophysiologic and histologic characterization of bortezomib-induced PN in a SwissOF1 mouse model (Bruna et al, J Peripher Nerv Syst 2009). By CTCAE grading, 41/64 (64%) patients in the clinical study developed sensory PN, and 7 (11%) had motor PN. In 35 of these patients, PN was also assessed using extensive neurophysiologic testing, including motor and sensory nerve conduction studies (NCS) and quantitative sudomotor axon reflex testing (QSART). Of these patients, 22/35 (63%) developed PN by modified consensus criteria (England et al, Neurology 2005), including 7 (20%) who had worsening of baseline MM-associated neuropathy, and 15 (43%) who developed new small-fiber (n=7) or both large- and small-fiber PN (n=8). Similarities between neurophysiologic changes seen in the 15 patients with new-onset PN and in 20 animals given bortezomib twice-weekly for 6 weeks in the mouse model are summarized in the Table. Histologic studies in mice showed significant reductions in myelinated fiber count (–9.9%) and myelin thickness (–17.5%), likely secondary to axonal damage, in bortezomib-treated vs control animals; however, PN with demyelinating features is uncommon clinically. Importantly, bortezomib-associated PN was reversible in both clinical and preclinical studies. By CTCAE grading, sensory PN resolved in 35 of the 41 (85%) patients who had PN in a median of 98 days (reversibility was not assessed by neurophysiologic testing). In the mouse model, after a 4-week wash-out period, complete normalization was seen in sensory NCS and histologic findings. Overall, there appears to be good correspondence between clinical manifestations of bortezomib-associated PN and evidence from the mouse model, with both showing a predominantly sensory PN that affects both large and small fibers and is reversible. Preclinical studies with bortezomib and other agents have suggested that this PN is a mechanism-based effect of proteasome inhibitors associated with cytoplasmic accumulations of ubiquitinated proteins in dorsal root ganglia neurons (Silverman et al, ASH 2008). Furthermore, histologic findings in mice of mild axonal loss and secondary loss of myelinated fibers are in accord with NCS findings, and may be due to the reversible disruption of proteasomal degradation of PMP22, a glycoprotein incorporated into myelin (Gilardini et al, Curr Med Chem 2008). Given the similarities with clinical findings, the SwissOF1 mouse model of bortezomib-induced PN appears to represent a promising vehicle for further exploration of this toxicity and the development of neuroprotective strategies. Table Neurophysiologic findings in clinical (vs baseline) and preclinical (vs controls) studies. Clinical Mouse model Sensory function Significant increase in Total Neuropathy Score (median 9.5) and sensory score (median 5) Significant impairment of sensory-motor function by Rotarod testing Large-fiber Significant reduction in sural (median –3.6 μV) and ulnar (median –1.8 μV) SNAP Significant reduction in compound SNAP and distal sensory NCV Small-fiber Significant reductions in QSART of foot (median –0.36 μL) and distal leg (median –0.36 μL) Algesimetry – significant increase in time to withdrawal from hot pain Motor function No change from baseline in median motor signs and symptoms No difference in compound motor action potential and motor NCV Autonomic function No change from baseline in median autonomic score No significant difference in sudomotor function and heart rate variability NCV, nerve conduction velocity; SNAP, sensory nerve action potential Disclosures: Richardson: Keryx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bruna:Millennium Pharmaceuticals, Inc.: Research Funding; Johnson and Johnson: Research Funding. Udina:Millennium Pharmaceuticals, Inc.: Research Funding; Johnson and Johnson: Research Funding. Mitsiades:Milllennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria. Hedley-Whyte:Millennium Pharmaceuticals, Inc.: Consultancy. Monbaliu:Johnson and Johnson: Employment, Equity Ownership. Vynckier:Johnson and Johnson: Employment, Equity Ownership. Silverman:Milllennium: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Navarro:Johnson and Johnson: Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding.

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JAK1/2 and BCL2 Inhibitors Synergize to Counter-Act Bone Marrow Stromal Cell-Induced Protection of AML

Blood ◽

10.1182/blood.v126.23.867.867 ◽

2015 ◽

Vol 126 (23) ◽

pp. 867-867

Author(s):

Riikka Karjalalainen ◽

Tea Pemovska ◽

Muntasir Mamun Majumder ◽

Bhagwan Yadav ◽

Jing Tang ◽

...

Keyword(s):

Bone Marrow ◽

Cell Viability ◽

Board Of Directors ◽

Dose Response ◽

Research Funding ◽

Stromal Cell ◽

Leukemic Cells ◽

Jak Inhibitors ◽

Advisory Committees ◽

Equity Ownership

Abstract BACKGROUND AND OBJECTIVES: The bone marrow (BM) microenvironment supports the survival of leukemic cells and influences their response to therapeutic agents by promoting drug tolerance and resistance. Novel therapeutic strategies are therefore needed that can override the BM mediated protection of AML cells in patients undergoing drug treatment. To address this we used a high-throughput drug screening method to identify novel drug combinations to reverse stromal-induced cytoprotection against the BCL2 antagonist venetoclax in primary AML samples. METHODS: Sensitivity of mononuclear cells collected from 18 AML BM aspirates or peripheral blood samples to a range of BCL2 inhibitors and tyrosine kinase inhibitors (TKIs) was assessed either in mononuclear cell medium (MCM, Promocell) or in a 25% HS-5 stromal cell-conditioned medium plus 75% RPMI medium mix (CM) to mimic cytoprotective bone marrow conditions. Cell viability was measured after 72 h and dose response curves generated for each tested drug. Drug sensitivity scores were calculated based on the area under the dose response curve. For the drug combination studies single agents (venetoclax, WEHI-539, ruxolitinib) were added simultaneously at fixed concentrations to AML cells and incubated for 72 h either in the MCM or CM medium. Cell viability was measured using the CellTiter-Glo assay. The expression of BCL2 genes was measured by qPCR after incubating the AML patient cells in either MCM or CM for 48 h. RESULTS: Incubation of primary AML cells in the CM culture condition led to reduced sensitivity to BCL2 family inhibitors, suggesting that stromal-derived factors in the CM promote cytoprotection. This effect was particularly pronounced for the selective BCL2 inhibitor venetoclax, where the CM-induced loss of sensitivity coincided with decreased BCL2 expression and increased BCL2L1 expression. In contrast, JAK inhibitors showed improved efficacy in CM compared to MCM culture conditions. To determine if the protective effects of CM stromal-like conditions against venetoclax could be diminished, the drug was tested in combination with the JAK1/2 inhibitor ruxolitinib using AML cells cultured in MCM or CM. When tested on AML cells from 4 patients with the FLT3-ITD alteration, we found that ruxolitinib rescued the sensitivity of venetoclax in leukemic cells in the presence of CM and the combination of two drugs exhibited synergistic effects in this setting. The combinatorial activity, however, was not recapitulated in the MCM condition. Since CM was found to induce BCL2L1 expression, venetoclax was also tested in combination with a BCLXLspecific inhibitor WEHI-539. Analogously to the ruxolitinib-venetoclax combination, synergistic activity between venetoclax and WEHI-539 was observed towards leukemic cells in the presence of CM. CONCLUSIONS: By applying a functional, drug-based approach to understand microenvironment-induced mechanisms of drug resistance in AML, we found that the activity of the selective BCL2 inhibitor venetoclax towards AML cells is adversely affected in stromal-based conditions, while JAK inhibitors, in contrast, exhibit increased efficacy in these conditions. Our results suggest stroma-derived cytokines induce JAK-STAT signaling in AML cells, which results in increased BCL2L1 expression and drives resistance to venetoclax. However, blocking JAK1/2 with ruxolitinib restores the sensitivity of AML cells to venetoclax. We found that JAK1/2 inhibitors such as ruxolitinib can act synergistically with BCL2/BCLXL inhibitors, suggesting clinically useful combination treatments. Disclosures Gjertsen: BerGenBio AS: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Porkka:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kallioniemi:Vysis-Abbot: Patents & Royalties; Medisapiens: Membership on an entity's Board of Directors or advisory committees; IMI-Project Predect: Research Funding; Roche: Research Funding; Pfizer: Research Funding. Wennerberg:Pfizer: Research Funding. Heckman:Celgene: Honoraria, Research Funding.

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A Murine Immunocompetent Acute Myeloid Leukemia (AML) Model for Testing Immunotherapies

Blood ◽

10.1182/blood-2020-141171 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 3-4

Author(s):

Hui Mei Lee ◽

Niloofar Zandvakilli ◽

Rhea Desai ◽

Purvi M Kakadia ◽

Peter Browett ◽

...

Keyword(s):

Immune System ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemic Cells ◽

Leukemia Cells ◽

Spleen Cells ◽

Advisory Committees ◽

Intact Immune System ◽

Acute Myeloid

Background With the emergence of immunotherapies as a promising cancer treatment, there is now a pressing need for pre-clinical animal models to test immunotherapy strategies for acute myeloid leukemia (AML). While murine xenotransplant models generated by transplanting human AML cells are frequently used to model AML, they require immune-deficient mice. Syngeneic murine bone marrow transplant leukemia models (MBMTLM), which are established in immune competent mice, usually require radiation of the recipient mouse before leukemic cells are transplanted. This radiation suppresses the immune system and makes it difficult to study how the immune system responds to leukemia cells. Aim Our aim was to establish immunocompetent MBMTLMs to understand how the immune system responds to leukemia cells expressing a highly immunogenic antigen. Method We first established MBMTLMs models driven by the CALM/AF10 or MLL/AF9 fusion genes. Then leukemia cells were transduced with a SIINFEKL expressing retrovirus (MSCV-DsRed-SIINFEKL). SIINFEKL is a highly immunogenic eight amino acid peptide from ovalbumin. Results The transduction efficiency of MSCV-DsRed-SIINFEKL was about 12.9% on leukemic cells of MLL/AF9 and 13% on CALM/AF10 cells. All primary MLL/AF9-SIINFEKL (n=3) developed leukemia with a latency of 22 days. SIINFEKL expression was detected on 75.5±3% of the spleen cells of these mice. These spleen cells were transplanted into irradiated (n=6) and non-irradiated (n=8) mice to establish secondary MLL/AF9-SIINFEKL leukemias (Table 1). In the irradiated recipients, five out of six mice developed leukemia within 22-29 days. In non-irradiated recipients, four out of eight developed leukemia with a latency of 29-45 days. Flow cytometry showed that SIINFEKL was expressed on 82.28±6% of the spleen cells in irradiated recipients. In contrast, fewer than 1% of the spleen cells in non-irradiated mice with secondary MLL/AF9-SIINFEKL leukemia expressed SIINFEKL. Similar experiments were performed with the CALM/AF10 model. All primary CALM/AF10-SIINFEKL transplanted mice (n=2) developed leukemia with a latency of 32 and 42 days and showed SIINFEKL positivity on 99% of the leukemia cells. Secondary CALM/AF10-SIINFEKL leukemias were generated by transplanting these AML cells into irradiated (n=6) and non-irradiated (n=8) mice. All six irradiated recipients developed leukemia within 21-40 days. However, only five of the eight non-irradiated recipients developed leukemia with a latency of 35-42 days. Strikingly, about 99% of the AML cells in the irradiated mice were SIINFEKL positive but fewer than 1% of the AML cells in the five non-irradiated mice who developed leukemia were SIINFEKL positive. Discussion We find that for both MLL/AF9 and CALM/AF10 AML an intact immune system (non-irradiated recipients) is able to largely eliminate AML cells which express the SIINFEKL antigen, even when challenged with a large number of AML cells. In the non-irradiated recipients only 50 to 60% developed leukemia, and the leukemias that did develop had hardly any SIINFEKL-positive cells. In contrast, nearly all irradiated mice (11 of 12) developed leukemia with a high percentage of SIINFEKL expressing AML cells. SIINFEKL was presented on the surface of leukemic cells by the murine MHC class I H-2Kb molecule. Our results differ slightly from research reported by Hasegawa et al. 2015, who showed that MLL/AF9 AML cells expressing ovalbumin caused leukemia after transplantation into non-irradiated mice. However, they did not investigate whether ovalbumin was still being expressed on the leukemia cells. Conclusion We have established syngeneic murine AML models in immunocompetent mice and have evidence that an intact immune system has the ability to suppress or even eliminate rapidly proliferating AML cells if they express a strong antigen. These models should be useful for developing immunotherapy strategies for AML. Disclosures Desai: University of Auckland: Current Employment. Kakadia:University of Auckland: Current Employment. Browett:University of Auckland: Current Employment; BeiGene: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Shire: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bohlander:University of Auckland: Current Employment; Family of Marijana Kumerich: Research Funding; Leukaemia and Blood Cancer New Zealand: Research Funding.

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pVAX14 DNA, An Immunoadjuvant, Extends Life-Span As Add-On Treatment To Azacitidine (AZA) In A Mouse Model Of High Risk Myelodysplastic Syndrome (HR-MDS)

Blood ◽

10.1182/blood.v122.21.1514.1514 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1514-1514

Author(s):

Gorombei Petra ◽

Le Pogam Carole ◽

Patricia Krief ◽

Satyananda Patel ◽

Guerenne Laura ◽

...

Keyword(s):

Mouse Model ◽

Immune Responses ◽

Life Span ◽

Board Of Directors ◽

Median Survival ◽

Survival Advantage ◽

Advisory Committees ◽

Kaplan Meier ◽

Equity Ownership ◽

Ifng Production

Abstract Background We have established animal models of MDS and acute myelogenous leukemia (AML) using NRASD12 and overexpression of BCL-2 (Omidvar Cancer Res 2007). These models have identified a novel MDS biomarker, the RAS:BCL-2 complex (Le Pogam, Leuk Res 2013) and a BH3 mimetic inhibitor, ABT-737, was shown to target leukemic cells and increase life span in both models (Beurlet, Blood 2013, Gorombei, EHA 2013). Here we have studied the benefit of an immunotherapeutic approach in HR-MDS by taking advantage of the reported immunomodulating effect of azacitidine (AZA) (Goodyear, Blood 2010) and of a non-specific DNA vaccine (pVAX14) we have designed. Methods pVAX14 is a non-specific DNA plasmid which, in an APL mouse model , gives similar survival to the specific PML-RARA DNA we previously designed (Padua, Nat Med 2003) (results submitted to ASH 2013). pVAX14 is a novel construct containing GC-rich sequences and coding for unique peptides, 3 of which we have shown to be immunogenic; ATRA was combined for its immunomodulatory properties. Survival efficacy was measured in mice treated with AZA alone (1mg/kg intraperitoneally 3 times per week until death), pVAX14 (6 weekly injections of 50 mg DNA injected intradermally, ATRA (10 mg 21-day release pellets) or their combinations, and untreated controls. MDS was monitored with biomarkers previously validated for this model, (Beurlet Blood 2013): peripheral blood (PB) counts, PB blasts (Mac1hi/Gr1lo) and spleen cell AKT, MEK1 and ERK1/2 levels by the Nanoimmunoassay (NIA) (Fan, Nat Med 2009). Immunomonitoring was based on lymphocyte subpopulations including Memory T-cells (CD4+/CD44hi/CD62Llo), IFNg production (ELISPOT) and Toll-like receptor-9 (TLR-9) activation measured on MYD88 transcripts by RQ-PCR. Results 1) Survival benefit: in a first cohort of HR-MDS mice, pVAX14 treatment significantly prolonged survival (median survival 100 days in treated versus 10 days in untreated mice) (Kaplan Meier p<0.0001, Fig. 1). In a second pilot cohort, AZA+pVAX14+ATRA appeared superior (4/4 mice alive at 70 days) to AZA+pVAX14 or AZA+ATRA (2/4 alive at 70 days in both groups) and AZA alone (1/5 alive at 70 days). In a third larger confirmatory cohort, median survival was 65 days with the AZA+pVAX14+ATRA combination, 40 days with AZA alone (Kaplan Meier p= 0.044 compared with AZApVAX14+ATRA) and 10 days in untreated mice (p<0.0001 compared with AZA alone) (Fig. 2). 2) Hematological parameters: survival advantage of mice treated with pVAX14 alone, AZA alone and AZA+pVAX14+ATRA was corroborated with lack of leukemic progression assessed on days 13, 32 and 55, as shown by stable platelet counts and peripheral blasts (Mac-1hi/GR-1lo population) and downregulation of RAS signaling proteins with dephosphorylation of AKT, MEK1 and ERK1/2. 3) Enhanced immune responses: survival advantage and absence of leukemic progression were correlated with enhanced immune responses: increased IFNg production (p<0.03) and expression of MYD88 (p<0.05) were seen in mice treated with pVAX14 alone compared to untreated mice); Tmem were significantly increased in treated mice, with values highest for AZA+pVAX14+ATRA (p<0.005 compared to untreated HR-MDS mice). Conclusions 1) AZA increases survival in this HR-MDS model. Immune mechanisms seem to be implicated but we are currently analyzing other potential mechanisms of action, including DNA methylation 2) pVAX14+ATRA as add-on therapy to AZA further improves survival, and potentiates the immune responses initiated by AZA. This adjuvant DNA immunotherapy may thus be a promising approach for MDS treatment. PG and CLP contributed equally to this work. Disclosures: Pierre: Celgene: Honoraria, Research Funding. Christine:VivaVacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study., I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees. Padua:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study., I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees.

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Crispr/Cas9-Mediated In Vivo Gene Targeting Corrects Haemostasis in Newborn and Adult FIX-KO Mice

Blood ◽

10.1182/blood.v128.22.1174.1174 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1174-1174 ◽

Cited By ~ 2

Author(s):

Lili Wang ◽

Yang Yang ◽

John White ◽

McMenamin Deirdre ◽

Peter Bell ◽

...

Keyword(s):

Mouse Model ◽

Board Of Directors ◽

Partial Hepatectomy ◽

Research Funding ◽

Advisory Committees ◽

Exon 2 ◽

Equity Ownership ◽

Donor Template ◽

Vector Approach

Abstract CRISPR/Cas9, a powerful genome editing tool, is promising for efficient correction of disease-causing mutations. We recently developed a dual AAV vector approach for in vivo delivery of three key components of the homology directed repair (HDR) mediated by CRISPR/Cas9: Cas9 enzyme from Staphylococcus aureus (SaCas9), a single-guide RNA (sgRNA), and a donor template. In a mouse model of ornithine transcarbamylase (OTC) deficiency caused by a single nucleotide change, we demonstrated HDR-based correction of the G-to-A mutation in 10% of OTC alleles in the liver of newborn OTC mice and clinical benefits following in vivogenome editing. However, most monogenic genetic diseases including hemophilia are caused by different mutations scattered in a specific gene rather than a single predominant mutation. The vector developed for one mutation would not be applicable for a patient with a different mutation. In this study, we aim to develop a more universal CRISPR/Cas9 gene targeting approach in which the vector system could be applied to majority of the patients with a specific disease, for example, hemophilia B. To validate this new approach, we performed the experiment in a factor IX knockout (KO) mouse model. In this two-vector approach, vector 1 expresses the SaCas9 gene from a liver-specific TBG promoter, same as it was in our previous approach. The difference lies in vector 2. Besides the sgRNA sequence expressed from a U6 promoter that specifically targets to a region in the 5' end of exon 2 of murine FIX and a 1.8-kb donor fragments, it also contains a partial human FIX cDNA sequence spanning the remaining exon 2 to exon 8 followed by the bovine growth hormone polyA inserted in between the left and right arms of the donor template. In addition, the partial human FIX cDNA is codon optimized and carrying the hyperactive FIX Padua mutation. Following double strand breaks generated by Cas9 and HDR, the partial human FIX cDNA would be fused to 5' end of murine FIX and a hybrid of murine-human FIX transcript would be expressed from the native murine FIX promoter. In the control vector 2, it contains everything except for the 20-nt target sequence. Co-injection of the two vectors with varying doses in newborn and adult FIX-KO mice resulted in stable FIX activity at or above the normal levels for over 4 months. Eight weeks after the vector treatment, a subgroup of the newborn and adult treated FIX-KO mice were subjected to a two third partial hepatectomy, and all of them survived the procedure without any complications or interventions. FIX levels persisted at similar levels after partial hepatectomy indicate stable genomic targeting. FIX-expressing hepatocytes were detected in liver samples collected at the partial hepatectomy. This study provides convincing evidence for efficacy in a hemophilia B mouse model following in vivo genome editing by CRISPR/Cas9. Disclosures Wilson: Dimension Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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DNA and All-Trans Retinoic Acid as Immunotherapy or Add-on Adjuvants to 5-Azacytidine In Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v116.21.2938.2938 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2938-2938

Author(s):

Carole Le Pogam ◽

Patricia Krief ◽

Stéphanie Beurlet ◽

Murielle Reboul ◽

Robert West ◽

...

Keyword(s):

Retinoic Acid ◽

Mouse Model ◽

Immune Responses ◽

Board Of Directors ◽

Dna Vaccination ◽

Advisory Committees ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Equity Ownership

Abstract Abstract 2938 Background: Myelodysplastic syndromes (MDS) are clonal stem cell hematological disorders characterized by ineffective hematopoiesis leading to cytopenia, which evolve to acute myeloid leukemia (AML). The ability of the DNA vaccination to induce effective immune responses has been demonstrated in different preclinical models of diseases. Having previously shown that DNA vaccination with a PMLRARaFrC plasmid in combination with all-trans retinoic acid (ATRA) induced long term remissions with appropriate immune responses in a mouse model of acute promyelocytic leukemia (APL), our aim was to use this strategy in a mouse model of MDS we have created using transgenic mice bearing a mutant NRAS and overexpression of human BCL-2. Methods: We used our MDS-like triple transgenic mice bearing NRASD12/MMTVLTRtTA/TetoBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). We cloned part of the kanamycin resistance gene and adjacent pVax1 plasmid sequences flipped so the antisense sequences were inserted into the pVax1 vector (named Flipper). These sequences code for 5 polypeptides. We treated the MDS mice with either DNA alone, ATRA alone (10mg daily release for 21 days) or a combination of ATRA+DNA or as add-on therapy with 5-azacytidine (5-aza). 5-aza (5mg/kg) was administered 3 times a week and continued until death. Treatment with ATRA and/or DNA was initiated after 12 courses of 5-aza injections. Results and Conclusions: Sequencing of the Flipper insert predicted 5 peptides, which were synthesized and shown not to affect growth in vitro of myeloid leukemic NB4, K562 and p39 cell lines. In vivo injections of healthy mice with these peptides had no effect on mortality. The inserted sequences did not appear to have detrimental effects. The studies on our MDS mouse model show that DNA vaccination, alone or in combination with ATRA induces a long-term survival of the treated mice compared to untreated controls (p<0,0001). DNA vaccination results in an increase of interferon-gamma secretion as measured by ELISPOT, increased CD4+/CD44hi/CD62-Llo memory T cells in the peripheral blood and maintains stable disease as determined by the persistence of low peripheral blood platelet counts and the of the Mac-1hi/GR-1lo populations, which mark the primitive cells and the ERK phosphorylation signatures measured by the NanoPro (Cell Biosciences). Memory T-cell levels increased 3-fold (at 6% compared to 2% in healthy controls) and remained high. The DNA vaccine stabilized the disease and its protective effect may persist for up to 10 months with increased expression of MYD88, which is downstream of toll-like receptors, in long-term survivors, suggesting the activation of this DNA pathway. ATRA alone also had efficacy in this model. As there was no evidence of differentiation as the Mac-1hi/Gr-1lo blast cell population remained unchanged after treatment compared to the levels before treatment, it is possible that in this setting where the leukemic stem cells are pro-apoptotic, that ATRA is acting as an immuno-adjuvant and the antigen shedding that occurs allows cross presentation and induces immune responses potentiated by ATRA. However, the disease in this group progresses and the mice die earlier than the other treatment groups (mean survival of 4.5 months compared with the untreated group of 1 month, p=0.0016). Vaccination with an adjuvant DNA alone or in combination with ATRA, results in a significant extension of lifespan in MDS mice. As add-on therapy to 5-aza the response rate increases from 20% with 5-aza alone to 50% with either ATRA or DNA and 100% with the 5-aza, ATRA and DNA combination. These studies suggest that adjuvant therapy with ATRA+DNA may have a role in addition to conventional therapy in prolonging remissions and may be promising for clinical trials. Disclosures: Fenaux: Novartis, Janssen, Cilag, Roche, Amgem, GSK, Merck and Cephalon: Honoraria, Research Funding. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Padua:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Targeting Metabolic Inputs into Epigenetic Regulations of Acute Leukemia

Blood ◽

10.1182/blood.v122.21.sci-26.sci-26 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-26-SCI-26

Author(s):

Craig B. Thompson

Keyword(s):

Acute Leukemia ◽

Board Of Directors ◽

Research Funding ◽

Cellular Differentiation ◽

Cancer Metabolism ◽

Leukemic Cells ◽

Advisory Committees ◽

Control Of Gene Expression ◽

Idh Mutations ◽

Equity Ownership

Abstract An essential feature of acute myeloid leukemia (AML) is the profound block to differentiation exhibited by leukemic cells. This AML-associated arrest in the normal process of cellular differentiation is not observed in more chronic forms of leukemia. Chronic forms of leukemia and lymphoma are marked by the increased propensity to enter the cell cycle as the malignant cells move to more mature states of differentiation and the increased resistance to apoptosis these cells exhibit. Considerable recent evidence has suggested that profound epigenetic remodeling occurs as either a result or a cause of cellular differentiation. Altered DNA methylation has now been established as a hallmark of acute leukemia and yet very little is known concerning the mechanisms through which this occurs. In 2010, in collaboration with others, we found that neomorphic mutations of the citrate metabolism genes IDH1 and IDH2 induce DNA hypermethylation and impair differentiation in hematopoietic cells. IDH mutations create a block to DNA and histone demethylation as a result of the production of 2-hydroxyglutarate (2HG). 2HG acts as a competitive inhibitor of α-ketoglutarate-dependent enzymes. The epigenetic effects of 2HG are caused in part though inhibition of TET2, a DNA demethylase enzyme also mutated in leukemia. IDH 1/2- and TET2-mutant primary AML cells displayed a similar defect in epigenetic programming consisting of global hypermethylation and a gene-specific methylation signature. This work identifies IDH1/2- and TET2-mutant leukemias as a biologically distinct disease subtype, and links cancer metabolism with epigenetic control of gene expression. The implications of this work and the identification of additional metabolic genes involved in epigenetic deregulation in leukemia will be discussed. Disclosures: Thompson: NIH: Research Funding; Keystone Symposia: Consultancy; NCI: Research Funding; Damon Runyon : Research Funding; Ludwig: Membership on an entity’s Board of Directors or advisory committees; HHMI: Membership on an entity’s Board of Directors or advisory committees; Merck: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Agios: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; MSKCC: Employment; Pfizer: Patents & Royalties; BMS: Patents & Royalties; Repligen: Patents & Royalties.

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Venetoclax in Combination with Hypomethylating Agents Induces Rapid, Deep, and Durable Responses in Patients with AML Ineligible for Intensive Therapy

Blood ◽

10.1182/blood-2018-99-117179 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 285-285 ◽

Cited By ~ 11

Author(s):

Daniel A Pollyea ◽

Keith W. Pratz ◽

Brian A. Jonas ◽

Anthony Letai ◽

Vinod A. Pullarkat ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Leukemic Cells ◽

Research Report ◽

Hypomethylating Agents ◽

Employment Equity ◽

Advisory Committees ◽

Transfusion Independence ◽

First Response ◽

Equity Ownership

Abstract BACKGROUND: Acute myeloid leukemia (AML) has a median age of diagnosis of 68 years; however, patients who are ineligible for intensive induction chemotherapy have limited therapeutic options. Venetoclax (Ven), an oral agent that targets the antiapoptotic protein, BCL-2, has shown synergistic antileukemic activity when combined with hypomethylating agents (HMA), decitabine (Dec) and azacitidine (Aza); this combination has resulted in high rates of durable remission, which are independent of cytogenetic and molecular characteristics. METHODS: This is an open-label, phase 1b, dose escalation and expansion trial (NCT02203773) studying the safety and efficacy of venetoclax in combination with decitabine or azacitidine. Patients had previously untreated AML and were ineligible for intensive chemotherapy due to comorbidities and age. Here, we present the data from the expansion cohort, where patients were treated with 400 mg venetoclax in combination with either HMA. Venetoclax was administered daily in a three day ramp-up from 100 to 200 to 400 mg and coadministered with either 20 mg/m2 of intravenous (IV) decitabine on days 1-5 or 75 mg/m2 of IV or subcutaneous azacitidine on days 1-7 within each 28 day cycle. Dose adjustments for venetoclax were implemented for concomitant medications routinely used for prophylaxis with known drug-drug interactions. Safety and efficacy were evaluated. Time to first response, complete remission (CR), CR with incomplete blood count recovery (CRi), CR with partial hematologic recovery, duration of response, achievement of transfusion independence, overall survival (OS) and adverse events (AEs) were assessed. Minimal residual disease (MRD) was evaluated centrally by multicolor flow cytometry at a cutoff of 10-3 leukemic cells. Enrollment of patients treated with Ven + Aza began December 2014 and the majority was enrolled between January 2017 and June 2017; enrollment of patients treated with Ven + Dec occurred between November 2014 and June 2016, resulting in significantly different median follow-up time for the two treatment arms. RESULTS: Data cutoff was December 22, 2017. Of 115 patients treated with the 400 mg dose of venetoclax, 84 were treated with Ven + Aza and 31 received Ven + Dec. The median ages for patients treated with Ven + Aza and Ven + Dec, respectively, were 75 (range: 61-90) and 72 (range: 65-86). Overall, 25% and 29% had secondary AML, and 39% and 48% had poor cytogenetic risk, in patients treated with Ven + Aza and Ven + Dec, respectively. Transfusion dependence for red blood cells (RBC) or platelets within 8 weeks prior to venetoclax treatment was 64% (54/81) and 74% (23/31) in patients treated with Ven + Aza and Ven + Dec, respectively. Key grade ≥3 AEs across all patients were febrile neutropenia (44%), anemia (28%), pneumonia (25%), thrombocytopenia (22%) and neutropenia (18%). Median time on study treatment was 6.4 and 5.7 months, and median follow up was 8.2 (range: 0.4-35.5) and 16.2 (range: 0.7-36.7) months for patients treated with Ven + Aza and Ven + Dec, respectively. Key efficacy results are shown in the Table. Seventy percent and 74% of patients achieved CR/CRi, and the median time to first response was 1.2 and 1.9 months for patients treated with Ven + Aza and Ven + Dec, respectively. The median overall survival was 14.9 months for patients treated with Ven + Aza and 16.2 months for those treated with Ven + Dec. Among patients transfusion dependent at baseline, 52% (40/77) achieved transfusion independence from both RBC and platelets, defined as not receiving RBC or platelet transfusion for ≥56 days. Across both treatment groups, among patients with CR/CRi, 45% achieved MRD response less than 10-3 leukemic cells. Patients who received venetoclax dose reduction for CYP3A inhibitors had similar responses compared to those without dose reduction. CONCLUSIONS: Venetoclax in combination with either azacitidine or decitabine led to high rates of rapid and deep responses that were durable in patients with AML ineligible for standard induction chemotherapy. A majority of patients who were transfusion dependent at baseline achieved transfusion independence after initiating venetoclax therapy. These results suggest that venetoclax, in combination with hypomethylating agents, may provide a potent therapeutic option for patients with AML who are not eligible for intensive chemotherapy. Disclosures Pollyea: Pfizer: 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, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Curis: Membership on an entity's Board of Directors or advisory committees. Pratz:AbbVie: Consultancy, Research Funding; Agios: Research Funding; Astellas: Consultancy, Research Funding; Boston Scientific: Consultancy; Millenium/Takeda: Research Funding. Letai:AbbVie: Consultancy, Other: Lab research report; AstraZeneca: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; Flash Therapeutics: Equity Ownership. Wei:Servier: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Konopleva:Stemline Therapeutics: Research Funding. Frankfurt:AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rizzieri:GlaxoSmithKline: Research Funding. Xu:AbbVie, Inc: Employment, Equity Ownership. Dail:Genentech: Employment, Equity Ownership. Chyla:AbbVie, Inc: Employment, Equity Ownership. Potluri:AbbVie: Employment, Equity Ownership. DiNardo:Celgene: Honoraria; Agios: Consultancy; Karyopharm: Honoraria; Abbvie: Honoraria; Medimmune: Honoraria; Bayer: Honoraria.

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