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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.

Bcl-2 and Mcl-1 play critical roles in AML stem/progenitor cell survival. Venetoclax (VEN), a highly selective Bcl-2 inhibitor, showed potent preclinical activity but limited clinical efficacy in AML. Expression of Mcl-1, a major VEN resistance factor is induced by VEN. Non-apoptotic activity of Mcl-1 has been reported but is not well understood in AML. We previously demonstrated that inhibition of Mcl-1 enhances VEN apoptogenic activity and overcomes intrinsic and acquired VEN resistance in vitro and in vivo in a PDX murine model of AML (Carter BZ et al., ASH 2018). Interestingly, CyTOF analysis of bone marrow PDX cells collected from mice treated with Bcl-2 inhibitor VEN, Mcl-1 inhibitor AZD5991, and the combination showed that AZD5991 or AZD5991+VEN, but not VEN alone greatly decreased CXCR4 in leukemia cells and stem/progenitor cell populations. Mcl-1 expression is to a large degree regulated by microenvironment clues, including CXCR4-CXCL12 axis. We found that inhibition of Mcl-1 also suppressed cell metabolic activities in AML cells suggesting that Mcl-1 may regulate leukemia-microenvironment interaction and cell metabolic functions, which may contribute to the efficacy of this combination against AML stem cells in the bone marrow microenvironment. To further investigate the roles of Mcl-1 in leukemia-stromal interactions, we determined the expression of proteins involved in cell migration and adhesion and found that Mcl-1 overexpressing (OE) or knockdown (KD) OCI-AML3 cells have increased/decreased cell surface expression of CXCR4 and CD44, both critical for leukemia-MSC (mesenchymal stroma cell) interactions. Mcl-1 OE or KD AML cells showed increased/decreased migration towards and adhesion to MSCs compared to their respective controls. Consistent with this observation, pharmacological inhibition of Mcl-1 with AZD5991 also decreased surface CXCR4 and CD44 levels in OCI-AML3 cells and decreased the interactions between leukemia and MSCs. Under the same conditions, Mcl-1 seems to exert a more profound effect on leukemia cell adhesion than migration to MSCs. Interestingly, inhibition of Bcl-2 with VEN did not significantly decrease CD44 or migration and adhesion of AML cells to MSCs, although a decrease in surface levels of CXCR4 was observed. To gain additional insight into the effects of Mcl-1 on cellular energetics and metabolism, we performed metabolomic analysis, employing a previously established method using ion chromatography-mass spectrometry (IC-MS) to trace 13C labels from 13C2-1,2-glucose and 13C5-glutamine in Mcl-1 genetically and pharmacologically modulated AML cells. Metabolomic analysis showed that overall 13C enrichment into key TCA cycle intermediates including citrate, fumarate, and malate was significantly lower in cells with Mcl-1 inhibition, by either AZD5991 or Mcl-1 KD, compared to the respective controls, both for 13C coming from glucose and glutamine, the two main carbon sources entering the TCA cycle. We observed a decrease in the total amount of secreted lactate in both the 13C2-1,2-glucose and 13C5-glutamine tracing experiments. Enrichment of 13C label in secreted lactate was mostly observed during 13C2-1,2-glucose tracing, but not during 13C5-glutamine tracing, indicating that lactate secretion is largely derived from glucose, and not from glutamine, suggesting a reduction in flux through glycolysis and/or pentose phosphate pathway (PPP). Flux through the oxidative PPP (OxPPP) is a rate limiting pathway in the generation of reductive equivalents of NADPH for ROS neutralization. Relative OxPPP flux calculation showed a decrease in OxPPP flux for both AZD5991 treatment and Mcl-1 KD. Levels of 6-Phospho-Gluconic acid (6PG) were also greatly reduced in Mcl-1 inhibited cells, in accordance with differential regulation of the OxPPP. Mcl-1 inhibition by AZD5991 or KD also decreased ATP levels. Collectively, inhibition of Mcl-1 results in a broad downregulation in cellular energetics and metabolism. Conclusion: data demonstrate that in addition to regulating apoptosis, Mcl-1 regulates leukemia-stromal interactions and metabolic activity in leukemia cells and that inhibition of Mcl-1 has anti-leukemia activity and enhance VEN activity through not only apoptosis induction, but also modulation of leukemia-stromal interactions and metabolic functions. Disclosures Carter: Ascentage: Research Funding; Amgen: Research Funding; AstraZeneca: Research Funding. Cidado:AstraZeneca: Employment. Drew:AstraZeneca: Employment. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; 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; Celgene: Consultancy; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership.

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.