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Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival, and they continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Cellular adhesion, growth, and apoptosis assays, along with measurements of expression of mitochondrial DNA and generation of mitochondrial reactive oxygen species indicated that direct interactions with BM stromal cells triggered compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, inhibition of OxPhos induced transfer of mitochondria derived from mesenchymal stem cells (MSCs) to AML cells via tunneling nanotubes under direct-contact coculture conditions. Inhibition of OxPhos also induced mitochondrial fission and increased functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, so we used electron microscopy to observe mitochondrial transport to the leading edge of protrusions of AML cells migrating toward MSCs. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased mitochondrial transfer of MSCs to AML cells triggered by OxPhos inhibition. Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells as well as endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

B-cell lymphoma extra large (BCL-XL) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis-targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL. The first BCL-XL-degrading PROTAC achieves safer and more potent antitumor activity than dual BCL-XL and BCL-2 inhibitor navitoclax because of reduced dose-limiting platelet toxicity and high target specificity.

The inferior cure rate of T-cell acute lymphoblastic leukemia (T-ALL) is associated with inherent drug resistance. The activating NOTCH1 gene mutations have been reported to cause chemoresistance at the stem cell level1. Direct NOTCH1 inhibition has failed in clinical trials due to a narrow therapeutic window but targeting key oncogenic and metabolic pathways downstream of mutated NOTCH1 may offer novel approaches. We previously reported that rapid transformation of thymocytes at the DN3 differentiation stage into preleukemic stem cells (pre-LSC) requires elevated Notch1 in addition to the presence of Scl/Lmo11. Notably, we showed that cellular metabolism of NOTCH1-mutated T-ALLs depends on Oxidative Phosphorylation (OxPhos) and that OxPhos inhibition using the complex I inhibitor IACS-010759 (OxPhos-i) is efficacious in NOTCH1-mutated T-ALL patient derived xenografts (PDXs)2. Here, we investigated the link between NOTCH1-mutated chemoresistance and OxPhos in pre-leukemic and leukemic cells, utilizing comprehensive molecular and functional assays. We hypothesized that chemotherapy aided by OxPhos-i overcomes chemoresistance, depletes LSCs and combats T-ALL. First, we analyzed the role of OxPhos in downstream Notch1 targets at the pre- and leukemic stage considering four stages of thymocyte differentiation (D1-D4), in a mouse model of human T-ALL1. Gene set enrichment analysis (GSEA) implicated increased expression of Notch1 target genes starting at DN1, and OxPhos target genes were the highest-ranked gene set at DN3. Next, activation of Notch1 by its ligand DL4 and inhibition of OxPhos reduced viability of pre-LSCs, indicating that ligand-dependent activation of Notch1 signaling upregulates the OxPhos pathway and sensitizes pre-LSCs to OxPhos-i. To clarify the role of Notch1 signaling, we examined the effect of IACS-010759 on pre-leukemic thymocytes harboring LMO1, SCL-LMO1, NOTCH1, LMO1-NOTCH1 and SCL-LMO1-NOTCH1 with and without DL4 stimulation. We found that in the absence of DL4, only thymocytes harboring the Notch1 oncogene responded to OxPhos-i, whereas all DL4-stimulated thymocytes responded regardless of Notch1 status (Fig. 1a). In addition, at the leukemic stage, we found elevation of the OxPhos pathway driven by oncogenic Notch1 when we compared transcriptomes of SCL-LMO1 induced T-ALL in the presence or absence of the NOTCH1 oncogene. In line with the murine T-ALL NOTCH1 model, we performed transcriptome analysis of two independent T-ALL patient cohorts prior to chemotherapy, COG TARGET ALL (n=263) and AALL1231 (n=75), comparing transcriptomes of NOTCH1-mutated vs NOTCH1-wt T-ALLs. We found co-segregation of NOTCH1 mutations with significant upregulation of OxPhos and TCA cycle genes and downregulation of apoptosis signaling. Aiming to reverse the NOTCH1-controlled anti-apoptotic program and chemoresistance, we next tested the combination of Vincristine, Dexamethasone and L-Asparaginase (VXL) with IACS-010759. When compared to vehicle, OxPhos-i or VXL alone, only the VXL-OxPhos-i treatment caused an energetic crisis indicated by decreased OCR and ECAR (Seahorse), which translated to a profound reduction of viability (CTG, flow cytometry) in T-ALL cell lines (n=9) and primary T-ALL samples (n=5). Additionally, the IACS-VXL combination in vivo resulted in pan-metabolic blockade, which caused metabolic shut-down and triggered early induction of apoptosis in leukemic cells in peripheral blood, spleen and bone marrow (Fig. 1b). Single cell Proteomic analysis (CyTOF) of spleen showed reduced expression of cell proliferation marker -ki67, c-myc, ERK and p38 proteins, and reduction in number of leukemic cells. Finally, this combination therapy resulted in reduced leukemia burden and extension of overall survival across all three aggressive NOTCH1-mutated T-ALL PDX models (p Disclosures Jabbour: Pfizer: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding. Teachey:Sobi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; La Roche: Consultancy. Rezvani:Takeda: Other: Licensing agreement; GemoAb: Membership on an entity's Board of Directors or advisory committees; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Virogen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Other: Educational grant; Affimed: Other: Educational grant; Formula Pharma: Membership on an entity's Board of Directors or advisory committees. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Lorenzi:Precision Pathways: Consultancy. Konopleva:Calithera: Research Funding; Kisoji: Consultancy; AbbVie: Consultancy, Research Funding; Sanofi: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Rafael Pharmaceutical: Research Funding; Eli Lilly: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Agios: Research Funding; AstraZeneca: Research Funding; Ablynx: Research Funding; Forty-Seven: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Ascentage: Research Funding.

Dual bromodomain BET inhibitors that bind with similar affinities to the first and second bromodomains across BRD2, BRD3, BRD4, and BRDT have displayed modest activity as monotherapy in clinical trials. Thrombocytopenia, closely followed by symptoms characteristic of gastrointestinal toxicity, have presented as dose-limiting adverse events that may have prevented escalation to higher dose levels required for more robust efficacy. ABBV-744 is a highly selective inhibitor for the second bromodomain of the four BET family proteins. In contrast to the broad antiproliferative activities observed with dual bromodomain BET inhibitors, ABBV-744 displayed significant antiproliferative activities largely although not exclusively in cancer cell lines derived from acute myeloid leukemia and androgen receptor positive prostate cancer. Studies in acute myeloid leukemia xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable with the pan-BET inhibitor ABBV-075 but with an improved therapeutic index. Enhanced antitumor efficacy was also observed with the combination of ABBV-744 and the BCL-2 inhibitor, venetoclax compared with monotherapies of either agent alone. These results collectively support the clinical evaluation of ABBV-744 in AML (Clinical Trials.gov identifier: NCT03360006).

Background: Malignant cells exploit cellular metabolism for their survival through various mechanisms. A significant dependency of malignant myeloid cells on glutamine metabolism has been demonstrated in vitro. Glutamine can be metabolized by glutaminase to form glutamate, a precursor for the citric acid cycle. CB-839 is an oral allosteric inhibitor of glutaminase that has been shown to reduce the level of glutamate in AML cells, with a consequent decrease in oxidative phosphorylation and survival. We report an updated interim analysis of a phase Ib/II clinical study evaluating the safety and efficacy of CB-839 in combination with azacitidine for the treatment of patients with MDS. Methods: This is a single arm phase Ib/II trial of CB-839 in combination with azacitidine (AZA) for patients with intermediate and high-risk MDS. Eligibility includes adult patients with high-risk MDS (IPSS Int-2 or high risk) or Int-1 risk MDS with high-risk mutations in TP53, ASXL1, EZH2, or RUNX1. Patients with prior hypomethylating agent (HMA) treatment were eligible. The primary outcome for the phase Ib portion was to identify the safety and recommended phase II dose of CB-839 in combination with AZA, confirmed as 600mg bid continuously with standard 7-day AZA dosing. The primary outcome for the phase II portion is clinical efficacy using IWG response criteria. Secondary endpoints include evaluation of pharmacokinetics (PK), drug exposure levels, and overall survival. Results: 23 patients with MDS were enrolled between December 2017 and June 2020. Baseline characteristics are listed in Table 1. The median age was 71 (range, 49-83). 7 patients had prior HMA exposure (median 6 prior cycles, range 4-9), and 1 patient was previously treated with a non-HMA regimen. 8 patients (34.7%) had therapy-related MDS. 9 patients (39.1%) had complex cytogenetics. The most frequent mutations were ASXL1 (43.5%), TET2 (43.5%), TP53 (34.8%), and RUNX1 (30.4%). The most common drug-related non-hematologic adverse events (AEs) of any grade were nausea (52.2%), constipation (39.1%), fatigue (26.1%), anorexia (17.4%), and generalized weakness (17.4%). The most common therapy-related grade >3 AEs were transaminitis (13%), thrombocytopenia (26%), and neutropenia (30.4%). Five patients (21.7%) required dose reduction(s) of CB-839, with reductions due to transaminitis (n=2), myelosuppression (n=2), weakness (n=2), nausea (n=1), and fatigue (n=1). 30 and 60 day mortality were 0% and 4.3%, respectively. Best responses are detailed in Table 2. Objective response was defined as CR, morphologic CR (mCR), or hematologic improvement (HI), and was achieved in 15 out of 23 (65.2%) patients, including 5 CR and 10 mCR with or without HI. CR/mCR/HI rate was 62.5% (5/8) in previously-treated patients. Median number of cycles received was 4 (range, 1-17). 5 patients proceeded to allogeneic stem cell transplant. At median follow-up of 10.3 months (range, 1.0-28.5), 6 patients remain on study. Median OS (mOS) has not been reached; 1 year overall survival is 53.1% (Figure 1). Patients with TP53 mutations had CR/mCR rate of 62.5% (5/8) and mOS of 8 months. Patients with complex cytogenetics had CR/mCR rate of 66.7% (6/9) and mOS of 10.1 months. Bone marrow samples from a subset of patients were analyzed by multiparameter flow cytometry to fractionate phenotypically defined malignant leukemic stem cells (LSCs) as well as blast populations. We isolated hematopoietic stem and progenitor cells (HSPC, Lin−CD34+CD38−) expressing at least one LSC marker (CD45RA, CD123, or IL1RAP). At the same time, we isolated HSPCs that were triple-negative for CD45RA, CD123, and IL1RAP to enrich for pre-malignant stem cells. Analysis of representative samples showed a reduction in LSCs after treatment. We also observed an increase in progenitor cells (Lin-CD34+CD38+), showing increased differentiation after CB-839 treatment. This pattern was seen in 5 responders examined. We did not observe a reduction in LSCs in patients that did not respond clinically. Conclusions: CB-839 in combination with AZA is a safe and effective regimen for patients with advanced MDS. Encouraging responses were seen in previously-treated and genomically high-risk patients. Of interest, CB-839 demonstrated in vivo activity against LSCs, with an increase in myeloid progenitor cells consistent with myeloid differentiation in responding patients. The trial continues to accrue. (NCT03047993) Disclosures Konopleva: Agios: Research Funding; Ablynx: Research Funding; Calithera: Research Funding; Cellectis: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Amgen: Consultancy; Forty-Seven: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Sanofi: Research Funding. Borthakur:PTC Therapeutics: Consultancy; GSK: Research Funding; PTC Therapeutics: Research Funding; BioTherix: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; Abbvie: Research Funding; Jannsen: Research Funding; Oncoceutics: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Curio Science LLC: Consultancy; FTC Therapeutics: Consultancy; Xbiotech USA: Research Funding; Polaris: Research Funding; AstraZeneca: Research Funding; BioLine Rx: Consultancy; Cyclacel: Research Funding; Argenx: Consultancy; BioLine Rx: Research Funding; BMS: Research Funding. Jabbour:Adaptive Biotechnologies: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding; Pfizer: Other: Advisory role, Research Funding. Pemmaraju:Stemline Therapeutics: Honoraria, Research Funding; Celgene: Honoraria; Pacylex Pharmaceuticals: Consultancy; Affymetrix: Other: Grant Support, Research Funding; Samus Therapeutics: Research Funding; Cellectis: Research Funding; DAVA Oncology: Honoraria; Plexxikon: Research Funding; Incyte Corporation: Honoraria; SagerStrong Foundation: Other: Grant Support; Daiichi Sankyo: Research Funding; MustangBio: Honoraria; AbbVie: Honoraria, Research Funding; Roche Diagnostics: Honoraria; Blueprint Medicines: Honoraria; Novartis: Honoraria, Research Funding; LFB Biotechnologies: Honoraria. Kadia:Novartis: Honoraria; Cyclacel: Research Funding; Pfizer: Honoraria, Research Funding; Cellenkos: Research Funding; Genentech: Honoraria, Research Funding; JAZZ: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Celgene: Research Funding; Incyte: Research Funding; Amgen: Research Funding; BMS: Honoraria, Research Funding; Astellas: Research Funding; Ascentage: Research Funding; Pulmotec: Research Funding; Astra Zeneca: Research Funding. Short:Takeda Oncology: Consultancy, Honoraria, Research Funding; Amgen: Honoraria; Astellas: Research Funding; AstraZeneca: Consultancy. Burger:AstraZeneca: Consultancy; Pharmacyclics, an AbbVie company: Consultancy, Research Funding, Speakers Bureau; Gilead Sciences: Consultancy, Research Funding; Janssen Pharmaceuticals: Consultancy, Speakers Bureau; Beigene: Research Funding, Speakers Bureau; TG Therapeutics: Research Funding, Speakers Bureau. Daver:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: 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; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: 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; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding. Kantarjian:Abbvie: Honoraria, Research Funding; Delta Fly: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria; Ascentage: Research Funding; Sanofi: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Research Funding; BioAscend: Honoraria; Adaptive biotechnologies: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; BMS: Research Funding; Aptitute Health: Honoraria. Garcia-Manero:AbbVie: Honoraria, Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Amphivena Therapeutics: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; H3 Biomedicine: Research Funding; Jazz Pharmaceuticals: Consultancy; Merck: Research Funding; Novartis: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onconova: Research Funding. Verma:Medpacto: Research Funding; acceleron: Consultancy, Honoraria; Janssen: Research Funding; BMS: Consultancy, Research Funding; stelexis: Current equity holder in private company. DiNardo:Jazz: Honoraria; ImmuneOnc: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Syros: Honoraria; Novartis: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; MedImmune: Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding. OffLabel Disclosure: CB-839, glutaminase inhibitor, investigation for use in advanced MDS

Leukemic blasts and stem cells in AML express high levels of CD123 compared to normal hematopoietic stem and progenitor cells, making CD123 a potential therapeutic target (Kovtun, Blood advances, 2018). IMGN632, a novel CD123-targeting ADC with a humanized IgG1 antibody with high affinity to CD123 and with a DNA single stranded break inducing alkylating payload, demonstrated clinical activity as a monotherapy in patients with relapsed/refractory AML and BPDCN (NCT03386513). We previously reported that the combination of IMGN632 with venetoclax (VEN, a BCL-2 inhibitor) induced additive or synergistic anti-leukemia effects in AML cell lines (EHA 2019, abstract #PF201). Given the FDA approval of the venetoclax and azacitidine (AZA, a hypomethylating agent) combination establishing this regimen as standard-of-care for elderly unfit AML patients, we further characterized the efficacy of the triple combination of IMGN632, VEN and AZA in AML models in vitro and in vivo. We first tested the cytotoxic effects of IMGN632 alone, VEN+AZA and the triple combination of IMGN632+VEN+AZA in AML cells lines. MOLM-13 and MV4-11 cells treated with varying doses of IMGN632 for 48 hours and VEN + AZA for 24 hours, reduced cell viability in a concentration- dependent manner. Treatment with the triple combination induced greater apoptosis and increased cell death when compared to IMGN632 alone or VEN+AZA, with the triple combination demonstrating synergism (CI=0.26) (Fig A-B). This was supported by western blot analysis in which MOLM-13 cells showed upregulation of apoptotic proteins such as cleaved-PARP, PARP, cleaved-caspase 3 and DNA damage markers, p-H2AX and ICAD in the triple combination. We next tested the efficacy of the triple combination of IMGN632, VEN and AZA in four AML PDX models. We injected AML PDX cells into NSG mice and upon engraftment in peripheral blood, randomized mice into four cohorts to receive vehicle, IMGN632 (0.120 mg/kg once a week x 3 weeks), VEN (100 mg/kg, 5 days/week x 3 weeks) with AZA (2.5 mg/kg, daily x 5 days) or the triple combination. The endpoints included circulating leukemia burden monitored by hCD45/hCD123 flow cytometry of serial peripheral blood (PB) samples and mice survival. WIMGN632 alone reduced leukemia burden and extended survival in all 4 CD123+ PDX models tested (median overall survival of 131 ± 74 days (range, 74-259) for IMGN632 vs. 59 ± 7 days (range 49-69) for vehicle). The triple combination showed superior anti-leukemic efficacy in all four PDX models, compared to IMGN632 or VEN+AZA alone (triple combination median overall survival of 152 days ± 99 days (range 41-313) vs. 131±74 days in IMGN632 (range 74-259) and 73 ± 22 days (range 51-110) in VEN+AZA. In two models refractory to VEN+AZA, triple therapy significantly exceeded efficacy of IMGN632 monotherapy, indicating synergy and potential to overcome VEN+AZA resistance with the triplet; no tumor cells were detected in the triple-treated cohort in PDX 3912018 when the experiment was terminated at day 313 (Fig C and D) . In one model sensitive to VEN+AZA, the triple combination showed superior anti-leukemic efficacy compared to VEN+AZA or IMGN632 monotherapy (Fig E). In DFAM-55517 PDX model, western blot analysis of enriched hCD45+ cells from the spleen of mice collected 48 hours after the administration of one dose of IMGN632 and two doses of VEN+AZA (n=3/group) showed an upregulation of apoptotic proteins, cleaved-PARP and cleaved caspase-3, and upregulation of the DNA damage marker p-H2AX in the triple combination group (Fig. F). In summary, combining IMGN632 with VEN and AZA induced synergistic cell death in AML cell lines and significantly improved survival in multiple AML PDX models compared with AZA+VEN doublet or IMGN632 monotherapy. These data support the addition of a CD123-targeted ADC with a novel DNA-damaging payload to the standard of care, AZA+VEN in AML patients. The combination of IMGN632 and Venetoclax and/or Azacitidine is being tested in an ongoing Phase Ib/II clinical trial (NCT04086264). Disclosures Daver: Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: 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; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: 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; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding. Sloss:ImmunoGen, Inc.: Current Employment. Zweidler-McKay:ImmunoGen, Inc.: Current Employment. Konopleva:Cellectis: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Stemline Therapeutics: Consultancy, Research Funding; Eli Lilly: Research Funding; Sanofi: Research Funding; Genentech: Consultancy, Research Funding; Kisoji: Consultancy; AstraZeneca: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Agios: Research Funding; Ablynx: Research Funding; Amgen: Consultancy; Forty-Seven: Consultancy, Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ascentage: Research Funding.

Despite recent approval of hypomethylating agent/venetoclax (HMA+VEN) therapy for older patients (pt) with AML unfit for induction chemotherapy, their outcomes remain suboptimal. Such pts have a median overall survival of only 14 months and only approximately 35% of pts enjoying long-term survival (DiNardo, EHA 2020). Approximately 20-40% of newly diagnosed older pts with AML do not respond to the HMA+VEN regimens, with higher rates of refractory disease as well as early relapse in high-risk pts. Metabolic reprogramming and dependence on mitochondrial oxidative phosphorylation (OxPhos) is a core feature of AML leukemia stem cells (LSC). Recent reports have shown that upregulation of OxPhos confers intrinsic and acquired resistance to VEN in AML, multiple myeloma and lymphoid malignancies, which can be reversed by disrupting OxPhos; mutated TP53 has been shown to confer intrinsic resistance to venetoclax through increased OxPhos (Nechiporuk T, et al. Cancer Discovery 2019). We hypothesized that combined blockade of mitochondrial fitness by OxPhos inhibitors and of BCL-2 with VEN/azacitidine (AZA) will perform synergistically in pre-clinical AML models. To test this hypothesis, we utilized a novel complex I inhibitor IACS-010759 that effectively inhibits cell respiration and leukemia progression in the in vitro and in vivo AML pre-clinical models (Molina, Nat Med 2018). Priming of MOLM-13 cells with 20nM VEN for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while single agents reduced OCR by 70%) of viable cell numbers in several AML cell lines tested (OCI-AML2, MV-4-11, and MOLM-13) (Figure 1). By co-immunoprecipitation VEN disrupted interaction of BCL-2 with the mitochondrial protein VDAC, known to regulate ADP/ATP exchange during electron transport across mitochondria membrane; this resulted in dramatic reduction of the intracellular ATP and CTP levels measured by MS-based metabolomics. Further, VEN increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of VEN and IACS-010759, possibly because of RNA degradation. In primary AML samples (n=3) and AML PDX cells (n=4) cultured ex vivo, combined VEN and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. We next tested the efficacy of the "triple" combination of VEN, AZA and IACS-010759 in the in vivo AML PDX models. We injected AML PDX cells 3871344 (with no mutations identified by targeted sequencing) and 4404778 (harboring IDH1, NPM1, FLT3-ITD mutations) into NRG mice and upon engraftment, randomized mice into 4 groups to receive 2 cycles of treatment with 3 weeks interruption between cycles: vehicle, VEN (50mg/kg daily, 5 days on/2 days off, day 1-21) with AZA (2.5 mg/kg daily, day 1-7), IACS-010759 (1mg/kg ?daily, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. In the less aggressive model 3871344, all therapies reduced circulating leukemia burden, and the triple treatment achieved best efficacy, with average circulating tumor burden at 10 weeks after cycle 2 of 90.8%, 38.7%, 68.8% and 7.4% in vehicle, VEN+AZA, IACS-010759, and triple-therapy cohorts, respectively. In the aggressive model 4404778, the treatments were less effective, but the combination offered highest activity, with average circulating tumor burden of 71.0%, 52.3%, 88.7% and 39.9% in vehicle, IACS-010759, VEN+AZA and triple-therapy cohorts, respectively (Figure 1). Analysis of survival and additional PDX models are ongoing and will be reported. In summary, our study demonstrates that BCL-2 modulates mitochondrial respiration and mitochondrial ATP generation in addition to its established anti-apoptotic role. VEN disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitro and in vivo. Disclosures Konopleva: Ascentage: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Ablynx: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Kisoji: Consultancy; AstraZeneca: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding.

Background The long-term survival of AML, especially of older AML patients, under the established therapies remains poor. A recent breakthrough therapy of selective BCL-2 inhibitor venetoclax with hypomethylating agents (HMA) azacitidine or decitabine is approved to treat older and unfit patients with AML (DiNardo et.al. EHA 2020). This combination induces responses in > 65% of older AML patients, but the responses in relapsed/refractory AML patients are sub-optimal. Upregulation of programmed death-1 (PD-1) in T-cells is associated with AML immune-suppression, and combination of anti-PD-1 with azacitidine has activity in relapsed/refractory AML (Daver et. al. Cancer Discovery 2019). Recent pre-clinical findings indicate that venetoclax preserved T-cells immunity by sparing central memory T-cells and enhanced activity of anti-PD-1 antibodies in immune-competent mouse models in vivo (Mathew et.al. Blood 2018). In this study, we tested the hypothesis that PD-1 inhibition facilitates the depth and duration of response to venetoclax and HMA combination by reactivating T-cells mediated immunity against AML. Results We collected peripheral blood (PB) samples from trial patients before and after receiving the treatment of decitabine and venetoclax (Dec_Ven) (NCT03404193) and examined the percentage of AML blasts and T-cells by multi-parameter flow cytometry. The combination of Dec_Ven effectively reduced PB CD33+/CD34+cells from 46 ± 15% at baseline (BL) to 27 ± 16% at 2.3 days (Day 1 - 3) (time point 1, or T1), and 15 ± 13% at 5.5 days (Day 3 - 8) (T2), and increased CD3+ T-cells: 28 ± 10% (BL), 54 ± 13% (T1) and 59 ± 19 % (T2), (BL vs.T1, CD33/CD34+, p = 0.001; CD3+, p = 0.02, n = 6). The reduction of CD33+/CD34+ positively correlated with the depletion of circulating blasts (R = 0.64, p = 0.0001). Further analyzing the T-cells subsets among CD4helper cells and CD8cytotoxic cells, we found that Dec_Ven therapy promoted an activated T-cells phenotype by upregulating CD69 and PD-1 in both CD4and CD8cells in early time point samples, and led to T-cells exhaustion, as indicated by persistent expression of PD-1 at later time-point when CD69 was undetectable (PD-1 in CD3+CD4+ cells: 13 ± 2%, n = 6 (BL), 15 ± 3%, n = 6 (T1) and 22 ± 7%, n = 5 (T2); PD-1 in CD3+CD8+ cells: 14 ± 3%, n = 6 (BL), 27 ± 5%, n = 6, (T1), 29 ± 9%, n =5 (T2), BL vs. T1, p = 0.05). The persistent expression of PD-1 is a hallmark of T-cells exhaustion and immunosuppression (Jia et.al. Blood Cancer J 2018). Mass cytometry CyTOF immune-profiling of various compartments of CD4and CD8cells on PB uncovered that Dec_Ven reduced naïve T-cells (CD45RA+CCR7+) but spared or even increased frequencies of the central (CD45RA-CCR7+) and effector memory (CD45RA-CCR7-) CD4 or CD8 cells. In parallel, we measured the serum cytokines levels, and found that treatment of Dec_Ven increased IL2, IL6 and INFg secretion in PB. IL2 and IL6 are two cytokines that regulate the proliferation and differentiation of effector memory T-cells; and INFg is a critical mediator of treatment-induced immune response. To further test if targeting PD-1 induced anti-leukemia effect and amplified the effect of Dec_Ven, we treated leukemia cells collected before and after Dec_Ven therapy with anti-CD3 and anti-PD-1 antibodies in vitro. Treatment reduced CD33+/CD34+ cells in 2 of 3 pre-treatment samples (untreated vs. anti-CD3_PD-1: 55% vs. 15%, pt #6; 70% vs. 52%, pt #7) and synergistically reduced/eliminated the residual CD33+/CD34+ cells in AML samples obtained after Dec_Ven therapy (untreated vs. anti-CD3_PD-1: 84% vs. 66% Day 3, pt #3; 18% vs. 8% Day 3, pt #6; 44% vs. 35% Day 1, 5% vs. 2% Day 4, pt #7) (Fig.1). Conclusions Our data indicate that Dec_Ven therapy induces PD-1 expression on T-cells but selectively preserves potentially important anti-tumor T-cells immune subsets. The "triple" combination therapy of HMA with BCL-2 and PD-1 antagonists may facilitate anti-leukemia responses by enhancing T-cells function. Clinical trial to test this hypothesis is ongoing in relapsed/refractory AML patients (NCT02397720). Disclosures DiNardo: Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Jazz: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Novartis: Consultancy; MedImmune: Honoraria; Takeda: Honoraria; Syros: Honoraria; ImmuneOnc: Honoraria; Notable Labs: Membership on an entity's Board of Directors or advisory committees. Daver:Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: 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; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: 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; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Konopleva:Agios: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Sanofi: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Eli Lilly: Research Funding; Calithera: Research Funding; Kisoji: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Ablynx: Research Funding; AbbVie: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Genentech: Consultancy, Research Funding.

Context IMGN632, a novel CD123-targeting ADC, demonstrates a favorable safety profile and complete remissions as a monotherapy in patients with relapsed/refractory AML and BPDCN ( NCT03386513 ). In pre-clinical models, IMGN632 has been shown to be synergistic with venetoclax (BCL-2 inhibitor) (EHA, 2019, abstract #PF201). Objective Given the recent approval of the combination of azacitidine (AZA, a hypomethylating agent) and venetoclax (VEN) in AML patients unfit for intensive chemotherapy, we investigated the combination of IMGN632 with AZA and the triple combination of IMGN632, AZA, and VEN in preclinical models. Results IMGN632, AZA, and VEN were highly active and prolonged survival in a cell line-derived AML model and patient-derived AML xenograft (PDX) models. In EOL-1, treatment with the triple combination IMGN632+AZA+VEN was the most active and resulted in 100% reduction in tumor growth inhibition (T/C) when compared to Vehicle, IMGN632, and AZA+VEN treatments. In PDX 4023126, the triple combination group survived the longest and was highly active with 74-day tumor growth delay (T-C) and 128% increased life span (% ILS) demonstrating highest survival benefit over IMGN632 (33-day T-C; 57% ILS), and AZA+VEN (52-day T-C; 90% ILS). In PDX model, 4079574, the triple combination was highly active (62-day T-C; 105% ILS) compared to both IMGN632 alone (15-day T-C; 25.4% ILS) and VEN+AZA (1-day T-C; 1.7% ILS). In model 3912018, VEN+AZA (2-day T-C; 3% ILS) was not very active, and strikingly, the triple combination (231-day T-C; 335% ILS) was highly active and prolonged survival the most compared to IMGN632 alone (190-day T-C; 275% ILS) with all the mice surviving for >300 days. Conclusion The addition of IMGN632 to AZA+VEN in multiple AML xenograft and PDX models led to improved survival. These data support the addition of a CD123-targeted ADC with a novel DNA-damaging payload to standard of care, AZA+VEN in AML patients. The combination of IMGN632 and Venetoclax and/or Azacitidine is currently being tested in a Phase1b/2 clinical trial ( NCT04086264 ).

Background: Genetic alterations in the osteoprogenitor cells has been shown to induce acute myeloid leukemia (AML) in several mouse models. Moreover, we have recently reported that AML cells induce osteogenic differentiation in mesenchymal stromal cells (MSC) to gain growth advantage in the bone marrow (BM; Battula et al., JCI Insight, 2017). However, the effect of AML cells on bone homeostasis/turnover and its impact on AML progression is unknown. Here, we hypothesize that AML cells expand osteoprogenitor cells and alter the balance between bone formation and resorption. Methods: To investigate the effect of AML cells on osteoprogenitor cells and mature osteoblasts, we used triple transgenic reporter mice with the genotype Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. Murine AML cells with MLL-ENL translocation were implanted into these transgenic mice, and Osteoprogenitor (Osx+) cells and mature osteoblasts (Ocn+) in femurs were measured by confocal microscopy. To investigate the effect of human AML cells on bone composition, patient-derived xenograft (PDX) cells were implanted into non-obese diabetic scid interleukin-2Rγnull (NSG) mice and bone histomorphometry was performed using H&E and Goldner's trichrome staining. Computed tomography (micro-CT) was used to measure bone volume (BV) and mineral density (BMD) in mice. Tartrate-resistant acidic phosphatase (TRAP) staining was performed to measure osteoclast number/activation. Finally, bone density on the vertebral bone (T12) was measured in 263 de-novo AML patients and 23 normal individuals by CT imaging. Results: In transgenic mice implanted with syngenic AML cells, we found a 3-4 fold increase in Osterix+, but not Osteocalcin+ cells, suggesting AML cells expand osteoprogenitor cells in the BM during short term exposure (2-3 weeks). To investigate the effects of AML on bone during a long term exposure, we implanted 10 different AML-PDX models in NSG mice (3 mice per model) and analyzed femurs by micro-CT and bone histomorphometry analysis. Interestingly, we observed a dramatic increase in new web-like medullary bone formation in 5/10 of the PDX models tested. Moreover, higher bone volume is associated with less aggressive PDX models (which takes 4-6 months to reach 90% circulating leukemia), but not aggressive PDX models (only 4-8 weeks to reach 90% circulating cells). These findings were also confirmed by micro-CT of mouse femurs. Interestingly, in some PDX models, CT images revealed large cavities in cortical bones close to epiphysis and metaphysis areas in the femur and tibia of mice with AML suggesting bone resorption. To validate bone resorption, we performed TRAP staining and found a significant increase in osteoclast activity on the endosteal surface and massive bone resorption in AML bone compared to normal bone. These data indicate high bone turnover in mice with AML compared to control mice. Next, we measured bone densities in AML patients and normal individuals by chest CT imaging. We found bone densities were gradually decrease with age in both healthy individuals and AML patients. However, compared to healthy individuals, bone densities are significantly higher in majority of AML patients (~70%) (p Conclusion: Our data suggest that AML cells expand osteoprogenitor-rich niche and alter BM microenvironment by high bone turnover. New bone induction by less aggressive AML-PDX models coupled with AML patient CT data suggests that high bone density and volume are associated with favorable prognostic factors in AML. Mechanisms underlying this observation are under investigation. Disclosures Andreeff: Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees. Konopleva:Ablynx: Research Funding; AstraZeneca: Research Funding; Kisoji: Consultancy; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Amgen: Consultancy; Agios: Research Funding; Sanofi: Research Funding; Cellectis: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding. Battula:Leukemia Lymphoma Society: Research Funding; Tolero Pharmaceuticals: Research Funding; Golfers Against Cancer: Research Funding.

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival and continually adapt to the bone marrow (BM) microenvironment. We investigated how the BM microenvironment impacts the response to energy-depriving OxPhos inhibition in AML using a novel complex I OxPhos inhibitor (OxPhosi), IACS-010759. We have reported that OxPhosi-resistant primary AML samples demonstrated higher baseline transcription of genes related to cell adhesion, integrin and/or Rho GTPase family genes that modulate intracellular actin dynamics. (Yang et al. ASH 2019) In this study, we performed Cap Analysis of Gene Expression (CAGE) transcriptome analyses using IACS-010759-sensitive and -resistant AML PDXs. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts (= transcription start sites) and allows investigating promoter structures necessary for gene expression. Primary AML cells from 9 AML PDXs were injected into irradiated NSG mice, which were randomized upon documented engraftment to receive IACS-010759 or vehicle (n = 3/group). The antileukemia efficacy of the treatment was monitored by serial measurements of circulating AML cells. Of the 9 models tested, we defined 4 PDXs as sensitive and 5 as resistant to OxPhos inhibitor therapy. In the resistant models, CAGE analysis of OxPhosi-induced changes (comparing pretreatment with posttreatment) identified upregulation of 77 promoters and downregulation of 207 promoters (log 2-fold change > 3.0, FDR < 0.05, EdgeR), including increased promoter expression (>3.0 fold) of genes associated with adhesion (CCR8,ADGRB2, LAG3, BMF, ATN1, PLXDC1), migration (CCR8, NKX3-2, TMEM123, IGLV7-43, FAM171A1, LBX2, TRAV21, PPP2R5C, BMF, PLXDC1), and actin cytoskeleton dynamics (FAM171A1, BMF, BEST1, PLXDC1). Of note, the 6 adhesion-associated promoters that were upregulated by OxPhosi in 5 of the OxPhosi-resistant mouse models were unchanged or downregulated in the 4 OxPhosi-sensitive models. We then used DEGseq, an R package for identifying differentially expressed genes, to identify promoters whose expression was different between OxPhosi-treated and vehicle-treated groups in the OxPhosi-resistant mouse models. DEGseq detected consistent changes of 214 upregulated and 626 downregulated promoters with OxPhosi treatment in all 5 mouse models. KEGG pathway enrichment analysis was performed with these consistently changed genes and revealed that OxPhos inhibitor treatment significantly upregulated the transcripts of cell adhesion pathway. We then confirmed that BM derived mesenchymal stem cells (MSC) protected OxPhosi-sensitive OCI-AML3 cells; the IC50 of IACS-010759 under MSC coculture was 80-fold higher than in monoculture conditions (IC50; 0.04 nM in monoculture vs. 3.25 nM in coculture), and IACS-010759 (10nM) induced 55% reduction of viable cells in coculture condition as compared to 70% reduction in monoculture. We further observed that OCI-AML3 cells adhered to MSCs were more profoundly protected from OxPhosi induced apoptosis than nonadherent cells. These results indicate that BM stromal cells, in particular those in direct contact with leukemia cells, play a key role in the microenvironment-mediated protection of AML cells from metabolic stress caused by OxPhos blockade. We further observed promoter upregulation of ASS1, coding Argininosuccinate Synthase 1 and of LRP1, coding LDL Receptor Related Protein 1. Argininosuccinate Synthase 1 is an epigenetically regulated key enzyme in the biosynthesis of arginine and energy starvation that induces adaptive transcriptional upregulation of ASS1. LDL Receptor Related Protein 1 plays a major role in lipid metabolism and has been reported to be responsible for hemin-induced autophagy in leukemia cells. These might contribute to intrinsic AML resistance to OxPhosi via activation of compensatory metabolic pathways, amino acid metabolism and lipid metabolism. Taken together, our data highlight the importance of direct interaction with BM stromal cells as well as complementally modification of amino acid- and lipid metabolism for the resistance of AML cells to OxPhos inhibition. While the mechanisms of stroma-leukemia interactions are likely complex, reducing the adhesion of AML cells to nurturing stromal cells ameliorates the resistance to the metabolic and energetic consequences of OxPhos inhibition. Disclosures Andreeff: Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding. Konopleva:Rafael Pharmaceutical: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Sanofi: Research Funding; AstraZeneca: Research Funding; Cellectis: Research Funding; AbbVie: Consultancy, Research Funding; Ablynx: Research Funding; Agios: Research Funding; Ascentage: Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Kisoji: Consultancy; Calithera: Research Funding.

Context We have reported that AML cells induce osteogenic differentiation in MSCs to facilitate AML growth in the bone marrow (BM) (Battula et al. JCI Insight, 2017). However, the effect of AML cells on various osteo-lineage cells in the BM microenvironment is not known. Objective i) To assess the stage-specific subsets of osteoblast lineage induced by AML cells in BME. ii) To investigate the effect of new bone formation using AML patient-derived-xenograft (PDX) mouse model. Design In this study, we used inducible triple transgenic mice with osterix (Osx) or osteocalcin (Ocn) reporter genes with genotype Osx-CreERt2; Ocn-GFP; ROSA-tdTomato to determine the effect of AML cells on various osteo-lineage cells. In addition, we used AML-PDX mouse models to investigate the impact of AML cell growth on new bone formation. Results In transgenic mice implanted with syngenic AML cells, we found a 3-4 fold increase in Osx+ cells, but no significant difference was observed in Ocn+ cells, suggesting AML cells expand osteoprogenitor cells but not mature osteoblasts in the BM. To determine whether the AML growth in the BM is associated with bone volume, we implanted AML-PDX cells (total 10 models) into NSG mice and analyzed femurs by micro-CT and histological analysis. Interestingly, we observed a dramatic increase in cortical bone thickness and new medullary bone formation in ∼50% of the PDX models tested. Interestingly, we found that increase in bone volume is associated with less aggressive PDX models (which takes 4-5 months to reach 90% PB engraftment). Aggressive PDX models (takes only 3-5 weeks to reach 90% PB engraftment) did not develop new bone. Masson-Goldner's trichrome staining confirmed weblike trabecular bone formation in mice with less aggressive PDX models, which was not observed in aggressive AML PDX models. Conclusion Our data suggest that AML cells induce supportive osteoprogenitor-rich niche in the BM. New bone formation in less aggressive AML-PDX models, but not in aggressive models suggests two different molecular mechanisms regulating AML growth in these two models. Finally, the role of osteoprogenitor niche in AML progression needs further investigation. Grant Acknowledgment Leukemia SPORE CDP, Emerson Collective, Golfers Against Cancer, LLS-TRP to VLB. RP to VLB.

Introduction: IMGN632, a novel CD123-targeting ADC, demonstrates a favorable safety profile and complete remissions as a monotherapy in patients with relapsed/refractory AML and BPDCN (NCT03386513). We have previously reported the synergy of combining IMGN632 with venetoclax (BCL-2 inhibitor) (EHA, 2019, abstract #PF201). Given the recent approval of azacitidine (AZA, a hypomethylating agent) and venetoclax (VEN) in AML patients unfit for intensive chemotherapy, we investigated the combination of IMGN632 with AZA and the triple combination of IMGN632, AZA and VEN. Methods: The IMGN632 and AZA +/- VEN combinations were evaluated in the following AML models: EOL-1, subcutaneous (SC); Molm-13, disseminated; MV4-11, disseminated; and one patient derived AML xenograft (PDX) model. In SC models, tumor volume was measured serially, complete regressions (CRs) were noted, and endpoint was determined by either clinical observations or when tumor volume reached 1000 mm3. Tumor growth inhibition (T/C) was calculated as (median tumor volume of the treated / median tumor volume of the control) x 100%. In disseminated models, the percent Increased Life Span (%ILS) was calculated as [(T-C, tumor growth delay) / C] x 100%, where T is the median survival time (days) of a treated group and C is the median survival time (days) of the vehicle control group. In AML PDX models, tumor burden was monitored by hCD45/hCD123 flow cytometry of serial peripheral blood (PB) samples. Results: In EOL-1, treatment with AZA (3 mg/kg, daily x5) was inactive, producing only a 4% reduction in T/C and 0/6 CRs treatment. IMGN632, at a sub-optimal single dose of 0.08 mg/kg resulted in a 36% reduction in T/C and 2/6 long-term CRs at study end. The combination of IMGN632 and AZA generated a robust 100% reduction in T/C and 4/6 CRs, while addition of VEN (100 mg/kg, daily x28) to AZA was not as active with a 54% reduction in T/C and 1/6 CRs. The triple combination with the addition of IMGN632 to the VEN + AZA doublet was the most active, with a 100% reduction in T/C and 5/6 CRs. Similarly, in Molm-13, AZA (3.5 mg/kg, daily x5) was minimally active, with in a 6-day T-C and 30% ILS. IMGN632 (0.024 mg/kg, weekly x3) was active and generated a 9.5-day T-C and a 47.5% ILS. Strikingly, the combination of IMGN632+AZA was highly active, resulting in a 61-day T-C and a 303% ILS (Figure 1A), demonstrating a clear survival benefit over single agent treatments. In a separate Molm-13 study, the combination of AZA+ VEN produced a 14.5-day T-C and a 67.4% ILS (highly active), while the triple combination including IMGN632 was clearly the most active combination, with a >116-day T-C and a >537% ILS (highly active) 50% of mice surviving beyond 4.5 months (Figure 1B). Similarly, in the MV4-11 model, the two AZA (either 4.5 mg/kg or 3 mg/kg, daily x5) + VEN (100 mg/kg, daily x28) combinations resulted in a 28-day T-C (62.2% ILS) and a 20-day T-C (44.4% ILS), respectively. The addition of IMGN632 (0.08 mg/kg) to these resulted in a superior regimen with a 64-day T-C (142.2% ILS) and a 76-day T-C (168.9% ILS), respectively. The PDX AML model, 4023126, responded very well to treatment with the triple combination (AZA (2.5mg/kg, daily x 5), VEN (100 mg/kg, 5 days/week x3 weeks) and sub-optimal IMGN632 (0.12 mg/kg once a week x3 weeks), with circulating leukemia burden lower than 1% after two weeks of treatment, compared to greater than 50% in the vehicle-treated group (Figure 1C). In model 4023126, IMGN632 was active and resulted in a 33-day T-C and 57% ILS, and AZA+VEN resulted in a 52-day T-C and 90% ILS. The triple combination group survived the longest and was highly active resulting in a 74-day T-C and 128% ILS demonstrating highest survival benefit (Figure 1D). Conclusions: The addition of IMGN632 to AZA alone and to AZA+VEN in multiple AML xenograft and PDX models leads to improved survival. These data support the addition of a CD123-targeted ADC with a novel DNA damaging payload to standard of care AZA and AZA+VEN in AML patients Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. McCarthy:ImmunoGen: Employment. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Sloss:ImmunoGen: Employment. Zweidler-McKay:ImmunoGen: Employment. Romanelli:ImmunoGen: Employment. Konopleva:Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

Previous studies have demonstrated that AML is BCL-2 dependent malignancy, and leukemia stem cell (LSC) rely on BCL-2 for survival (Pan, Cancer Discovery 2014;Lagadinou, Cell Stem Cell, 2013). Selective Bcl-2 inhibitor venetoclax (ABT-199) combined with azacitidine was reported to inhibit complex II of the mitochondrial transport chain in AML (Pollyea, Nat Med 2018). IACS-010759, a novel complex I inhibitor, demonstrated effective inhibition of cell respiration and potent anti-leukemia effect in AML pre-clinical models (Molina, Nat Med 2018). We designed the experiments to study the combined efficacy and mechanisms of action of venetoclax and IACS-010759 in AML. In vitro, priming of MOLM-13 cells with 20nM venetoclax for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while only partial inhibition (70%) reduction of viable cell numbers (OCI-AML2, MV-4-11, and MOLM-13). We have further shown by co-immunoprecipitation studies that venetoclax disrupts interaction of BCL-2 with the mitochondrial protein VDAC known to regulate ADP/ATP exchange during electron transport across mitochondria membrane. In addition, an MS-based metabolomics analysis indicated that ATP and CTP intracellular levels dropped to undetectable levels following treatment with ABT-199 (with or without IACS). ADP, GDP and UDP levels were unchanged with ABT-199; however, GDP levels dropped to undetectable levels following the combined treatment. Moreover, ABT-199 significantly increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of ABT-199 and IACS-010759. In primary AML samples (n=3) and PDX cells (n=4) cultured ex vivo, combined venetoclax and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. To better understand the role of BCL-2 in cellular respiration, we examined the oxygen consumption rates (OCR) in control or Bcl-2-overexpressing HL-60 cells (a kind gift of Dr. K. Bhalla, MDACC). The HL-60/BCL-2 cells had higher basal and maximal OCR than the control cells by Seahorse analysis, and higher mitochondrial ROS production by H2DCFDA and MitoSOX Red flow cytometry. BCL-2 inhibition with 100nM venetoclax for 2 hrs induced ROS production in control HL-60 cells but not in cells with BCL-2 overexpression. Further, cells with BCL-2 overexpression were less sensitive to IACS-010759. These data suggest that BCL-2 facilitates cellular respiration and reduces efficacy of the mitochondrial inhibitors. Given recent accelerated FDA approval of venetoclax and azacitidine combination for elderly unfit AML, we next tested the efficacy of the "triple" combination of venetoclax, azacitidine and IACS-010759 in the in vivoAML PDX model. We injected AML PDX cells 3747422 harboring IDH1, NMP1, NRAS, CEBPA, FLT3-ITD mutations into NRG mice and upon engraftment, randomized mice into 4 groups to receive vehicle, venetoclax (50mg/kg, 5 days on/2 days off, day 1-21) with azacitidine (1.25mg/kg daily , day 1-7), IACS-010759 (1mg/kg, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. All therapies reduced circulating leukemia burden with the best efficacy seen in the triple-therapy cohort, with average circulating tumor burden of 31.2%, 6.9%, 5.1% and 0.4% in vehicle, IACS-010759, venetoclax/azacitidine and triple-therapy cohorts, respectively. Survival analysis and additional PDX models are ongoing and will be reported. In summary, these findings indicate that BCL-2 modulates mitochondrial respiration in addition to its established anti-apoptotic role. Venetoclax disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor IACS-010759. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitroand in vivo. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Konopleva:Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

Acute myeloid leukemia (AML) is initiated and maintained by a relatively rare leukemia stem cells (LSCs) capable of self-renewal and proliferation. Recent data showed that LSCs (Lagadinou et al. Cell Stem Cell 2013) and residual cytarabine (Ara-C)-resistant AML cells (representing minimal residual disease, MRD) (Farge et al. Cancer Discovery 2017) are highly dependent on mitochondrial function for survival. This unique metabolic biology makes chemoresistant LSCs and AML cells vulnerable to pharmacological blockade of the oxidative phosphorylation (OXPHOS). We have reported that a novel OXPHOS inhibitor IACS-010759 potently inhibits mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells in vitro and in vivo (Molina et al. Nat Med 2018). In this study, we aimed to determine the effects of OXPHOS inhibition with IACS-010759 on residual AML cells surviving standard chemotherapy (Doxorubicin/Ara-C, DA) in cell line and patient-derived xenograft (PDX) AML models. Consistent with our hypothesis, OCI-AML3 cells treated with DA in vitro induced elevated levels of reactive oxygen species, higher mitochondrial mass and membrane potential (Fig. 1A), indicating reliance on the mitochondrial metabolism. Further, Ara-C treatment resulted in significantly increased basal and maximal oxygen consumption rates (OCR) (36%±8%, p=0.03; 36%±3%, p=0.003, respectively) compared to control. In turn, targeting OXPHOS with IACS-010759 at 30 nM fully inhibited basal and Ara-C-induced OCR. These findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor. To test the efficacy of combining IACS-010759 (5 mg/kg) and standard chemotherapy (Doxorubicin: 1.5 mg/kg; Ara-C: 50 mg/kg) in vivo, we injected NRG mice with genetically engineered OCI-AML3/Luc/GFP cells. Bioluminescent imaging demonstrated significantly reduced leukemia burden in DA/IACS-010759 combination group compared to vehicle on days 15 and 42 (p We next examined the efficacy of IACS-010759 on leukemia cells surviving chemotherapy in a chemosensitive PDX AML model of minimal residual disease (Fig. 1D). Treatment of mice inoculated with a human AML PDX harboring FLT3-ITD mutation with DA reduced circulating leukemia burden (0.8 ± 0.6% vs 45.8 ± 8.2% blasts in vehicle-treated mice, p=0.001). The residual AML cells in DA-treated mice expanded and caused rapidly progressive leukemia (78.2 ± 6.2% vs 95.3 ± 1.0% in vehicle-treated mice, p=0.047) on week 6 post DA. Daily oral treatment of mice with IACS-010759 (7.5 mg/kg) as a single agent reduced leukemia burden, and delayed leukemia recurrence when administered post completion of DA (Fig. 1E). A SPADE tree was built based on 13 surface markers and colored by expression intensity of CD34 using CyTOF mass cytometry data (Fig. 1F). The data demonstrated reduced frequency of CD34+CD38lowCD123+ AML LSCs and increase in CD11c+ differentiated cells in both IACS and IACS/DA groups (Fig. 1G&H). In contrast, chemotherapy alone failed to significantly reduce fractions of LSCs or induce differentiation. Proliferation measured by Ki67 was greatly reduced by IACS/DA combination in all populations including LSCs (1.4 ± 0.3% vs 5.5 ± 0.4% in vehicle group, p In conclusion, minimal residual AML cells surviving chemotherapy depend on OXPHOS for survival. OXPHOS inhibition with complex I inhibitor IACS-010759 is effective in reducing LSCs and MRD, alone and in combination with chemotherapy in vivo. Our data advocate for combining IACS-010759 with chemotherapy for improved control of MRD upon identification of a recommended Phase II dose in a clinical trial of IACS-010759 in AML (NCT02882321). Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Kantarjian:Pfizer: Honoraria, Research Funding; Cyclacel: Research Funding; AbbVie: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Ariad: Research Funding; Novartis: Research Funding; Agios: Honoraria, Research Funding; BMS: Research Funding; Astex: Research Funding; Amgen: Honoraria, Research Funding; Jazz Pharma: Research Funding. Daver:Jazz: Consultancy; Hanmi Pharm Co., Ltd.: Research Funding; Agios: Consultancy; Immunogen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; Karyopharm: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy; Servier: Research Funding; NOHLA: Research Funding; Glycomimetics: Research Funding; Otsuka: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy. Konopleva:Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. First generation BET inhibitor ABBV-075 and Venetoclax were recently shown to be synergistic in AML cell lines (Bui MH,Cancer Res 2017). ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia efficacy of the concomitant BCL-2 blockade by venetoclax and of BDII inhibition with ABBV-744 in primary AML samples. Anti-leukemia activity of venetoclax and ABBV-744 was examined in 21 primary AML samples with diverse genomic alterations. The combination significantly enhanced cell death (57.0 ± 6.3%) compared to the single agent treatment (43.9 ± 5.7% in ABT-199 10 nM group, p In two AML primary samples tested, combination treatment of ABBV-744 and ABT-199 induced apoptosis with caspase-3 activation and PARP cleavage through regulation of the key proteins regulating survival and proliferation pathways (e.g. (BCL-2, BCL-XL, MCL-1, c-Myc)). To identify biomarkers of response to therapy, we performed the baseline transcriptome analysis of AML cells used for in vitro response assessment (n=25) by RNA-sequencing (RNA-seq), and correlated baseline gene expression levels with in vitro response to therapy. Based on response to venetoclax or combination, samples were divided into 3 groups: sensitive to venetoclax (n=10), samples with no response to single agent or combination ("low apoptosis", n=7) and samples resistant to venetoclax as a single agent but responsive to venetoclax/ ABBV-744 combination ("synergy", n=4). AML samples sensitive to venetoclax and venetoclax/ABBV-744 combination were characterized by high level of BCL2 and lower levels of MCL1 and BCL2L1 transcripts, consistent with known inability of venetoclax to inhibit MCL-1 and BCL2L1 (Fig.1C).The resistant samples additionally expressed higher levels of anti-apoptotic genes such as GADD45, BCL2L10, PMAIP1. AML cells that showed synergy between venetoclax/ABBV-744 expressed low levels of AR, IL1R1 genes and had high CCND1 expression. The gene expression analysis indicated that the genes differentially expressed in the synergy vs. low apoptosis samples overlap with the genes inhibited by dual BCL-2/BCL-XL inhibitor ABT-737. To test the efficacy of this regimen in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, EGFR, IDH1, NPM1, TET2 mutations in NSG mice. Upon engraftment, mice were randomized to receive vehicle; single agent venetoclax at 50 mg/kg; ABBV-744 at 9.4 mg/kg; or venetoclax plus ABBV-744 for 21 days. After 21 days of therapy, flow cytometry data demonstrated significantly reduced leukemia burden in venetoclax treated group (9.5% ± 1.7%) but not in ABBV-744 group (22.3% ± 5.8%) compared to controls (30.8% ± 3.9%), with lowest tumor burden in the combination group (5.0% ± 0.8%, p In summary, combinatorial blockade of BDII bromodomain and of BCL-2 anti-apoptotic pathway facilitates apoptotic cell death, suppresses proliferation in the majority of primary AML cells and produces anti-AML activity in AML PDX models in vivo at tolerable doses of both agents. This combination is currently undergoing testing in a Phase I clinical trial in AML (NCT03360006). Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Lin:AbbVie: Employment. Uziel:AbbVie: Employment, Other: stock or other options. Lu:AbbVie: Employment. Zhang:AbbVie: Employment. Huang:AbbVie: Employment. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Shen:AbbVie: Employment. Konopleva:Astra Zeneca: Research Funding; Ablynx: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Agios: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Genentech: Honoraria, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria.

Background: Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy caused by differentiation arrest and suppression of apoptosis of immature myeloid cells. The long-term survival of AML under the established therapies remains poor. Differentiation therapy has been developed to promote the normal process of hematopoietic maturation from self-renewing progenitors to terminally differentiated effector cells. The recent discovery of a novel target, the enzyme dihydroorotate dehydrogenase (DHODH), offers differentiation-promoting therapy for the majority of AML (Sykes, D.B., et al.Cell, 2016). DHODH is the rate-limiting enzyme in the pyrimidine biosynthesis pathway. DHODH inhibition was reported to efficiently relieve the differentiation block caused by HoxA9 overexpression in 70% of AML, making this discovery potentially universally applicable for AML patients with diverse genomic alterations. AG-636 is a novel, potent, selective DHODH inhibitor developed by Agios Pharmaceuticals. This small molecule inhibitor has favorable pharmacokinetic properties and is in dose-finding Phase I clinical trials in lymphoma patients (NCT03834584) and is ready to enter a Phase I study in acute leukemia and myeloid dysplasia syndrome. Here, we investigated single agent activity of AG-636 in pre-clinical AML models. Results: AG-636 inhibited cell proliferation, induced apoptosis in AML cell lines, primary blasts and CD34+ leukemic stem/progenitor cells from AML patients with various genomic alterations cultured under physiologic conditions of stromal support (Fig. 1A). Flow cytometry and multi-parametric mass cytometry (CyTOF) analysis demonstrated that AG-636 reduced bulk AML and facilitated emergence of the differentiated myelo-monocytic cell subset co-expressing CD11b, CD11c and CD14 (Fig. 1B). Both cytotoxic and differentiating effects were rescued by supplementing the DHO downstream metabolite uridine, supporting on-target activity of AG-636 through DHODH inhibition and dependency of AML survival and stemness on the pyrimidine biosynthesis (Fig 1A top). Mass spectrometric analysis of 166 metabolites confirmed that targeting DHODH by AG-636 resulted in accumulation of the upstream L-dihydroorotic acid and ureidosuccinic acid, and depletion of the downstream metabolites, such as uridine 5'-diphsophate, uridine 5'-monophsophate, CDP and dCMP in pyrimidine biosynthesis pathway. Metabolic profiling further demonstrated the depletion of 5'-phosphoribosyl-N-formylglycinamide in treated cells, indicating the sequential effect of AG-636 on purine biosynthesis and metabolism. Seahorse-based metabolic assay showed inhibition of basal oxygen consumption and ATP generation in AG-636-treated cells, suggesting a contribution of DHODH in coupling of the mitochondria function. Proteomic profiling and immunoblots analysis revealed that AG-636 triggered AMPK activation in response to metabolic stress, and upregulated the expression of TP53, PUMA and NOXA known to regulate mitochondrial integrity. A role for DHODH inhibition in impairment of mitochondria function is of note given the key metabolic dependence of AML cells on OXPHOS/mitochondria function as shown by us and others (Molina J.R. et al. Nat Med, 2018). In vivo, twice daily administration of AG-636 significantly extended survival in a xenograft MOLM13-GFP-luciferase mouse model (Fig. 1C). Flow cytometry and CyTOF analysis demonstrated that AG-636 induced differentiation of CD11b+CD14+ and CD11b+CD11c+CD14+ monocytes in the bone marrow of treated mice (Fig. 1D). Administration of AG-636 significantly reduced tumor burden, induced differentiation and delayed leukemia progression in two AML patient-derived xenograft mouse models, one harboring mutations EZH2, NRAS and TET3, the other with mutations in ASXL1, BCOR and U2AF1. Daily treatment of AG-636 was well tolerated in all xenograft AML models tested, with a minimal effect on body weight and no significant toxicity recorded over the course of regimen. Conclusions: Our preliminary findings demonstrate that AG-636 is highly active against ex-vivo stroma-supported AML and AML stem/progenitor cells and in the in vivo AML xenograft models with diverse genetic subtypes. The significant monotherapy efficacy observed in pre-clinical studies provides the strong rationale for a clinical evaluation of AG-636 in myeloid malignancies. Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Kantarjian:Daiichi-Sankyo: Research Funding; Agios: Honoraria, Research Funding; Astex: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria; Ariad: Research Funding; Cyclacel: Research Funding; Pfizer: Honoraria, Research Funding; Immunogen: Research Funding; Jazz Pharma: Research Funding; Novartis: Research Funding. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees. DiNardo:celgene: Consultancy, Honoraria; daiichi sankyo: Honoraria; jazz: Honoraria; medimmune: Honoraria; syros: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; abbvie: Consultancy, Honoraria; agios: Consultancy, Honoraria. Murtie:Agios Pharmaceuticals, Inc.: Employment. Ulanet:Agios: Employment, Equity Ownership. Konopleva:Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy with historically poor outcomes and no established standard of care. Nearly 100% of patients with BPDCN overexpress CD123, and targeting CD123 has therefore emerged as an attractive therapeutic target. UCART123v1 is an allogeneic "off the shelf" product composed of genetically modified T-cells expressing an anti-CD123 CAR and a RQR8 depletion ligand, which confers susceptibility to rituximab. The expression of the T-cell receptor αβ (TCRαβ) is abrogated through the inactivation of the TRAC gene, using Cellectis' TALEN® gene-editing technology. We have previously reported the selective in vitro anti-tumor activity of UCART123v1 cells against primary BPDCN samples using cytotoxicity and T-cell degranulation assays, as well as the secretion of IFNγ and other cytokines (IL2, IL5, IL6, IL-13 and TNF-α) by UCART123v1 cells when cultured in the presence of BPDCN cells (Cai et al, 2017 ASH). To evaluate anti-tumor activity of UCART123v1 cells in vivo, we established two relapsed BPDCN patient-derived xenografts (PDX1 and 2) in NSG-SGM3 mice. In PDX-1 model, mice were randomized upon tumour engraftment (D21 after primary BPDCN injection) into 4 groups and received an IV injection of either vehicle, 10×106 TCRαβ KO control T-cells, or UCART123v1 cells (3×106 or 10×106 cells). Mice from vehicle group died by D53 after BPDCN injection with high tumor burden in PB, spleen and BM. 3 out of 9 (33%) mice treated with 3×106 and 6 out of 9 (67%) mice treated with 10×106 UCART123v1 were alive and disease-free at the end of the study (D299 after primary BPDCN injection). In PDX-2 model, which received the same treatment as PDX-1 (at D19 after primary BPDCN cell injection), all vehicle-treated mice died by D49. UCART123v1 therapy extended survival of treated mice to 104-241 days, but tumors relapsed at 90-155 days (Fig. 1A). The relapses in UCART123v1 treated mice were associated with the emergence of CD123-, CD56+CD45+ BPDCN cells infiltrating spleens and BMs (Fig. 1B). To understand the molecular basis for CD123 loss, we isolated RNA from CD123+ cells from two of the vehicle-treated mice and CD123- cells from four of the UCART123v1-treated mice and performed RT-PCR and RNA-sequencing. The cells from all samples were hCD45+ and hCD56+, indicating leukemic origin. These analyses detected the presence of full-length transcripts (exons 2-12) in both CD123+ control samples (Sample 1 and 2in Fig. 1C). In 2 of the 4 CD123- samples, CD123 transcripts were absent, as were transcripts of neighbouring genes (samples 3 and 9 in Fig. 1C). RNA-sequencing reads aligned to Genome Browser tracks for CD123 and housekeeping gene GPI showed no reads present for CD123 but reads present for GPI in the two samples with CD123 loss. The aCGH (Array‐Based Comparative Genomic Hybridization) results showed that samples 3 and 9 (CD123-) had large regional deletions on chromosome X, which includes the CD123 gene. In another sample (sample 5), the splicing analysis algorithm MAJIQ detected CD123 transcripts containing only exons 2-9, indicating premature transcription termination. If translated, this truncated transcript would produce a protein isoform lacking the transmembrane domain in exon 10. Finally, MAJIQ also revealed canonical splicing of exon 2 to exon 3 in all CD123+ samples but a sharp increase in skipping from exon 2 to exon 5 in sample 16 (Fig. 1D). This exon-skipping event preserves the open-reading frame and yields the previously reported transcript variant 2. Per UniProt, the resultant protein will retain the ligand-binding domain but lack several glycosylation sites and two beta sheets in the extracellular domain, potentially compromising recognition by UCART123v1 cells. The aCGH and FISH results further showed that this patient sample harbored TP53 deletion, which could have contributed to DNA instability observed in different mice engrafted with these tumor cells. In summary, allogeneic anti-CD123 CAR T therapy resulted in eradication of BPDCN in vitro and in increased disease-free survival in primary BPDCN PDX models. However, CD123 loss was observed in one PDX model harboring a TP53 deletion. These results provide preclinical proof-of-principle that UCART123v1 cells have potent anti-BPDCN activity, and indicate potential mechanisms leading to antigen loss and disease relapse. Disclosures Galetto: Cellectis Inc: Employment. Gouble:Cellectis: Employment. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Neelapu:Cellectis: Research Funding; Celgene: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; BMS: Research Funding; Poseida: Research Funding; Novartis: Consultancy; Karus: Research Funding; Acerta: Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Merck: Consultancy, Research Funding; Unum Therapeutics: Consultancy, Research Funding; Precision Biosciences: Consultancy; Cell Medica: Consultancy; Allogene: Consultancy. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. Kantarjian:BMS: Research Funding; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; Agios: Honoraria, Research Funding; Novartis: Research Funding; Immunogen: Research Funding; Jazz Pharma: Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Takeda: Honoraria; Astex: Research Funding; Daiichi-Sankyo: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding. Guzman:Cellectis: Research Funding; Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay; SeqRx: Consultancy. Pemmaraju:Stemline Therapeutics: Consultancy, Honoraria, Research Funding; samus: Research Funding; plexxikon: Research Funding; incyte: Consultancy, Research Funding; affymetrix: Research Funding; sagerstrong: Research Funding; Daiichi-Sankyo: Research Funding; cellectis: Research Funding; celgene: Consultancy, Honoraria; abbvie: Consultancy, Honoraria, Research Funding; novartis: Consultancy, Research Funding; mustangbio: Consultancy, Research Funding. Konopleva:Genentech: Honoraria, Research Funding; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties.

Acute myeloid leukemia (AML) cells highly depend on oxidative phosphorylation (OxPhos) to satisfy their heightened demands for energy, and the complex I OxPhos inhibitor IACS-010759 (Molina, Nat. Med. 2018) is currently in Phase 1 clinical trial in AML. In this study, we investigated how the bone marrow (BM) microenvironment affects the response to OxPhos inhibition in AML. To characterize the molecular mechanisms of sensitivity to OxPhos inhibition, we performed Cap Analysis of Gene Expression analysis (CAGE) on 31 genetically diverse primary AML samples (20 were defined as sensitive and 11 as resistant to IACS-010759; cut off >3.0 fold annexin V(+) by 100 nM IACS-010759/DMSO at 72 hours). CAGE identified higher expression of transcription start sites (TSS) for 17 genes in IACS-010759 resistant AML samples compared to sensitive (fold change >2.0, FDR < 0.05, EdgeR), which were related to cell adhesion, integrin and/or Rho GTPase family genes that modulate intracellular actin dynamics. We next investigated the interactions between IACS-010759 sensitive OCI-AML3 cells and BM-derived mesenchymal stem cells (MSC). Under conditions mimicking the BM microenvironment, IACS-010759 upregulated the pathways of focal adhesion and ECM-receptor interaction in OCI-AML3 cells (KEGG analysis based on CAGE). In turn, MSC co-culture increased oxygen consumption by AML, induced generation of mitochondrial ROS (control 4.4% vs IACS 44.4%), increased mtDNA (2-fold by q-PCR) and upregulation of mitochondrial proteins VDAC and cytochrome C, translating into dampened growth-inhibitory effects of IACS-010759. We further demonstrated that OCI-AML3 cells adhering to MSCs were fully protected from IACS-010759 induced apoptosis (IACS-induced specific apoptosis: non-adherent cells 16.2% ± 1.6% vs adherent cells 1.6% ± 0.7%, p=0.008, 30nM, 72hours). Similarly, adherent cells were fully protected from apoptosis induced by combination of IACS and AraC. These findings indicate that direct interactions with MSC trigger compensatory activation of mitochondrial respiration, increase in mitochondrial mass and resistance to OxPhos inhibition in AML. We next hypothesized that the trafficking of mitochondria from BM stroma cells to AML cells could represent a putative mechanism of an acquired resistance to OxPhos inhibition. To visualize mitochondria, OCI-AML3 and MSC were stably transfected with mitochondria-targeted PDHA1-GFP and -dsRed, respectively. We discovered that IACS-010759 induced transfer of MSC-derived mitochondria to OCI-AML3 cells (% of GFP/dsRed double-positive OCI-AML, control 4.1 ± 1.7 vs IACS 26.2 ± 13.4, p=0.002) via tunneling nanotubes (TNTs) detected by confocal and electron microscopy (Fig.1). Mitochondria transfer was only observed in the direct contact but not in the transwell co-cultures, and was abrogated by ICAM-1 neutralizing antibody and TNT blockade with Cytochalasin B. Likewise, combination of IACS with AraC increased mitochondrial transfer. We further found that IACS-010759 induced autophagy in OCI-AML3 cells co-cultured with MSC, as noted by increased conversion of LC3-I to LC3-II, which was further enhanced by the lysosome inhibitor Bafilomycin. Additionally, we observed autophagosome formation enwrapping MSC-derived mitochondria (Fig.1F), along with the degradation of an outer mitochondrial membrane protein Tom20. Finally, IACS-010759-induced transfer of mtDNA in BM-resident AML cells was confirmed in vivo in humanized AML PDX models (n=2). Daily oral treatment of mice harboring human AML with IACS-010759 (5.0 mg/kg/day, 21 days) increased the ratio of murine/human mtDNA in human AML cells isolated from BM, in 5 days on/2 days off PDX models tested (2.1 ± 0.3 fold, n=2). In conclusion, the findings of this study indicate an important role of mitochondria trafficking from BM stromal cells to AML cells in a compensatory adaptation to OxPhos inhibition in BM microenvironment. We propose that blocking of mitochondrial transfer could enhance the anti-AML efficacy of OxPhos targeting agents. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Reata: Equity Ownership; Aptose: Equity Ownership; 6 Dimensions Capital: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees. Konopleva:Astra Zeneca: Research Funding; Agios: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding.

T-ALL is an aggressive hematologic malignancy arising from immature T-cell precursors. Previous studies identified dependence of T-ALL (with a notable exception of early T-cell precursor (ETP) ALL) on BCL-XL (Chonghaile, Cancer Discovery 2014; Khaw, Blood 2016). However, BCL-XL specific inhibitors exhibit on-target toxicity of thrombocytopenia, restricting the use in acute leukemias (Vogler, Blood 2011). DT2216, a novel BCL-XL specific proteolysis targeting chimera (PROTAC), targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase, leading to BCL-XL ubiquitination and degradation selectively in cells express VHL (Khan, ASH 2018). Platelets lack VHL expression and therefore are spared from destruction by DT2216. Here we studied the pre-clinical efficacy of DT2216 in T-ALL cell lines in vitroand in vivousing T-ALL patient-derived xenograft (PDX) models. We first analyzed anti-apoptotic proteins (BCL-XL, BCL-2, MCL-1) expression in 4 B-ALL (LAZX2, MUTZ5, RS4:11, BALL1) and 6 T-ALL cell lines (SUPT1, KOPT1, Loucy, CCRF-CEM, PF384, Jurkat) by immunoblotting. This analysis demonstrated that ALL cell lines generally co-express BCL-XL and BCL-2 (Figure 1A). To identify functional dependencies, we utilized BH3 profiling that measures cytochrome C release after priming cells with BH3 peptides selectively targeting pro-survival BCL-2 family proteins in 4 B-ALL and 3 T-ALL cell lines. Similarly, cells were co-dependent on several anti-apoptotic members as shown by higher cytochrome c release in response to BIM, BID and BMF peptides targeting multiple anti-apoptotic proteins, and lower response to FS-1, ABT-199, HRK, MS-1, targeting individual anti-apoptotic members (Figure 1B). Analysis of the 3 B-ALL and 3 T-ALL PDX lines identified similar patterns that ALL cells are co-dependent on several anti-apoptotic members. Notably, we observed high cytochrome C release in response to mBAD that targets BCL-2 and BCL-XL; in addition, two of the three T-ALL PDXs, but none B-ALL PDX, responded to BCL-XL specific peptide HRK and to DT2216 confirming a functional role of BCL-XL in T-ALL survival. Next, we studied the sensitivity of ALL cells to ABT-199, DT2216 and the combination, in comparison with dual BCL-2/BCL-XL inhibitor ABT-263. DT2216 treatment (24hrs) caused a dose-dependent reduction of cellular viability in all 6 T -ALL and 3 B-ALL lines (except for BALL1 with complex karyotype refractory to all agents) measured by Cell TiterGlo assay, with T-ALL cells demonstrating a log higher sensitivity compared to B-ALL. In contrast, 5 out of 6 T-ALL lines (all besides ETP line Loucy) had no response to ABT-199, while 3 B-ALL lines showed dose-dependent response. All lines except BALL1 responded to ABT-263 (Figure 1C). Notably, the combination of DT2216 with ABT-199 synergistically reduced cell viability, with average CI of 0.3 (range 0.1-0.7 in all lines besides BALL1) (Figure 1D). Immunoblotting of DT2216 treated cells confirmed dose-dependent, on-target BCL-XL degradation as early as 6 hrs (Figure 1E). We next tested the therapeutic efficacy of DT2216 alone or combined with chemotherapy in T-ALL PDX models. NSG mice were engrafted with T-ALL PDX CU76 and D115. After documenting bone marrow (BM) engraftment by flow cytometry in BM aspirates on Day 14 post cell injection, mice were randomized to receive vehicle, chemotherapy ("VDL", VCR 0.15mg/kg, Dexa 5mg/kg, L-ASP 1000U/kg, ip., qw), DT2216 (15mg/kg, ip., q4d) or their combination for 3 weeks. Mice tolerated DT2216 therapy well, with no platelet toxicity by whole blood count 24hrs post the first and last DT2216 dosing. DT2216 reduced leukemia burden, delayed leukemia progression (Fig 1G) and significantly extended mice survival in both models. VDL chemotherapy had no effect on ALL progression in CU76 model and showed efficacy similar to DT2216 in D115 model; of importance, VDL+ DT2216 combination resulted in significant extension of survival in both chemoresistant and chemosensitive models (Figure 1F). In summary, T-ALL cells are functionally dependent on BCL-XL for survival and are highly sensitive to DT2216, while B-ALL are largely BCL-2 dependent and respond to BCL-2 inhibitors such as ABT-199. DT2216 alone and in particular when combined with chemotherapy reduced leukemia burden and prolonged survival in T-ALL PDX models. This study suggests targeting BCL-XL by DT2216 represents highly effective and safe adjunct therapeutic modality in T-ALL. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Zhang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Ghotbaldini:CPRIT Research Grant: Research Funding. Zheng:Dialectic Therapeutics: Equity Ownership, Other: Co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents. Konopleva:Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Forty-Seven: Consultancy, Honoraria; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

Bcl-2 and Mcl-1 play critical roles in AML stem/progenitor cell survival. Venetoclax (VEN), a highly selective Bcl-2 inhibitor, showed limited clinical efficacy in AML as a single agent. FLT3 is the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients can adapt to TKI treatment by reactivating the MEK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 can be induced by VEN treatment and could be a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML and synergize with VEN, targeting AML cells and stem/progenitor cells. We treated FLT3-ITD positive AML cells with a selective inhibitor of Mcl-1 (AMG 176) and FLT3 TKIs and found that inhibition of Mcl-1 induced cell death and significantly enhanced the activity of sorafenib or gilteritinib in cell lines including cells acquired resistance to VEN (CI We previously showed that overexpression/knockdown of Mcl-1 greatly protected/sensitized AML cells from VEN induced cell death (Carter BZ, ASH 2018) supporting Mcl-1 as a key VEN resistance factor. We treated primary AML cells (n=5) with VEN (10 nM) or AMG 176 (250 nM) alone, or in combination and found that VEN+AMG 176 synergistically induced cell death in AML blasts and AML stem/progenitor cells even in samples clinically resistant to or relapsed after VEN containing regimen (CI To investigate the antileukemia activity in vivo, we tested combined inhibition of Mcl-1 and Bcl-2 using two PDX models in NSG mice. The first model was developed from a resistant/relapsed patient with FLT3-ITD mutation and complex karyotype. The combination showed the most significant antileukemic activity and extension of survival, followed by AMG 176 and VEN treatment alone (median survival for the combination, 146 d, p=0.004; AMG 176, 137 d, p=0.032; VEN, 102 d, p>0.05 vs. control, 85.5 d; respectively). The second PDX model was developed from a patient who first responded and then became resistant to the combination of VEN and decitabine and harbors FLT3-ITD, NRAS, and GATA2 mutations and complex karyotype. VEN or AMG 176 monotherapies marginally prolonged survival (median survival 127 or 129 vs. control 124 d). The combination was highly effective in this model and greatly decreased circulating blasts (Fig. 1) and leukemia tissue infiltration, measured by flow cytometry and spleen size. CyTOF analysis demonstrated that only the combination strongly reduced blasts as well as the AML stem/progenitor cell populations. Median survival for the combination group currently has not been reached (>325 d) (Fig. 2). Collectively, these data demonstrate that inhibition of Mcl-1 enhances the efficacy of TKIs in FLT3 mutated AML. Furthermore, it synergizes with VEN, targeting not only AML blasts but also AML stem/progenitor cells, both in vitro and in vivo in PDX models with the potential of significantly improving treatment outcome, which warrants clinical evaluation. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Konopleva:Kisoji: Consultancy, Honoraria; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Ablynx: Research Funding; Agios: Research Funding; Astra Zeneca: Research Funding. Caenepeel:Amgen Inc.: Employment. Canon:Amgen Inc.: Employment. Hughes:Amgen Inc.: Employment. Morrow:Amgen Inc.: Employment. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees.

Notch1-mutated T-ALL is an aggressive hematologic malignancy lacking targeted therapeutic options. Genomic alterations in Notch1-gene and its activated downstream pathways are associated with metabolic stress response and heightened glutamine (Gln) utilization to fuel oxidative phosphorylation (OxPhos) (Kishton at al., Cell Metabolism 2016, 23:649, Herranz at al., Nat Med, 2015, 21(10): 1182-1189). Hence, targeting NOTCH1-associated OxPhos and/or Gln dependency could constitute a plausible therapeutic strategy for T-ALL. In this study we examined metabolic vulnerabilities of NOTCH1-driven T-ALL and tested pre-clinical efficacy of novel mitochondrial complex I (OxPhosi) IACS-010759 and of glutaminase inhibitor CB-839 (GLSi) in T-ALL models including Notch1-mutated T-ALL cell lines, patient-derived xenograft (PDX) and primary T-ALL cells. We have previously reported and confirmed in this expanded study the anti-leukemia efficacy of IACS-010759 (EC50s 0.1-15 nM) (Molina at al., Nat Med, 2018, 24: 1036; Baran at al., Blood, 2018, 132:4020). Metabolic characterization demonstrated that OxPhosi caused striking dose-dependent decrease in basal and maximal oxygen consumption rate (OCR), ATP and NADH generation in T-ALL cell lines and primary T-ALL samples (p OxPhosi, similar to knockout of complex I subunit NDUFS4 using CRISPR-CAS9, induced profound changes in T-ALL mitochondria, with induction of mitochondrial reactive oxygen species (ROS), DNA damage, activation of AMPK and inhibition of mTOR pathway. OxPhosi altered cellular energy homeostasis by reduction of TCA cycle intermediates, glutathione and reduction of intracellular nucleotides ATP, CTP, GTP, and UTP, translating into inhibition of DNA and RNA synthesis (p To confirm that blockade of Gln entry into TCA cycle with GLSi synergistically reduced viable ALL cell numbers, we studied potential synergy of OxPhosi and GLSi. The key role of Gln in maintaining energy production and cell proliferation via OxPhos in Notch1-mutated T-ALL cells was confirmed by the findings that Gln starvation or pharmacological GLS inhibition by CB-839 reduced ATP production and OCR and decreased cell proliferation by more than 50% in vitro (Fig.2, Fig.3). Dual blockade of OxPhos together with GLS induced DNA damage response via accumulation of ROS upon glutathione deprivation, induced AMPK signaling through profound reduction of all adenosine related intermediates and inhibited mTOR signaling. This translated into significant reduction of leukemia burden and extension of overall survival in vivo (p In summary, our findings indicate that dual blockade of metabolic processes by inhibiting complex I of mitochondria and restricting Gln utilization results in metabolic catastrophe in Notch1-mutated T-ALL associated with energy depletion and oxidative stress, which combined severely inhibit T-ALL growth and survival. We postulate that targeting this unique metabolic vulnerability of Notch1-mutated T-ALL cells constitutes a novel therapeutic modality in this aggressive malignancy. Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Jabbour:AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Konopleva:Agios: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Astra Zeneca: Research Funding; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Eli Lilly: Research Funding; Cellectis: Research Funding; Forty-Seven: Consultancy, Honoraria; Ablynx: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; AbbVie: Consultancy, Honoraria, Research Funding.

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia efficacy of the concomitant BCL-2 blockade by venetoclax and of BDII inhibition with ABBV-744 in primary AML samples. First, anti-leukemia activity of venetoclax and ABBV-744 was examined in 12 primary AML samples with diverse genomic alterations. The combination significantly enhanced cell death (61.4% ± 8.7%), as compared to the single agent treatment (51.4% ± 9.3% in venetoclax 10 nM group and 22.2% ± 3.4% in ABBV-744 50 nM group, p To test the efficacy of this regimen in vivo, we established a patient-derived xenograft (PDX) from an AML patient in NSG mice. After 21 days of therapy, flow cytometry data demonstrated significantly reduced leukemia burden in venetoclax treated group (9.5% ± 1.7%) but not in ABBV-744 group (22.3% ± 5.8%) compared to controls (30.8% ± 3.9%), with lowest tumor burden in the combination group (5.0% ± 0.8%, p Next, the anti-leukemia efficacy of ABBV-744 was tested in 7 additional AML PDX models. In all of the 7 models, Combination of ABBV-744 and venetoclax treatment delayed AML progression compared to untreated mice (survival days: 141 vs 105, 275 vs 153, 62 vs 46, 136 vs 119, 138 vs 77, 129 vs 116, 94 vs 86). In summary, combinatorial blockade of BDII bromodomain and of BCL-2 anti-apoptotic pathway facilitates apoptotic cell death and suppresses proliferation in the majority of primary AML cells and produces anti-AML activity in AML PDX models in vivo. Citation Format: Tianyu Cai, Vinitha Kuruvilla, Xiaoyu Lin, Tamar Uziel, Xin Lu, Lu Zhang, Qi Zhang, Lina Han, Antonio Cavazos, Yu Shen, Marina Konopleva. Selective targeting BET family BDII bromodomain with ABBV-744 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3831.

Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy characterized by limited therapeutic options and a high rate of treatment failure due to chemoresistance. T-ALL is largely driven by activating NOTCH1 mutations, where oncogenic NOTCH1 facilitates glutamine oxidation, induces metabolic stress, and facilitates reliance on oxidative phosphorylation (OXPHOS)1. In other malignancies, the shift toward OXPHOS-dependent high-energy status is associated with acquired chemoresistance. In this study, we found that the novel inhibitor of mitochondrial complex I (OXPHOSi) IACS-0107592 has preclinical activity in NOTCH1-mutated T-ALL; we also characterize the cellular and metabolic responses to OXPHOS inhibition and propose that an OXPHOSi be incorporated into standard-of-care therapy to improve outcomes in patients harboring NOTCH1-mutated T-ALL. Exposure to IACS-010759 (0-370 nM) in vitro drastically reduced T-ALL viability, with EC50 ranging from 0.1-10 nM for cell lines (n=7) and from 13-60 nM for patient-derived xenograft (PDX)-derived and primary T-ALL cells (n=10) (Fig.1). Oral administration of IACS-010759 (7.5 mg/kg/day) significantly reduced leukemia burden and extended overall survival (p In summary, our findings indicate that OXPHOSi, alone and particularly in combination with standard chemotherapy and GLS inhibition, constitutes a novel therapeutic modality that targets a unique metabolic vulnerability of NOTCH1-mutated T-ALL cells. References:Kishton RJ, Barnes CE, Nichols AG at al., AMPK Is Essential to Balance Glycolysis and Mitochondrial Metabolism to Control T-ALL Cell Stress and Survival, Cell Metabolism, 2016, 23(4):649-62Molina JR, Sun Y, Protopopova M et al., An inhibitor of oxidative phosphorylation exploits cancer vulnerability, Nat Med, 2018, 24: 1036-1046 Disclosures Lorenzi: NIH: Patents & Royalties; Erytech Pharma: Consultancy. Konopleva:Stemline Therapeutics: Research Funding.

The BET family proteins consist of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two highly conserved bromodomains (BDI and BDII). First generation BET inhibitors, including ABBV-075, are pan-BET inhibitors that bind with nearly equimolar affinity to BDI and BDII. Pan-BET inhibitors generally block transcription and inhibit the proliferation of a large spectrum of tumor types, but exhibit a narrow therapeutic index. ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia activities of ABBV-744 in acute myelogenous leukemia (AML) cell lines and primary samples. In AML cell lines, ABBV-744 induced G1 cell cycle arrest and apoptosis with caspase-3 activation and PAPR cleavage through regulation of the key proteins that are involved in cell cycle and apoptosis pathways (e.g. (BCL-2, BCL-XL, MCL-1, c-Myc). Mechanistically, ABBV-744 displaced BRD4 from the regulatory region of BCL2, RUNX1, BCL2L1 and IL8 genes by ChiP-Seq. MLL-rearranged cells were most sensitive to BDII inhibition, and ABBV-744 displaced both, BRD4 and MLL from the regulatory regions of BCL2 and c-Myc genes in MV4;11 (MLL-AF4) and Nomo-1 (MLL-AF9) cells. We examined anti-leukemia activity of ABBV-744 in 12 primary AML samples with diverse genetic alterations. ABBV-744 inhibited cell growth in 8/12 primary samples (20.2%-77.9%) and induced apoptosis (26.7 ± 9.6% in ABBV-744 group vs 14.1± 3.9% in DMSO group, p=0.007, Fig 1A). We next performed the whole genome transcriptome analysis of pre-treatment AML cells by RNA-sequencing (RNA-seq). The samples which were sensitive to ABBV-744 tend to have higher pro-apoptosis (TXNIP, DR5) and lower anti-apoptosis (BCL2, EXPO) than resistant samples. For efficacy assessment in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, IDH1 and NPM1 mutations in NSG mice. Upon engraftment, mice were randomized to receive vehicle or single agent ABBV-744 at a well-below MTD dose of 9.4 mg/kg for 21 days. The median survival time of mice treated with ABBV-744 (Median survival: 76 days) was significantly higher compared to untreated mice (Median survival: 67.5 days, p=0.007, Fig 1B). Next, the anti-leukemia efficacy of ABBV-744 was tested in 7 additional AML PDX models. NSG mice were injected with 7 AML PDX (one mouse per cohort design). After engraftment was confirmed by retro-orbital bleeding, mice were treated with vehicle or ABBV-744. Circulating tumor burden was measured by flow cytometry in peripheral blood samples from mice collected on indicated days of therapy. In five of the 7 models, ABBV-744 treatment delayed AML progression compared to untreated mice (survival days: 135 vs 105, 205 vs 153, 62 vs 46, 118 vs 77, 97 vs 86). No significant impact on mice' weight was noted (Fig 1C), and no clinical signs of toxicity recorded over the course of therapy. In summary, blockade of BET family bromodomain BDII promotes apoptotic cell death and suppresses proliferation in the majority of primary AML cells. Importantly, ABBV-744 therapy reduced AML tumor burden and extended survival in AML PDX models at a well-below MTD dose. Together, these results provide proof of concept that highly selective inhibitors of the second bromodomain of BET family may maintain robust anti-leukemia efficacy in AML while exhibiting improved tolerability relative to pan-BET inhibitors. Disclosures Uziel: AbbVie Inc.: Employment. Shen:AbbVie Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding. Lin:AbbVie Inc: Employment. Lu:AbbVie Inc: Employment. Zhang:AbbVie Inc: Employment.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy of plasmacytoid dendritic precursor cells. Lacking standard therapy due to limited understanding of biology and historical rarity of the disease, clinical outcomes for BPDCN patients remain poor. Surface overexpression of CD123/IL3Rα is seen 70-80% of patients with AML (Han et.al, Clin Cancer Res, 2017) and in nearly 100% of BPDCN cases, with a markedly high intensity compared to normal hematopoietic stem cells, thus making CD123 an attractive target for BPDCN treatment. IMGN632 is a CD123-targeting antibody-drug conjugate (ADC) comprised of a novel humanized anti-CD123 antibody G4723A linked to a DNA mono-alkylating payload of the indolindobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. IMGN632 demonstrates potent activity in AML samples at low concentrations with minimal impact on normal bone marrow progenitors, and anti-leukemia effects in AML xenograft models (Kovtun et.al., Blood Advances, 2018). A phase I clinical trial of IMGN632 in relapse/ refractory AML (NCT033865) is now being performed and achieved encouraging results. We thus explored the anti-tumor effect of IMGN632 in BPDCN. We first tested the in vitro activity of IMGN632 in a BPDCN cell line CAL-1. Cells were exposed to control antibody or IMGN632 at various concentration for 96 hours, after which viable cell numbers were measured using AnnexinV /DAPI assay with counting beads by flow cytometry. IMGN632 demonstrated cytotoxic activity at 0.2μg/ml, with stronger effects at concentrations of 2μg/ml and above (Figure 1A). Next, the in vivo efficacy of IMGN632 was evaluated in BPDCN patient-derived-xenograft (PDX) models. NSG mice (6-8 weeks old) were injected with 1e6/mouse BPDCN cells by tail vein 24 hours after 250cGY sublethal irradiation. Upon confirmation of the bone marrow engraftment of hCD45/hCD123 cells by bone marrow aspiration, mice were randomized to 5 or 8 mice/group and received weekly intravenous vehicle, control antibody or IMGN632 by tail vein injection 24 hours following FcR blockade (400mg/kg naked antibody by intraperitoneal injection) for 3 weeks. Bone marrow engraftment and survival were monitored. For model A, the mice received either 24μg/kg or 240μg/kg of IMGN632. For model B, the mice received 240μg/kg IMGN632. Both samples responded to the IMGN632 treatment, with the engraftment lower than 1% in the bone marrow after 3 doses, compared with over 90% in vehicle and control antibody group (Figure 1B). All mice from vehicle and control antibody groups in model A died by 52 days after cell injection; the 24μg/kg IMGN632 treated mice had prolonged survival with median survival of 111 days (p In summary, our preliminary data demonstrate pre-clinical proof of targeting CD123 in BPDCN using a novel antibody-drug conjugate. IMGN632 exhibited promising anti-tumor effects in BPDCN in vitro and in vivo, even at a dose 10-fold lower than the anticipated therapeutically active dose. This large therapeutic window would be expected to be advantageous when used in combination with other treatments in BPDCN. Disclosures Adams: ImmunoGen Inc.: Employment. Callum:ImmunoGen Inc.: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-one: Consultancy. Kovtun:ImmunoGen Inc.: Employment. Daver:Daiichi-Sankyo: Research Funding; Novartis: Consultancy; Novartis: Research Funding; Incyte: Consultancy; BMS: Research Funding; Kiromic: Research Funding; Otsuka: Consultancy; Karyopharm: Consultancy; Alexion: Consultancy; Sunesis: Consultancy; Pfizer: Research Funding; Sunesis: Research Funding; Incyte: Research Funding; ARIAD: Research Funding; Pfizer: Consultancy; ImmunoGen: Consultancy; Karyopharm: Research Funding. Pemmaraju:Affymetrix: Research Funding; SagerStrong Foundation: Research Funding; plexxikon: Research Funding; daiichi sankyo: Research Funding; samus: Research Funding; celgene: Consultancy, Honoraria; abbvie: Research Funding; cellectis: Research Funding; stemline: Consultancy, Honoraria, Research Funding; novartis: Research Funding. McKay:ImmunoGen Inc.: Employment. Konopleva:Stemline Therapeutics: Research Funding.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy with historically poor outcomes and no established standard of care. Nearly 100% of patients with BPDCN overexpress CD123, and targeting CD123 emerged as an attractive therapeutic target given its differential expression on BPDCN cell surface. UCART123 product (Cellectis) uses genetically modified allogeneic T-cells (derived from healthy donors, so-called “off the shelf”) containing an anti-CD123 CAR and a RQR8 depletion ligand that confers susceptibility to rituximab. The expression of the T-cell receptor (TCR) is abrogated through the inactivation of the TCRα constant gene, using Cellectis' TALEN® gene-editing technology. We have previously reported the selective in vitro anti-tumor activity of UCART123 cells against CD123+ primary BPDCN samples using cytotoxicity assays, T-cell degranulation assay and the secretion of IFNγ and other cytokines (IL2, IL5, IL6, IL-13 and TNF-α) by UCART123 cells when cultured in the presence of BPDCN cells (Tianyu Cai, 2017 ASH). However, UCART123 had minimum toxicity against normal bone marrow cells. To evaluate in vivo anti-tumor activity of UCART123 cells, we established two patient-derived xenografts (PDX1-2) from patients with relapsed BPDCN in NSG-SGM3 mice. In PDX-1 model, all mice in vehicle-treated group died by D53, with high tumor burden in peripheral blood, spleen and bone marrow. Three out of 9 (33%) mice treated with 3×106 UCART123 and Six out of 9 (67%) mice treated with 10×106 UCART123 were alive and disease-free at the end of the study (D299). In PDX-2 model, while UCART123 similarly extended survival of the mice (D104-241), relapses occurred in all treatment cohorts at D90-155. Flow cytometric analysis showed that all of the relapses were associated with emergence of CD123- BPDCN clones (95-96% CD123-). To understand the molecular basis for loss of CD123 surface expression, we isolated RNA from two CD123 positive samples from vehicle group and two CD123 negative samples from 1×106 UCART123 group. RT-PCR and RNA-seq detected the presence of full-length transcripts containing exons 1-12 in both CD123 positive samples. In one of the two CD123 (-) samples, CD123 transcripts were completely absent, along with loss of transcripts of neighboring genes. In another CD123 (-) sample, CD123 transcripts containing only exons 1-9 were detected, indicating the presence of a truncation. Interestingly, if translated, this transcript would produce a protein isoform lacking the transmembrane domain (Ex 10). In summary, UCART123 therapy results in BPDCN eradication and long-term disease-free survival in a subset of primary BPDCN PDX models. However, loss of CD123 through diverse genetic mechanisms could lead to escape from UCART123 therapy and cause relapses. A phase I trial of UCART123 in BPDCN is opened for enrollment (NCT03203369). Citation Format: Tianyu Cai, Kathryn L. Black, Ammar Naqvi, Roman Galetto, Agnès Gouble, Julianne Smith, Antonio Cavazos, Lina Han, Qi Zhang, Vinitha Kuruvilla, Sergej Naumovich Sergej Konoplev, Sattva S. Neelapu, Andrew A. Lane, Monica L. Guzman, Hagop Kantarjian, Andrei Thomas-Tikhonenko, Naveen Pemmaraju, Marina Konopleva. Preclinical efficacy of allogeneic anti-CD123 CAR T-cells for the therapy of blastic plasmacytoid dendritic cell neoplasm (BPDCN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2560.

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABBV-075 is a potent and selective BET family bromodomain inhibitor that entered phase I clinical trials. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. ABBV-075 and Venetoclax were recently shown to be synergistic in AML cell lines (Bui MH, Cancer Res 2017). In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 blockade by venetoclax in combination with BET inhibitor ABBV-075 in primary AML samples. First, anti-leukemia activity of venetoclax and ABBV-075 was examined in 12 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death (71.2 ± 29.3 %), as compared to the single agent treatment (51.4 ± 32.2 % in venetoclax 10 nM group and 32.8 ± 21.7% in ABBV-075 20 nM group). ABBV-075 inhibited cell proliferation in the majority of AML cases (51.3 ± 30.9 %) and the cell growth suppression was more profound in the combination group (87.9 ± 18.8 %, p To test the efficacy in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, IDH1 and NPM1 mutation in NSG mice. Upon engraftment, mice were randomized to receive vehicle, single agent venetoclax at 50 mg/kg for 21 days, ABBV-075 at 0.5 mg/kg for 21 days; or venetoclax at 50 mg/kg plus ABBV-075 at 0.5 mg/kg for 21 days. After 21 days treatment, flow cytometry data demonstrated significantly reduced leukemia burden in treated groups (7.9 ± 5.9 % in venetoclax group and 12.2 ± 5.1 % in ABBV-075 group) compared to controls (33.0 ± 12.2 %), more prominently in the combination group (1.9 ± 0.8 %). In summary, combinatorial blockade of BET family bromodomain and BCL-2 pathways promotes apoptotic cell death and suppresses proliferation in the majority of primary AML cells. Ongoing studies will evaluate efficacy of this combination therapy in 10 additional primary human AML xenografts and elucidate mechanisms of synergy. Citation Format: Tianyu Cai, Vinitha Mary Kuruvilla, Tamar Uziel, Qi Zhang, Lina Han, Antonio Cavazos, Yu Shen, Marina Konopleva. Targeting BET Family Bromodomain with ABBV-075 and BCL-2 with venetoclax (ABT-199) is synergistic in primary acute myeloid leukemia models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-261.

Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with limited treatment options, largely driven by the activating Notch1 mutations. Oncogenic Notch1 facilitates c-Myc signaling and glutamine oxidation, induces metabolic stress and increased reliance on oxidative metabolism maintained by AMPK and modulates metabolism under energy stress by mTOR (Kishton, Cell Metabolism 2016; Chan, Blood 2007). In this study, we report pre-clinical activity of the novel OXPHOS inhibitor (OXPHOSi) IACS-010759 in NOTCH-mutated T-ALL, and characterize the cellular and metabolic responses to OxPhos inhibition. Exposure to IACS-010759 (0-370 nM) in vitro for 5 days drastically reduced T-ALL viability, with EC50 ranging from 0.001-10 nM for T-ALL cell lines and 13-45 nM for T-ALL PDX models (n=5). Oral administration of IACS-010759 at 7.5 mg/kg daily was tolerable in both, aggressive T-ALL PDX and in Notch-1 mutated murine T-ALL model, significantly reduced leukemia burden and extended survival. Functional metabolic characterization of T-ALL confirmed that IACS-010759 effectively inhibited mitochondrial respiration and caused striking dose-dependent decrease in basal and maximal OCR, ATP and NADH production. Pharmacological inhibition of Complex I with IACS-010759, similar to knockout of Complex I subunit NDUSF4 using CRISPR-CAS9, induced catastrophic changes in mitochondria, with induction of ROS, DNA damage and compensatory mTOR pathway activation. Further, OXPHOSi led to downregulation of mitochondrial Complex I, II, III and IV, decrease of wide range of TCA cycle enzymes and proteins involved in the mitochondrial transport. This translated into decrease of TCA cycle intermediates and reduction in ATP and NADH content by metabolomic analysis. Using stable isotope-resolved metabolomics (SIRM) flux analysis, IACS-010759 (30 nM at 24 hr) significantly decreased flux of glucose through the TCA cycle and redirected it towards glycolysis, additionally increased utilization of glutamine for fueling the TCA cycle, in particular through reductive metabolism, uncovering reliance on glutaminolysis as an additional therapeutic target. Consistent with this hypothesis, combined therapy of OXPHOSi with Glutaminase (GLS-i) or mTOR inhibitors caused additive or synergistic reduction of viability of T-ALL cells, and elicited anti-leukemia activity in T-ALL resistant to Complex I inhibitor alone. Ongoing in vivo studies will address the impact of Complex I Inhibition in the context of genetic GLS knockout utilizing Notch1-mutated GLS fl/fl murine model (Herranz, Nat Med 2016). Taken together, our findings indicate that OXPHOSi, alone and more so in combination with GLS inhibition, constitutes an novel therapeutic modality that targets unique metabolic vulnerability of Notch1- mutated T-ALL cells. Citation Format: Natalia Baran, Alessia Lodi, Shannon Sweeney, Vinitha Mary Kuruvilla, Antonio Cavazos, Anna Skwarska, Sriram Shanmuga Velandy, Karine Harutyunyan, Ningping Feng, Jason Gay, Marcin Kaminski, Elias J. Jabbour, Adolfo Ferrando, M. Emilia Di Francesco, Joseph R. Marszalek, Stefano Tiziani, Marina Konopleva. Oxidative metabolism as a novel therapeutic target to eradicate T-ALL with mitochondrial complex I inhibitor IACS-010759 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1875.