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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.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2–containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation “armored” chimeric antigen receptor (CAR) engineering of cord blood–derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15–secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy. Key Points: • CRISPR-Cas9 CISH deletion enhances the metabolic fitness and antitumor activity of armored IL-15–secreting CB-derived CAR-NK cells.

Spleen tyrosine kinase (SYK) is an important oncogene and signaling mediator activated by cell surface receptors crucial for acute myeloid leukemia (AML) maintenance and progression. Genetic or pharmacologic inhibition of SYK in AML cells leads to increased differentiation, reduced proliferation, and cellular apoptosis. Herein, we addressed the consequences of SYK inhibition to leukemia stem-cell (LSC) function and assessed SYK-associated pathways in AML cell biology. Using gain-of-function MEK kinase mutant and constitutively active STAT5A, we demonstrate that R406, the active metabolite of a small-molecule SYK inhibitor fostamatinib, induces differentiation and blocks clonogenic potential of AML cells through the MEK/ERK1/2 pathway and STAT5A transcription factor, respectively. Pharmacological inhibition of SYK with R406 reduced LSC compartment defined as CD34+CD38−CD123+ and CD34+CD38−CD25+ in vitro, and decreased viability of LSCs identified by a low abundance of reactive oxygen species. Primary leukemic blasts treated ex vivo with R406 exhibited lower engraftment potential when xenotransplanted to immunodeficient NSG/J mice. Mechanistically, these effects are mediated by disturbed mitochondrial biogenesis and suppression of oxidative metabolism (OXPHOS) in LSCs. These mechanisms appear to be partially dependent on inhibition of STAT5 and its target gene MYC, a well-defined inducer of mitochondrial biogenesis. In addition, inhibition of SYK increases the sensitivity of LSCs to cytarabine (AraC), a standard of AML induction therapy. Taken together, our findings indicate that SYK fosters OXPHOS and participates in metabolic reprogramming of AML LSCs in a mechanism that at least partially involves STAT5, and that SYK inhibition targets LSCs in AML. Since active SYK is expressed in a majority of AML patients and confers inferior prognosis, the combination of SYK inhibitors with standard chemotherapeutics such as AraC constitutes a new therapeutic modality that should be evaluated in future clinical trials.

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.

Mitochondria play a central and multifunctional role in the progression of tumorigenesis. Although many recent studies have demonstrated correlations between mitochondrial function and genetic makeup or originating tissue, it remains unclear why some cancers are more susceptible to mitocans (anticancer drugs that target mitochondrial function to mediate part or all of their effect). Moreover, fundamental questions of efficacy and mechanism of action in various tumor types stubbornly remain. Here we demonstrate that cancer type is a significant predictor of tumor response to mitocan treatment, and that acute myeloid leukemias (AML) show an increased sensitivity to these drugs. We determined that AML cells display particular defects in mitochondrial metabolism that underlie their sensitivity to mitocan treatment. Furthermore, we demonstrated that combinatorial treatment with a mitocan (CCCP) and a glycolytic inhibitor (2-deoxyglucose) has substantial synergy in AML cells, including primary cells from patients with AML. Our results show that mitocans, either alone or in combination with a glycolytic inhibitor, display anti-leukemia effects in doses much lower than needed to induce toxicity against normal blood cells, indicating that mitochondria may be an effective and selective therapeutic target.

Background DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in 1q21 MM. Method We continuously treated MM cells with an ILF2-targeting antisense (ILF2-ASO) or control non-targeting antisense (NT-ASO) for 3 weeks to see if ILF2-ASO exposure leads to the selection of MM clones intrinsically resistant to DNA damage or activates compensatory mechanisms to overcome ILF2 depletion-induced DNA damage. Results Single-cell RNA sequencing (scRNA-seq) analysis revealed that DNA damage-resistant ILF2-ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Metabolomic analysis of MM cells after long-term exposure to ILF2-ASO showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates. Consistent with these results, ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells’ metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 screening strategy to identify DNA repair genes whose loss of function suppresses MM cells’ ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs in ILF2-ASO-treated JJN3 cells were significantly depleted after 3 weeks of treatment, suggesting that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO- and ILF2-ASO-treated MM cells exposed to NSC for 3 days showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. Conclusion In conclusion, our study has revealed a novel mechanism through which MM cells counteract oxidative DNA damage and maintain mitochondrial respiration after metabolic reprogramming.

B-cell–activating factor (BAFF) mediates B-cell survival and, when deregulated, contributes to autoimmune diseases and B-cell malignancies. The mechanism connecting BAFF receptor (BAFFR) signal to downstream pathways and pathophysiological functions is not well understood. Here we identified DYRK1a as a kinase that responds to BAFF stimulation and mediates BAFF-induced B-cell survival. B-cell–specific DYRK1a deficiency causes peripheral B-cell reduction and ameliorates autoimmunity in a mouse model of lupus. An unbiased screen identified DYRK1a as a protein that interacts with TRAF3, a ubiquitin ligase component mediating degradation of the noncanonical nuclear factor (NF)-κB–inducing kinase (NIK). DYRK1a phosphorylates TRAF3 at serine-29 to interfere with its function in mediating NIK degradation, thereby facilitating BAFF-induced NIK accumulation and noncanonical NF-κB activation. Interestingly, B-cell acute lymphoblastic leukemia (B-ALL) cells express high levels of BAFFR and respond to BAFF for noncanonical NF-κB activation and survival in a DYRK1a-dependent manner. Furthermore, DYRK1a promotes a mouse model of B-ALL through activation of the noncanonical NF-κB pathway. These results establish DYRK1a as a critical BAFFR signaling mediator and provide novel insight into B-ALL pathogenesis.

Activating FLT3 internal tandem duplication (FLT3-ITD) mutations, present in ~30% of AML patients, are associated with poor prognosis. Given that efficacy of FLT3 inhibitors used as monotherapy is limited due to development of secondary FLT3 mutations or activation of alternative survival pathways, new combinatorial strategies are needed to improve outcome in FLT3-ITD AML patients. Constitutive activation of mutated FLT3 often leads to elevated expression of MCL-1, an anti-apoptotic member of the BCL-2 family. Upregulation of MCL-1 underlies the resistance of AML to initially successful targeted therapies, including Bcl-2 inhibition with venetoclax, placing MCL-1 as an attractive target for combinatorial therapies. We have recently shown for the first time that a novel MCL-1 inhibitor S63845 elicited synergistic activity with FLT3 inhibitors AC220, sorafenib and with a multi-kinase inhibitor midostaurin at nanomolar doses in pre-clinical in vitro models of FLT3-ITD AML, including cells resistant to venetoclax (Skwarska A, et al. Cancer Res 2019;79 Suppl 13, Abstract#342). Here, we further explored the therapeutic potential and mechanisms-of-action of dual targeting of FLT3-ITD and MCL-1 in vitro and in vivo. S63845 and midostaurin co-treatment significantly increased apoptosis in FLT3-ITD cells with caspase-3 activation and PARP cleavage within 6 hours of treatment. Analysis of drug combinations using Bliss independence model revealed strong synergistic effect of S63845/midostaurin combination in murine isogenic Ba/F3-FLT3-ITD, Ba/F3-FLT3-ITD-D835Y and Ba/F3-FLT3-ITD-F691L dual mutants with minimal effect in Ba/F3-wt FLT3 cells, suggesting that inhibition of FLT3-ITD could mechanistically contribute to the synergy between midostaurin and S63845. Midostaurin monotherapy caused de-phosphorylation of FLT3-ITD and its downstream targets STAT5, AKT and MAPK, known to upregulate MCL-1 expression and stability (Fig 1A). Consequently, midostaurin significantly reduced Mcl-1 T163 phosphorylation and protein levels exclusively in FLT3-ITD cells. Midostaurin also decreased inhibitory phosphorylation of GSK3, suggesting the involvement of ubiquitin/proteasome axis in MCL-1 degradation. Additionally, pre-treatment with pan-caspase inhibitor zVAD-fmk partially reversed MCL-1 downregulation, implying contribution of caspase-mediated degradation to overall decrease of MCL-1 after midostaurin treatment. While midostaurin alone enhanced degradation of MCL-1, it did not fully deplete the pool of MCL-1. Surprisingly, midostaurin also induced the loss of proapoptotic NOXA, an endogenous inhibitor of MCL-1, which can potentially increase a fraction of free MCL-1. These results provide a strong rationale for targeting residual MCL-1 with selective MCL-1 inhibitor S63845 with the goal to facilitate deeper therapeutic responses in FLT3-ITD AML. Mechanistically, midostaurin increased expression of apoptosis activator BIM and blocked BIM phosphorylation required for its inhibitory sequestration by MCL-1. BIM shRNA knockdown cells were less sensitive to midostaurin/S63845 combination. Similarly, CRISPR/Cas9-mediated knock-out of BAK, a BIM-activated executor of apoptosis, resulted in reduced efficacy of tested combination. These results indicate that activation of BIM/BAX axis has a functional role in the response of AML cells to dual FLT3-ITD and MCL-1 inhibition (Fig 1B). To validate the efficacy of combination in vivo, we used xenotransplantation model of MOLM-14 cells expressing luciferase. Mice were treated for 3 weeks with low doses of midostaurin (25 mg/kg/5 days per week) and with MIK665, structurally optimized version of S63845 Mcl-1 inhibitor with improved pharmacokinetics in mice (Halilovic E, et al. Cancer Res 2019;79 Suppl 13, Abstract#4477). Administration of MIK665 at 25 mg/kg or 35 mg/kg IV 2 days per week in combination with oral midostaurin resulted in a marked delay in the progression of MOLM-14 cell line-derived xenograft and significantly reduced leukemia tumor burden (Fig 1C). The long-term effect of combination on mice survival is currently being tested and will be reported. Altogether, our results indicate that efficacy of FLT3-ITD inhibitors can be enhanced through combination with low doses BH3 mimetics targeting MCL-1 and provide a rationale for the clinical evaluation of such combinations in FLT3 mutant AML patients. Figure 1 Figure 1. Disclosures Skwarska: Halilovich E, Wang Y, Morris E, Konopleva M, Skwarska A.: Patents & Royalties: Combination of a MCL-1 inhibitor and midostaurin, uses and pharmaceutical composition thereof.. Halilovic: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Mistry: Novartis: Current Employment, Current holder of individual stocks in a privately-held company. Derréal: Servier: Current Employment. Banquet: Servier: Current Employment. Daver: Genentech: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Novimmune: Research Funding; Glycomimetics: Research Funding; Trovagene: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Astellas: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Hanmi: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Andreeff: Medicxi: Consultancy; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Glycomimetics: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy; Aptose: Consultancy; Oxford Biomedica UK: Research Funding; Amgen: Research Funding; Breast Cancer Research Foundation: Research Funding; Senti-Bio: Consultancy. Wei: Novartis, Astellas, Pfizer, MacroGenics, AbbVie, Genentech, Servier, Celgene, Amgen, AstraZeneca, Janssen: Honoraria; Novartis, Celgene, AbbVie, Servier, AstraZeneca, and Amgen: Research Funding; Walter and Eliza Hall Institute: Ended employment in the past 24 months. Konopleva: Cellectis: Other: grant support; Stemline Therapeutics: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; AstraZeneca: Other: grant support, Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Ascentage: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; KisoJi: Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding.

Metabolic reprogramming is recognized as one of the key hallmarks in acquiring aggressive phenotype and chemoresistance in solid tumors and hematologic malignancies. We have previously demonstrated that T-ALL are characterized by significant dependency on oxidative phosphorylation (OxPhos) with ability to utilize glutamine either in oxidative or reductive directions of TCA cycle, when mitochondria are blocked by Complex I Inhibitor (Baran N, et al. ASH 2020). To survive upon Complex I blockade leukemic cells require functional monocarboxylate transporter MCT1, that enables excretion of lactate and permissive pyruvate flux (Fig.1 a). Here we show that metabolic intervention utilizing OxPhos blockade can be potentiated by targeting MCT1 transporter and propose a novel metabolic synthetic lethality that could be exploited to eradicate T-ALL and other OxPhos-dependent malignancies. We first demonstrated that Complex I inhibition leads to increased MCT1 expression; on the contrary, MCT1 transporter blockade forces cells to increase OxPhos. In turn, the combinatorial therapy with Complex I inhibitor (IACS-010759) and MCT1 inhibitor (AZD3965) causes loss of ATP content (Fig. 1b), significant reduction of cell number and massive induction of apoptosis. Mechanistically, the combination treatment further reduced oxygen consumption rate (OCR) (Fig. 1c) and increased extracellular acidification rate, as measured by Seahorse. In concert with those results, dual inhibition led to TCA blockade, accumulation of intracellular lactate and depletion of glutamine, cystathionine and glutathione, indicating severe disruption of redox balance as measured by mass spectrometry and confirmed by significant accumulation of intracellular and mitochondrial reactive oxygen species (ROS) (Fig. 1d), loss of mitochondrial membrane potential (ΔΨ) (Fig. 1e) and subsequent mitochondria swelling. RNAseq data showed simultaneous upregulation of glycolysis and glutathione-related processes as possible mechanisms of metabolic compensation, yet strong upregulation of genes regulating apoptosis related to mitochondria dysfunction (Fig. 1f). Real-time hyperpolarized MRI based metabolic imaging studies with [1-13C]-pyruvate in patient-derived xenografts in vivo revealed significant decrease of lactate-to-pyruvate ratio in mice treated with AZD3965 or IACS-010759 alone, and in mice treated with drug combination. [13C]-Glucose isotope tracing analysis in patient-derived xenografts in vivo revealed an increased intracellular trapping of lactate as a marker of treatment effectiveness in mice subjected to dual blockade. While MCT1 inhibition induced only moderate reduction of leukemia growth in vitro and tumor burden in vivo, combination with IACS-010759 depleted significantly both, circulating and marrow/spleen/liver resident leukemia cells. Mechanistically, inhibition of MCT1 by AZD3965 therapy in leukemia-bearing mice led to lactate accumulation, OCR increase, moderate ROS production and mitochondrial membrane hyperpolarization, while Complex I blockade resulted in upregulation of MCT-1, reduction of OCR, lactate production and increase of ROS ; consequently, combinatorial therapy caused complete mitochondria shut-down and drastic inhibition of tumor growth both in vitro and in vivo in two xenografts models and led to significant extension of overall survival (p Figure 1 Figure 1. Disclosures Skwarska: Halilovich E, Wang Y, Morris E, Konopleva M, Skwarska A.: Patents & Royalties: Combination of a MCL-1 inhibitor and midostaurin, uses and pharmaceutical composition thereof.. Konopleva: Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Ascentage: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Cellectis: Other: grant support; Sanofi: Other: grant support, Research Funding; KisoJi: Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support.

Background: Presence at diagnosis or acquisition of activating RAS pathway mutations is a pervasive mechanism of resistance to therapy in AML. Efforts to directly target mutant RAS have been unsuccessful and the efficacy of BRAF and MEK inhibitors has been limited due to compensatory reactivation of MAPK signaling. ERK1/2 (ERK) is a key downstream component in the MAPK pathway and therefore represents an attractive target for inhibiting MAPK signaling. Compound 27 (1) is a dual-mechanism inhibitor of ERK that inhibits both the catalytic activity of ERK and its phosphorylation by MEK. It is a close analog of ASTX029, a dual-mechanism ERK inhibitor currently under clinical investigation in solid tumors (NCT03520075). Objectives: We analysed the preclinical activity of Compound 27 in AML, investigated its mechanism of action and ability to overcome resistance. Results: Using a panel of 9 AML cell lines, the IC50 value for single agent Compound 27 was in the low to intermediate nanomolar range (1.89-388 nM). Decreased ERK phosphorylation was confirmed by Western blot analysis. To better characterize the biological effects of Compound 27, we performed mass cytometry (CyTOF) analysis of NRAS-mutated OCI-AML3 cells. This experiment showed approximately 75% downregulation of CyclinB1 and cMyc in 250 nM drug-treated cells versus untreated cells (Figure 1a). The expression of anti-apoptotic proteins, including MCL1, BclXL and Bcl2, were also decreased. Western blot analysis confirmed increased cleaved PARP, and reduced cMyc and cell cycle-related proteins CyclinB1, CyclinD1 and CDK4 with Compound 27 treatment. In isogenic cells, p53 knock-down had no effect on the efficacy of Compound 27. We next investigated the efficacy of simultaneous inhibition of ERK and Bcl-2 in AML cells. Compound 27 sensitized OCI-AML3 cells, which are intrinsically resistant to ABT-199 (a Bcl-2 inhibitor), to treatment with ABT-199 and shifted the cytostatic effect of the single agents to a cytotoxic effect with a combination index (CI) of 0.008 (cell death 91% for combination versus 20% with ABT-199 alone). This suggests strong synergistic effects of combination treatment (Figure 1b). In OCI-AML2 cells with acquired resistance to ABT-199, the combination increased apoptosis to 80% as compared to 20% with ABT-199 alone. Compound 27 sensitized bulk CD45+ as well as CD34+CD38-leukemia progenitor cells to ABT-199. Compound 27 also sensitized FLT3-ITD mutant human AML cell lines MOLM13, MOLM14, MV-4-11 and murine Ba/F3-ITD cells to the FLT3 inhibitor AC220 (CI in MOLM13=0.3). Synergy of Compound 27 and 5-azacitidine was also observed (p=0.009). Leukemia microenvironment-mediated resistance to therapy is partly mediated by MAPK activation. We co-cultured OCI-AML3 and MOLM13 cells with normal bone marrow-derived mesenchymal stromal cells (NMSCs) to mimic the bone marrow microenvironment and analysed the effect of Compound 27 in combination with either ABT-199 or AC220. Combination drug treatment were more effective in terms of cytoreduction and apoptosis induction in coculture. However, neither combination was able to completely overcome stroma-mediated resistance (Figure 1b). Analysis of other stroma-relevant molecules in coculture showed that CXCR4 was increased while CD44 was decreased in response to ERK inhibition. Effective reactive oxygen species (ROS) mitigation and hyper-active mitochondrial fission is important for maintaining "stemness" of AML cells (2). ERK phosphorylates DRP1, which is necessary for mitochondrial fission. Treatment of OCI-AML3 cells with Compound 27 led to increased mitochondrial ROS, decreased levels of pDRP1(Ser616) and increased mitochondrial length, suggesting impaired fission and reduced "stemness" of AML cells (Figure 1c). Conclusion: ERK inhibition by Compound 27 synergizes with 5-azacitidine, ABT-199 and AC220 and can overcome primary or acquired resistance. The impact on mitochondrial dynamics suggests a potential impact on leukemia stem cells. Additional mechanistic confirmatory work is in progress. References: 1. Heightman TD, Berdini V, Braithwaite H, et al. Fragment-based discovery of a potent, orally bioavailable inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2. J Med Chem. 2018;61(11):4978-4992. 2. Schimmer AD. Mitochondrial Shapeshifting Impacts AML Stemness and Differentiation. Cell Stem Cell. 2018;23(1):3-4. Figure 1 Figure 1. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Dao: Astex Pharmaceuticals, Inc.: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Andreeff: Medicxi: Consultancy; Syndax: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Oxford Biomedica UK: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees. Borthakur: University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; ArgenX: Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; GSK: Consultancy.

Background: De novo nucleotide synthesis is necessary to meet the enormous demand for nucleotides, other macromolecules associated with acute myeloid leukemia (AML) progression 1, 2, 3 4. Hence, we hypothesized that targeting de novo nucleotide synthesis would lead to the depletion of the nucleotide pool, pyrimidine starvation and increase oxidative stress preferentially in leukemic cells compared to their non-malignant counterparts, impacting proliferative and differentiation pathways. Emvododstat (PTC299) is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Objectives: The goals of these studies were to understand the emvododstat-mediated effects on leukemia growth, differentiation and impact on Leukemia Stem Cells(LSCs). Comprehensive analyses of mitochondrial function, metabolic signaling in PI3K/AKT pathways, apoptotic signatures, and DNA damage responses were carried out. The rationale for clinical testing emvododstat was confirmed in an AML-PDX model. Results: Emvododstat treatment in cytarabine-resistant AML cells and primary AML blasts induced apoptosis, differentiation, and reduced proliferation, with corresponding decreased in cell number and increases in annexin V- and CD14-positive cells. Indeed, the inhibition of de novo nucleotide synthesis compromises the dynamic metabolic landscape and mitochondrial function, as indicated by alterations in the oxygen consumption rate (OCR) and mitochondrial ROS/membrane potential and corresponding differentiation, apoptosis, and/or inhibition of proliferation of LSCs. These effects can be reversed by the addition of exogenous uridine and orotate. Further immunoblotting and mass cytometry (CyTOF) analyses demonstrated changes in apoptotic and cell signaling proteins (cleaved PARP, cleaved caspase-3) and DNA damage responses (TP53, γH2AX) and PI3/AKT pathway downregulation in response to emvododstat. Importantly, emvododstat treatment reduced leukemic cell burden in a mouse model of AML PDX ( Complex karyotype ,mutation in ASXL1, IDH2, NRAS), decreased levels of leukemia stem cells frequency (1 in 522,460 Vs 1 in 3,623,599 in vehicle vs emvododstat treated mice), and improved survival. The median survival 40 days vs. 30 days, P=0.0002 in primary transplantation and 36 days vs 53.5 days, P=0.005 in secondary transpantation in a PDX mouse model of human AML. This corresponded with a reduction in the bone marrow burden of leukemia and increased expression of differentiation markers in mice treated with emvododstat (Fig. 1). These data demonstrate effect of emvododstat on mitochondrial functions . Conclusion: Inhibition of de novo pyrimidine synthesis triggers differentiation, apoptosis, and depletes LSCs in AML models. Emvododstat is a novel dihydroorotate dehydrogenase inhibitor being tested in a clinical trial for the treatment of myeloid malignancies and COVID-19. Keywords: AML, emvododstat, DHODH, apoptosis, differentiation References: 1 Thomas, D. & Majeti, R. Biology and relevance of human acute myeloid leukemia stem cells. Blood 129, 1577-1585, doi:10.1182/blood-2016-10-696054 (2017). 2 Quek, L. et al. Genetically distinct leukemic stem cells in human CD34- acute myeloid leukemia are arrested at a hemopoietic precursor-like stage. The Journal of experimental medicine 213, 1513-1535, doi:10.1084/jem.20151775 (2016). 3 Villa, E., Ali, E. S., Sahu, U. & Ben-Sahra, I. Cancer Cells Tune the Signaling Pathways to Empower de Novo Synthesis of Nucleotides. Cancers (Basel) 11, doi:10.3390/cancers11050688 (2019). 4 DeBerardinis, R. J. & Chandel, N. S. Fundamentals of cancer metabolism. Sci Adv 2, e1600200, doi:10.1126/sciadv.1600200 (2016). Figure 1 Figure 1. Disclosures Weetall: PTC therapeutics: Current Employment. Sheedy: PTC therapeutics: Current Employment. Ray: PTC therapeutics: Current Employment. Andreeff: Karyopharm: Research Funding; AstraZeneca: Research Funding; Oxford Biomedica UK: Research Funding; Aptose: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Syndax: Consultancy; Breast Cancer Research Foundation: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Senti-Bio: Consultancy; Medicxi: Consultancy; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Glycomimetics: Consultancy. Borthakur: ArgenX: Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; Astex: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Ryvu: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy.

DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in MM (Marchesini et. al., Cancer Cell 2017). We continuously treated JJN3 and KMS11 cells with an ILF2-targeting antisense oligonucleotide (ILF2 ASOs) or control non-targeting antisense oligonucleotide (NT ASOs). Whereas KMS11 cells maintained a high level of DNA damage activation and a significantly increased rate of apoptosis after 3 weeks of ILF2 ASOs treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and became resistant to ILF2 ASOs treatment. To evaluate whether continuous ILF2 ASOs exposure could lead to the selection of MM clones intrinsically resistant to ILF2 ASO-induced DNA damage, we performed single-cell RNA seq (scRNA-seq) analysis of JJN3 cells treated with NT or ILF2 ASOs for 3 weeks. Our analysis divided JJN3 cells into 2 main clusters that were independent of treatment (Fig. 1A), suggesting that persistent exposure to ILF2 ASOs did not induce clonal selection. Differential gene expression analysis of NT ASO- and ILF2 ASO-treated cells in each of these clusters revealed that DNA damage resistant ILF2 ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Consistent with these results, metabolomic analysis of JJN3 cells after long-term exposure to ILF2-ASOs showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates (Fig. 1B). ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 library screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs were significantly depleted after 3 weeks of treatment in ILF2-ASO-treated JJN3 cells but not in ILF2-ASO-treated KMS11 cells. These data suggest that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis in JJN3 cells. These data gain added significance in light of previous findings that DNA2 is a nuclear and mitochondrial DNA nuclease/helicase that enables cancer cells to counteract the DNA replication stress and mitochondrial oxidative DNA damage induced by DNA-damaging agents. Accordingly, we observed that DNA2 was mainly localized into the mitochondria of MM cells. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO-and ILF2-ASO-treated MM cells exposed to NSC for 72 hours showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. These data suggest that DNA2 is essential to counteract oxidative DNA damage and maintain mitochondrial respiration after MM cells' metabolic reprogramming. In conclusion, our study has revealed a novel mechanism through which MM cells can overcome DNA damage activation. Further studies will clarify whether targeting DNA2 is synthetically lethal in tumors with increased demand of mitochondrial metabolism. Figure 1 Figure 1. Disclosures Konopleva: Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Rafael Pharmaceuticals: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Stemline Therapeutics: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; KisoJi: Research Funding.

Background: Acute myeloid leukemia (AML) stem cells (LSC), the likely source of relapsed disease, are capable of surviving current standard chemotherapy. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression datasets and identified CD200 as significantly over-expressed in LSCs when compared to paired blast cells, as well as when compared to their normal hematopoietic stem cell (HSC) counterparts (Fig 1A; Herbrich et al Blood. 2018; 130:3962). CD200 can identify AML cells with LSC activity in vivo (Ho et al Blood. 2016; 128:1705). Functionally, CD200 has been shown to have an immunosuppressive effect on macrophages (Hoek et al Science. 2000; 290:1768) and NK cells (Coles et al Leukemia. 2012; 26:2148), and correlates with a high prevalence of FOXP3+ regulatory T cells (Coles et al Leukemia. 2012; 26:2146). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget. 2015; 6:30212). To date, we have screened 40 primary AML patient samples by flow cytometry, 95% of which are positive for CD200. Methods: To study the functional role of CD200 in AML, we generated a CD200 overexpression model in the human OCI-AML3 cell line (with no basal expression) and characterized changes in proliferation, survival, and gene expression. To examine the immune function of CD200 in AML in vitro, we performed a series of mixed lymphocyte reactions with isolated effector immune cells and target isogenic AML cell lines to assess immune cell-mediated apoptosis, proliferation, and cytokine secretion. To understand the contribution of CD200 immune protection in a physiological setting, we developed a peripheral blood mononuclear cell (PBMC)-humanized mouse in which we tracked the engraftment and overall survival of the CD200+/- OCI-AML3 cells. Lastly, the utility of CD200-blockade using a fully humanized anti-CD200 monoclonal antibody (CD200-IgG1) was evaluated both in vitro and in vivo. Results: In vitro, CD200+ AML significantly inhibited the secretion of inflammatory cytokines and cytotoxic enzymes from healthy PBMCs; a phenomenon that could be largely reversed by blocking the CD200/CD200R interaction with the CD200 antibody (Fig 1B). In vivo, OCI-AML3 CD200+/- cells showed no difference in engraftment, progression, and overall survival in immunodeficient NSG mice (Fig 1C). However, when mice were humanized using healthy PBMCs, CD200+ leukemia progressed rapidly, escaping T cell-mediated elimination, compared to CD200- control leukemic cells (Fig 1D). Cytokine production in PBMC-humanized mice was significantly compromised in those with CD200-expressing leukemia. Transcriptome analysis revealed that T cells from humanized mice exposed to CD200 expressing disease were metabolically quiescent. In humanized mice, CD200-IgG1 therapy eliminated CD200+ AML disease (Fig 1E). The novel CD200-IgG1 antibody also induced potent, specific NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody dependent cellular phagocytosis (ADCP; Fig 1F). Conclusion: We have identified CD200 as a putative stem cell-specific immunomodulatory target that aids in establishing an immunosuppressive microenvironment by significantly suppressing cytokine secretion in response to AML. In a PBMC-humanized mouse model, the presence of cell-surface CD200 was sufficient to protect AML cells from immune-mediated clearance and could be reversed using a blocking anti-CD200 mAb. These findings indicate a utility of CD200 as a novel immune checkpoint target for the development of therapeutic strategies in AML. Disclosures Konopleva: Calithera: Research Funding; Kisoji: Consultancy; 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; Ablynx: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; AstraZeneca: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Forty-Seven: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ascentage: Research Funding.

The antileukemic effect of inhibiting bromodomain and extra-terminal domain-containing (BET-containing) proteins (BETPs) such as BRD4 has largely been largely attributed to transcriptional downregulation of cellular anabolic and antiapoptotic processes, but its effect on the bone marrow microenvironment, a sanctuary favoring the persistence of leukemic stem/progenitor cells, is unexplored. Sustained degradation of BETP with the small-molecule BET proteolysis-targeting chimera (PROTAC) ARV-825 resulted in a marked downregulation of surface CXCR4 and CD44, key proteins in leukemia-microenvironment interactions, in acute myeloid leukemia (AML) cells. Abrogation of surface CXCR4 expression impaired SDF-1α–directed migration and was mediated through transcriptional downregulation of PIM1 kinase, which in turn phosphorylates CXCR4 and facilitates its surface localization. Downregulation of CD44, including isoforms CD44v8–10 impaired cystine uptake, lowered intracellular reduced glutathione, and increased oxidative stress. More important, BETP degradation markedly decreased the CD34(+)CD38(–)CD90(–)CD45RA(+) leukemic stem cell population and, alone or in combination with cytarabine, prolonged survival in a mouse model of human leukemia that included AML patient-derived xenografts (AML-PDX). Gene expression profiling and single-cell proteomics confirmed a downregulation of the gene signatures associated with “stemness” in AML and Wnt/β-catenin and Myc pathways. Hence, BETP degradation by ARV-825 simultaneously targets cell-intrinsic signaling, stromal interactions, and metabolism in AML.

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.

Anti-PD1 therapy in hematologic malignancies has shown clinically inferior effects when compared to solid tumors.1 However, immunotherapy has been part of the standard of care in the treatment of acute leukemia for over 40 years. Recently, loss of MHCII was shown to be a mechanism of Immune escape in patients with acute myeloid leukemia after stem cell transplantation.2 Here we demonstrate, in pre-clinical models, that responses to anti-PD1 can be enhanced by increased antigen presentation through induction of MHCII. We used a Philadelphia chromosome positive (Ph+) Acute Lymphoblastic Leukemia (ALL) syngeneic mouse model and treated 4 cohorts of mice with the following regimens: 1) vehicle 2) single agent Dasatinib; 3) single agent anti-PD1; 4) combination anti-PD1 + Dasatinib (Figure 1a). Single agent Dasatinib, was highly active against this model compared to vehicle. Although anti-PD1 therapy showed little or no activity as a single agent, when combined with sub-therapeutic doses of Dasatinib, we observed significantly enhanced survival of mice compared to single agent anti-PD1 or to single agent Dasatinib. (Figure 1b) In our mouse model, Dasatinib increased the MHCII on the surface of the antigen presenting cells in the tumor microenvironment, most notably in dendritic cells (CD11c+ cells) from mouse tumor-infiltrating lymphocytes collected from the mice bone marrow after treatment (Fig 1c). To prove whether Dasatinib-induced MHCII expression can occur independently of a tumor microenvironment, we treated antigen presenting cell lines (KG1) with Dasatinib and were able to confirm an increased MHCII expression by flow cytometry and by western blotting. (Fig 1d) Since Dasatinib is approved for the treatment of Philadelphia chromosome positive ALL, CML, and has been used in a variety of other settings in the treatment of both malignant hematology and solid tumors, this data has immediate translational potential. In fact, Dasatinib in combination with anti-PD1 therapy is currently being tested in a variety of phase I/II trials in various kinds of malignancies (NCT04284202, NCT03516279). Here we show that induction of MHCII by Dasatinib may serve as a biomarker and could predict the potential benefit from the combination treatment. Additionally, targeting anti-PD1 following Dasatinib administration may increase response rates of patients treated with Dasatinib, providing a rationale for sequential treatment design in patients with persistent minimal residual disease. Figure 1. (a) C57BL/6J mice were injected via tail vein injection with a transplantable, immunocompetent BCR-ABL+ B-ALL model and treated with vehicle/Dasatinib/or anti-PD1 as described in the schema. (b) Overall survival of the mice treated in the previous experiment. (c) MHCII cell surface expression on dendritic cells (CD11+ cells) from the bone marrow of treated mice. (d) relative Cell surface expression of MHCII in the KG1 cell line (APC cell line) treated with Dasatinib vs. vehicle. (e) protein expression of MHCII in the KG1 cell line (APC cell line) treated with Dasatinib vs. vehicle. 1. Masarova L, Kantarjian H, Ravandi F, Sharma P, Garcia-Manero G, Daver N. Update on Immunotherapy in AML and MDS: Monoclonal Antibodies and Checkpoint Inhibitors Paving the Road for Clinical Practice. Adv Exp Med Biol 2018;995:97-116. W2. Christopher MJ, Petti AA, Rettig MP, et al. Immune Escape of Relapsed AML Cells after Allogeneic Transplantation. New England Journal of Medicine 2018;379:2330-41. Figure Disclosures Koller: Jazz Pharmaceuticals, Inc.: Consultancy. Konopleva:Stemline Therapeutics: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; Sanofi: Research Funding; Genentech: Consultancy, Research Funding; Ablynx: Research Funding; Cellectis: Research Funding; Agios: Research Funding; AbbVie: Consultancy, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy; 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; AstraZeneca: Research Funding; Amgen: Consultancy; Calithera: Research Funding; Eli Lilly: Research Funding; Rafael Pharmaceutical: Research Funding. OffLabel Disclosure: anti-pd1 therapy in acute lymphoid leukemia

Isocitrate dehydrogenases (IDH) are involved in redox control and central metabolism. We hypothesized that key metabolic fluxes are selectively reprogrammed to maintain biosynthetic homeostasis and lower drug responses in IDH mutant acute myeloid leukemia cells. Here we show that metabolic reprogramming initiated by IDH1 mutation leads to marked increases in glucose, glutamine and fatty acid catabolism that along with enhancement of wild-type IDH enzyme activity contribute to provision of α-KG required for 2-HG synthesis and to replenish Krebs cycle intermediates for biosynthetic reactions, oxygen consumption and ATP production. Mechanistically, this occurs through both methylation-driven CEBPα activation of FAO and reprogramming of systemic metabolic fluxes through other pathways that augment catabolic flexibility. Consequently, this catabolic flexibility enhances Krebs cycle and OxPHOS activities that are not necessarily rescued by IDH mutant inhibitors or 2-HG reduction. This renders IDH1 mutant cells more resistant to chemotherapeutics but more susceptible to mitochondrial inhibition. Our findings provide a scientific rationale for innovative combinatory targeted therapies to treat this subgroup of patients, especially those unresponsive to or relapsing from IDH mutant-specific inhibitors.

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.

Immune checkpoint-based therapies, which target the regulatory pathways of immunocompetent cells to enhance anti-tumor responses, have been at the heart of many recent clinical advances and have led to long-term remissions and possible cures. Most of this success was achieved with T-cells, but there are compelling reasons to predict that checkpoint ablation could modify NK cells in ways that would facilitate their antitumor activity. The suppressor of cytokine signaling (SOCS) family of proteins plays an important role in NK cell biology by attenuating cytokine signaling and effector function against cancer. One of its members, cytokine inducible SH2 containing protein (CIS), encoded by the CISH gene, is as an important checkpoint molecule in NK cells and is upregulated in response to IL-15. We hypothesized that CIS may act as a potent checkpoint in our iC9/CAR19/IL15 NK cells given the fact that they continuously produce IL-15, and that targeting this pathway would enhance their potency against B cell malignancies. In a series of in vitro studies, we showed that CISH is induced in iC9/CAR19/IL15 NK cells in a time dependent manner. To examine the functional consequences of CISH deletion in our CAR-NK cells, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing to silence CISH and retroviral transduction with the iC9/CAR19/IL15 construct. On day 7 we nucleofected the CAR transduced NK cells with Cas9 alone (Cas9 control) or Cas9 pre-loaded with crRNA:tracrRNA duplex targeting CISH exon 4. Gene editing efficiency was >90% as quantified by PCR and western blot. CISH knockout induced a phenotype characterized by the increased expression of markers of activation and cytotoxicity. These included granzyme-b, perforin, TRAIL and CD3z; transcription factors such as eomesodermin and T-bet; adaptor molecules such as DAP12; and activating coreceptors/proliferation markers such as DNAM, CD25 and Ki67. CISH knockout resulted in significantly enhanced function of iC9/CAR19/IL15 NK cells against Raji lymphoma evident by increased cytokine production (TNFa p=0.007, IFNg p=0.033) and degranulation (CD107a p=0.003) compared to Cas9 control cells. Moreover, CISH KO iC9/CAR.19-IL15 NK cells killed Raji lymphoma more efficiently than Cas9 control cells and formed a stronger immunologic synapse (p=0.037). RNA sequencing with gene set enrichment analysis (GSEA) confirmed enrichment of JAK/STAT signaling, TNFα and IFN-γ inflammatory response, mTORC1, and MYC hallmark pathways in CISH KO iC9/CAR19/IL15 NK cells compared to Cas9 control counterparts, providing a molecular mechanism for their enhanced effector function. Moreover, in an in-vivo NSG mouse model of Raji lymphoma, the antitumor activity of a single dose of CISH KO iC9/CAR19/IL15 transduced CB NK cells was significantly better than that of Cas9 control cells leading to a significant survival advantage (p=0.003) without evidence of increased toxicity. Thus, we demonstrate for the first time, that silencing a critical checkpoint in CAR-NK cells improves their potency, permitting greater cytotoxic effector function than seen with unmodified CAR-NK cells. Our data support the merging of CAR-engineering and immune checkpoint gene editing to enhance the therapeutic potential of NK cells. We are in the process of scaling up this approach in our GMP facility for translation to the clinic for the treatment of relapsed/refractory B cell hematologic malignancies. Disclosures 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. Champlin:Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

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.

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.

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.

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.

Imidazoacridinone C-1311 is a small molecule inhibitor of topoisomerase II and receptor tyrosine kinase FLT3. We have previously shown that C-1311 induces apoptosis in acute myeloid leukemia (AML) cells with FLT3-ITD mutations (Skwarska A. et al. Biochem Pharmacol 2015; 95:238-52). Oncogenic internal tandem duplication mutations (ITD) of FLT3 occur in 25% cases of AML and are associated with aggressive clinical course. Although preclinical studies indicate that inhibition of receptor tyrosine kinases often induces protective autophagy, little is known of the role of FLT3-ITD in this process. Recent studies indicate that unlike wild-type FLT3, FLT3-ITD mutant facilitates basal autophagy in AML suggesting that this subset of leukemia may highly depend on autophagic activity. The role of autophagy in leukemia cell biology and treatment is complex as depending on the nature, intensity and duration of the drug treatment, autophagic cells may survive or die. Here we investigated the functional impact of autophagy on the cytotoxic activity of C-1311 in a panel of AML cell lines including FLT3-wt (OCI-AML3, OCI-AML2, HL60) and mutant FLT3-ITD (MV4-11, MOLM13, MOLM14). In FLT3-ITD MV4;11 AML cells but not in FLT3-wt HL-60 cells, C-1311 treatment led to significant activation of autophagy as confirmed by acridine orange staining that selectively labels autophagic vesicular organelles (Fig 1A). Further, autophagy flux (monitored as a decrease of autophagy-related p62 protein associated with concentration-dependent conversion of LC3-I to LC3-II) was observed in FLT3-ITD but not in FLT3-wt cells. Western blot analysis showed that C-1311 triggered autophagy by blocking a major repressor of autophagy, mammalian target of rapamycin (mTOR) and its downstream phosphorylated effectors such as S6, 4EBP1 and eIF4E. Importantly, the elevated autophagy observed in FLT3-ITD AML was associated with downregulation of the anti-apoptotic Mcl-1 and Bcl-XL proteins, PARP cleavage and phosphatidylserine exposure consistent with apoptosis induction (Fig. 1B). In contrast, C-1311 induced minimal autophagy and apoptosis in FLT3-wt AML cells. Pharmacological inhibition of late stages of autophagy with bafilomycin A1 markedly decreased viability of C-1311 co-treated FLT3-ITD cells as well as primary FLT3-ITD AML cells and enhanced apoptotic response as compared to single agent therapy. On the contrary, combination treatment with bafilomycin A1 only moderately sensitized FLT3-wt cells to C-1311 with no effect on healthy peripheral blood mononuclear cells. These results suggests that autophagy induced by C-1311 is a pro-survival cellular response enhanced in mutated FLT3-ITD cells. Recent evidence indicate that transcription factor ATF4, which regulates expression of autophagy genes in response to stress, serves as an essential mediator of FLT3-ITD-induced autophagy as its levels are highly dependent on FLT3-ITD activity (Heydt Q. et al. Oncogene 2017;1-11). Consistent with C-1311-mediated inhibition of mutated FLT3, the level of ATF4 significantly decreased in FLT3-ITD mutant cells (Fig 1C). In contrast, C-1311 induced increased expression of ATF4 in FLT3-wt cells. Given that ATF4 is also a key regulator of the mitochondrial stress in mammalian cells, ATP production and oxygen consumption rate (OCR) were measured using Seahorse XF24 analyzer. Both, OCR and ATP were profoundly reduced in FLT3-ITD mutants in response to C-1311 (Fig 1D, E). Importantly, bafilomycin A1 further facilitated C-1311-caused decrease in mitochondrial respiration and ATP, suggesting that suppression of autophagy impairs mitochondrial function in FLT3-ITD mutants. In FLT3-wt cells, C-1311 treatment increased ATP production, which correlated with elevated ATF4 expression and reduced apoptosis (Fig 1D, E). Our data suggest that C-1311-mediated inhibition of FLT3-ITD triggers the induction of pro-survival autophagy, which cannot be sustained due to loss of autophagy regulator ATF4 accompanied by mitochondrial dysfunction and apoptosis. Importantly, the decrease in ATF4 and induction of massive apoptosis was characteristic for FLT3-ITD mutants exposed to C-1311, whereas cells with FLT3-wt upregulated expression of ATF4 as an adaptive response preventing cell death. These results indicate on a cross-talk between FLT3-ITD, ATF4 and autophagy which may dictate the fate of cells exposed to FLT3 inhibitors like C-1311. Disclosures Konopleva: Stemline Therapeutics: Research Funding.

Introduction Anti-apoptotic Bcl2 family members mediate resistance to therapies in acute myeloid leukemia (AML)1. The small molecule Bcl2 inhibitor ABT-199 (venetoclax) promotes mitochondria driven intrinsic apoptosis, and in combination with hypomethylating agents or chemotherapy, has been highly promising in the clinic as treatment of AML2-4. The response rate to ABT-199 is very impressive, but acquired resistance is a major problem. Compensatory upregulation of Mcl1 is an important mechanism of such acquired resistance to mitochondrial apoptosis5. Autophagy is vital for mitochondrial health, mediates resistance to apoptosis and is induced by Bcl2 inhibition6. We performed mechanistic studies to address our hypothesis that disabling autophagy by targeting the apical autophagy kinase ULK1 can reverse resistance to ABT-199. Methods ULK1 was genetically modified in OCIAML3 (human AML cell line), by shRNA knockdown (KD) or CRISPR-Cas9 knockout (KO). In addition, AML cell lines (including ABT-199 resistant) and patient samples were treated with ABT-199 and ULK1 inhibitor SBI-02069657. Combination index (CI) for drug synergy was calculated based on Chou-Talalay method8. Drug-treated or genetically manipulated cells were profiled by reverse phase protein array (RPPA), mass cytometry (CyTOF) and gene expression profiling (GEP). Autophagy was detected by LC3 quantification by western blot (WB) and flow cytometry, and monodansylcadaverine assay. Mitochondrial functions were analyzed by Seahorse Cell Mito Stress test, and MTG, TMRE and ROS assays (flow cytometry). For in vivo studies ULK1 KO and corresponding control cells were injected in NSG mice and monitored by bioluminescent imaging (BLI) and quantification of human CD45 cells. Results ABT-199 induced autophagy in OCIAML3 (increase by 175±27%, p=0.01 - LC3 flow; 4X increase in LC3 II/I ratio - WB). Apoptosis induction by ABT-199 was enhanced by ULK1 KD (36±1.9% over control, p Mcl1 was significantly downregulated by ULK1 inhibitor alone and in combination with ABT-199. ULK1 inhibition lowered Mcl1 transcription, as measured by qRT-PCR: 43±0.03% with SBI-0206965 and 63±0.3% in KO cells (both p Since ABT-199 modulates mitochondrial function, we examined the effect of inhibiting ULK1 in this context. By Seahorse assay, the combination decreased basal OCR and ATP production by 62 and 58% respectively, p Corroborating our earlier data, the ABT-199 resistant cells (OCIAML2R & MOLM13R) show enhanced autophagy as compared to parental cells (OCIAML2: 83%, MOLM13: 35% increase; p=0.001 & 0.009). SBI-0206965 reversed ABT-199 induced autophagy and restored ABT-199 sensitivity in these cells (Fig 4). In a pilot in vivo experiment control and ULK1 KO cells were injected in NSG mice and leukemia engraftment was markedly delayed in the ULK1 KO group (Fig 5). The therapeutic combination study is ongoing. Conclusion Results indicate concomitant targeting of autophagy by ULK1 inhibition and Bcl2 inhibition by ABT-199 can overcome acquired resistance to ABT-199. Hence, with the emergence of Bcl2 inhibitors in frontline therapy for AML and efforts at developing ULK1 inhibitors, this study informs the development of novel apoptosis/autophagy targeting approaches to improve AML therapy. Disclosures Konopleva: Stemline Therapeutics: Research Funding; abbvie: Research Funding; Immunogen: Research Funding; cellectis: Research Funding. Andreeff:Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Jazz Pharma: Consultancy; SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Celgene: Consultancy; Reata: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding.

Background: Persistence of leukemia initiating cells (LIC) in T acute lymphoblastic leukemia (T-ALL) results in relapse in 20- 25% of pediatric and over 50% of adult patients1. LIC are characterized by high CD44 expression and low reactive oxygen species (ROS) levels 2, 3. T-ALL LIC maintain low ROS by cystine/glutamate anti-porter complex of which CD44 is a key component 2, 4. CD44 also interacts with microenvironmental components; hyaluronic acid, osteopontin, fibronectin etc. NOTCH1 transcriptionally upregulates CD44/MYC by binding the upstream 'super-enhancer' sites. BRD4, a member of the bromodomain and extra terminal domain (BET) family, is a critical scaffold of super-enhancer complexes that binds acetylated histones (3 and 4) and drives NOTCH1 mediated MYC/CD44 transcription 5, 4, 1. We hypothesized that degradation of BRD4 with hetero-bifunctional PROteolysis TArgeting Chimera (PROTACTM) such as ARV-825, will lead to efficient and sustained downregulation of NOTCH1/MYC /CD44 transcription and disrupt cell intrinsic and extrinsic pathways for persistence of T-ALL LIC. Methods: We confirmed the anti-leukemia effect of ARV-825 (IC50 pico to low nanomolar) against T-ALL cells including early T-cell phenotype (ETP) and Gamma-secretase inhibitor (GSI) resistant T-ALL8. To confirm disruption of NOTCH1/MYC/CD44 axis in-vivo, we specifically tested the impact of BRD4 degradation on NOTCH1 and its target genes including MYC, CD44 and as functional readout, intra-cellular ROS, in a T-ALL/ patient-derived xenograft (PDX) mouse model of disseminated leukemia possessing a constitutively active NOTCH1 mutation. Mass cytometry based proteomic analysis (CyTOF) studies were done to quantitatively assess T-ALL LICs and suppression of NOTCH1-MYC-CD44 axis, and secondary transplantation was carried out into sub-lethally irradiated mouse recipients to functionally evaluate LIC elimination. Results: ARV-825 mediated sustained BRD4 degradation resulted in profound down-regulation of MYC, CD98, CD44 and its variants (CD44v). Moreover, we observed down-regulation of cell intrinsic anti-apoptotic proteins Bcl-2, Bcl-XL. As a functional correlate of down-regulation of CD98/CD44/CD44v (glutathione anti-porter system), flow cytometry confirmed increased intracellular ROS and decreased reduced glutathione (GSH). In a PDX mouse model of human T-ALL, ARV-825 treatment improved survival compared to mice treated with vehicle (P=0.002) (Fig.1). CyTOF analysis of mouse bone marrow cells showed quantitative reduction of phenotypically defined LIC (CD4+CD8+CD7+ CD19- ) 6,7 with down regulation of the NOTCH1-MYC-CD44 axis along with oncogenic molecules (transcription factors Myc and NFkB, cell cycle regulators, activated PI3K/Akt, and anti-apoptotic Bcl2 family proteins) in mice treated with ARV-825 (Fig.2). Finally, secondary transplantation of equal number of human CD45+ cells from Vehicle and ARV-825 treated mice in to NSG mice led to delayed leukemia development and extended survival of mice engrafted from ARV-825 treated mice (Vehicle:38 days Vs ARV-825: 58 days P=0.0001/ Vehicle:36.5 days Vs ARV-825: 50 days P=0.0001) (Fig.3), providing functional confirmation of LIC elimination. Conclusion: Degradation of BRD4 with PROTAC (ARV-825), modulates the NOTCH1/MYC/CD44 axis and has the potential of therapeutically targeting the LIC in T-ALL. Reference Pui CH, Carroll WL, Meshinchi S, Arceci RJ. Journal of clinical oncology 2011; 29(5): 551-565. Ishimoto T, Nagano O, Yae T, Tamada M, Motohara T, Oshima H et al.Cancer cell 2011; 19(3): 387-400. Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN et al.Nature 2009; 458(7239): 780-783. Garcia-Peydro M, Fuentes P, Mosquera M, Garcia-Leon MJ, Alcain J, Rodriguez A et al.The Journal of clinical investigation 2018. doi: 10.1172/JCI92981 Sanchez-Martin M, Ferrando A. Blood 2017; 129(9): 1124-1133. Cox CV, Martin HM, Kearns PR, Virgo P, Evely RS, Blair A. Blood 2007; 109(2): 674-682. Gerby B, Clappier E, Armstrong F, Deswarte C, Calvo J, Poglio S et al.Leukemia 2011; 25(8): 1249-1258. Sujan Piya, Hong Mu, Seemana Bhattacharya, Teresa McQueen, R. Eric Davis, Vivian Ruvolo, Natalia Baran, Yimin Qian, Craig M. Crews, M. James You , Patrick Zweider-McKay, Marina Konopleva, Hagop Kantarjian , Michael Andreeff1, Gautam Borthakur. 59 th Annual Meeting &Exposition, Atlanta GA: December 9-12, 2017. Disclosures Qian: Arvinas LLC Inc: Employment. Raina:Arvinas LLC Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding. Andreeff:Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Jazz Pharma: Consultancy; Celgene: Consultancy; Amgen: Consultancy, Research Funding.

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.

Metabolic reprogramming of the key energy-generating pathways has been long recognized as one of the key oncogenic properties of cancer including leukemia. While accelerated glycolysis is considered to be most common feature of tumors, reliance on oxidative phosphorylation (Oxphos) as a major energy source has been reported for various tumor types. IACS-010759 is a novel OxPhos inhibitor(OxPhosi) that blocks cellular respiration through inhibition of complex I (Molina et al., AACR2016 Abstract #335) and considered as validated drug with clinical relevance in AML and solid tumors. Treatment of adult T-ALL remains unsatisfactory, with approximately one-third of patients experience disease relapse, and novel treatment strategies are warranted. In this study, we report pre-clinical activity of IACS-010759 in T-ALL models and characterize a cellular metabolic profile of T-ALL. Analysis of a panel of T-ALL cell lines showed that IACS-010759 significantly reduced viability measured by CTG assay in all cell lines tested (Notch mutant: Pf382, 1301, Jurkat, MOLT-4, P12-Ichikawa and Notch wt: T-ALL1). T-ALL cells displayed high sensitivity pattern to OxPhos inhibition with EC50 between 0,001 and 10 nM at day 5 analyzed by CTG assay (Fig.1). This reduction of cell viability was primarily due to cell cycle arrest demonstrated by reduction in EdU uptake, and moderate induction of apoptosis in selected T-ALL cell lines. In primary T-ALL samples from patients with newly diagnosed or relapsed/refractory ALL (n=2), in vitro 5-day treatment with IACS-010759 reduced viable cell number at EC50 of 13 nM and 45 nM, respectively. In primary human T-ALL PDX xenografts study, daily oral administration of IACS-010759 at 7.5mg/kg/qd was well tolerated, caused significantly reduced circulating leukemia burden and extended median survival duration (Fig.2). The mitochondrial fuel usage that characterizes Oxphos dependency in T-ALL cell line PF382 was analyzed by Mito fuel Test using the Seahorse Bioscience XF96 Analyzer. Among all three energy sources, PF382 depends most on free fatty acids (FA), indicating strong coupling to Oxphos and TCA cycle (Fig.3). Treatment of T-ALL with IACS-010759 had effectively inhibited FA-stimulated mitochondrial respiration indicated by decreased oxygen consumption rates (OCR) (Fig.4A). However, the cells maintain an ability to generate energy via glycolysis, indicated by high extracellular acidification rate (ECAR) in both, control and IACS-treated groups (Fig.4B). Next, mitochondrial function of T-ALL cells (PF382, Jurkat, 1301, P12Ischikawa, MOLT4, TALL1) was investigated using Mito Stress Test in Seahorse Bioscience XF96 Analyzer. IACS-010759 exposure for 2 hrs caused a striking dose-dependent decrease in basal and maximal OCR, reduction of proton leak and ATP production (Fig.5A, B, C), confirmed by the decreased ATP/ADP and NADH/NAD ratios measured by luminescence assays (ADP/ATP Glow assay, NADH/NAD Glow assay), consistent with inhibition of Oxphos. Conclusions: Taken together, these data provide information about metabolic profiling of T-ALL and indicate that Oxphos inhibition constitutes a novel therapeutic approach that targets a unique metabolic vulnerability of T-ALL cells. Further preclinical evaluation of Oxphos inhibitors in T-ALL is warranted. Disclosures Jabbour: ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Background: Mutational or non-mutational epigenetic events that aberrantly modify the chromatin regulatory machinery to enhance oncogene expression are a hallmark of myeloid malignancies. BRD4, a member of the bromodomain and extra terminal domain (BET) family, is a transcriptional coactivator that co-occupies super enhancer complexes associated with transcription of oncogenes (MYC, SOX2, NF-kB etc.) - and apoptosis regulators (Bcl-2, Bcl-XL, etc.) and has been validated as a target in AML therapy1. ARV-825 is a hetero-bifunctional PROteolysis TArgeting Chimera (PROTAC) that recruits BRD4 to the E3 ubiquitin ligase cereblon and leads to efficient and sustained degradation of BRD4 resulting in down-regulation of MYC2. Objectives: We examined the anti-leukemic effect of ARV-825 against AML cell lines, primary AML blasts and a mouse model of disseminated leukemia. Since tumor-stroma interactions driven by oncogene activation play a major role in resistance to AML therapy, we tested whether ARV-825 could overcome stroma-mediated drug resistance. As MYC harmonizes nutrient acquisition by cancer cells through regulation of the metabolites antiporter systems (SLC7A11, SLC5A5) 3, we profiled changes in a few important amino acids and organic acids in AML cell in response to ARV-825. Results : The IC50s for all tested cell lines and primary AML cells at 72 hours were in the low nanomolar range (2-50 nM) and 10-100 times lower than JQ1, a small molecule BRD4 inhibitor. ARV-825 induces sustained BRD4 degradation accompanied by down-regulation of targets such as MYC, anti-apoptotic BCL-2 family molecules and an increase in apoptosis and DNA damage4. In an in vitro tumor-stroma co-culture model including hypoxic conditions, bone marrow derived mesenchymal stromal cells (MSCs) protected OCI-AML3 cells from cytarabine ( 55.4% apoptosis with vs. 35.0% without MSCs in normoxia and 50.6% vs. 32.8%, respectively, in hypoxia), but no such protection was observed against ARV-825 (58.7% apoptosis vs. 55.2% in normoxia and 57.4% vs. 58.3%, respectively, in hypoxia). Mass cytometry based proteomic analysis (CyTOF) (Fig. 1), immunoblotting and flow cytometry showed that among apoptotic, cell adhesion and signaling proteins, MYC, CD44 and surface CXCR4 were the most down-regulated proteins in AML cells. The functional relevance of surface CXCR4 down regulation was confirmed in migration assays against the CXCR4 ligand SDF-1. Phosphorylation of CXCR4 by PIM1 kinase is necessary for surface expression of CXCR4, ARV-825 treatment reduced PIM1 levels and phosphorylation of CXCR4 in AML cells while overexpression of PIM1 or Myc reversed the phenomena. Quantitative PCR and immunoblotting analysis confirmed the transcriptional down regulation of total CD44 and CD44 variants 8-10 (2-fold change treated vs. untreated). As a functional correlate of CD44 variants, mass spectrometry based intracellular metabolomics and flow cytometry confirmed reduction in cysteine uptake and increased reactive oxygen species (ROS) generation (Fig. 2). Additional metabolic readouts using the Sea horse system revealed inhibition of mitochondrial respiration as indicated by decreased in oxygen consumption rate and production of ATP upon treatment of ARV-825. Furthermore, array based gene expression profiling showed down-regulation of additional amino acid transporters and the Wnt/β-catenin pathway. Finally, in a mouse model of human leukemia, the leukemia burdens were significantly lower in the ARV-825 treated mice as confirmed by luciferase imaging, flow cytometry, spleen size and survived longer compared to control mice (p=0.0005) (Fig.3). Conclusion : ARV-825 has substantial, broad anti-AML activity and importantly modulates the tumor microenvironment and metabolism to overcome stroma-mediated drug resistance. Together, our data suggest that ARV-825 is an effective in targeting BET family of proteins for the treatment of AML Reference: 1. Nature 2011; 478(7370): 524-528. doi: 10.1038/nature10334 2. Chem Biol 2015; 22(6): 755-763. doi: 10.1016/j.chembiol.2015.05.009 3. Cancer Res 2015; 75(9): 1782-1788. doi: 10.1158/0008-5472.CAN-14-3745 4. 604(675): ASH 2015,San franscisco,USA. Disclosures Lorenzi: Erytech Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: NIH-held patent related to L-asparaginase. Qian:Arvinas, LLC: Employment. Kantarjian:Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

Separation of leukemic stem cells (LSC) and residual hematopoietic stem cells (HSC) from the same individual patient with acute myeloid leukemia (AML) is essential for a proper understanding of the leukemic driving mechanisms. We have studied the role of aldehyde dehydrogenase (ALDH) for this purpose and have defined the functional properties of ALDHbright cells in specific subgroups of AML. We have examined the ALDH activity by flow cytometry in bone marrow samples (BM) from 14 healthy donors and 73 patients with de novo AML. The median frequency of cells with high ALDH activity (ALDHbright cells) in the healthy subjects was 1.92% with a range from 0.58 to 3.16%. For patients with AML, the median number of ALDHbright cells was 0.25% with a broad range from 0.004 to 33.57%. Whereas the majority of patients with AML (n = 56) had low frequencies of ALDHbright cells (median 0.11%; range 0.004 – 1.77%; defined as ALDH-low AML), 17 patients had relatively numerous ALDHbright cells (median 9.01; range 3.54 – 33.57%; defined as ALDH-numerous AML). In both groups, ALDHbright cell populations were highly enriched for CD34+CD38- cells. The ALDHbright cells derived from ALDH-low AML did not contain chromosomal and molecular aberrations characteristic of the original leukemia, and were able to induce multi-lineage hematopoiesis in NSG mouse models. Thus, genetically and functionally normal HSC could be successfully isolated in the ALDHbright subset, whereas LSC were enriched in ALDHdimCD34+CD38- subset for patients with ALDH-low AML. For 17 patients with ALDH-numerous AML, the ALDHbright subset was consistently contaminated with LSC. In clinical follow-ups, patients with ALDH-numerous AML showed resistance to induction chemotherapy and were characterized by a very poor long-term outcome that was comparable to patients with high-risk cytogenetic or molecular genetic markers. In four patients with ALDH-numerous AML we demonstrated that the ALDHbrightCD34+CD38- subset contained chemotherapy-resistant clones with repopulating ability. Furthermore, such ALDHbright cells were characterized by a lower cell-cycle activity and an increased resistance to cytarabine in comparison with ALDHdim blasts in in vitro assays. Our data have provided evidence that LSC and residual HSC can be separated using ALDH in patients with low frequencies of ALDHbright cells. In patients with ALDH-numerous AML, the ALDHbright subset is associated with leukemic features both in vitro and in animal models. Thus our data demonstrated the feasibility of appropriate comparisons of LSC versus HSC from the same patient with specific subtypes of AML and the impact of LSC properties on clinical outcome. Disclosures: Buss: Novartis: Travel support Other; Micromet/Amgen: Reimbursements for participation in a clinical study , Reimbursements for participation in a clinical study Other. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genzyme: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Background: Salvage options for patients with relapsed T cell acute lymphoblastic leukemia (T-ALL) are limited, with less than 25% of these patients achieving second remission 1, 2. 70% of T-ALL cases have activating mutations of the NOTCH1 pathway, which transcriptionally activates MYC by binding to its `superenhancer' region 3, 4. Other deregulated oncogenic pathways in T-ALL include PI3K/Akt, the anti-apoptotic Bcl-2 family, and CDKN2A/2B cell cycle regulators 5, 6. The NOTCH1-MYC regulatory circuit is an attractive therapeutic target, but clinical development of gamma-secretase inhibitors (GSI) to target NOTCH1 has been limited by 'on target' toxicities. A better target may be BRD4, a critical component of superenhancer complexes that binds to acetylated histone (3 and 4) and drives NOTCH1 mediated MYC transcription7. ARV-825 is a hetero-bifunctional PROteolysis TArgeting Chimera (PROTAC) that has 3 components: a thienodiazepine-based BRD4 ligand, a linker arm, and a cereblon-binding ligand. ARV-825 recruits BRD4 to the E3 ubiquitin ligase cereblon and leads to efficient and sustained degradation of BRD4, resulting in down-regulation of MYC. Methods: We investigated the effectiveness of ARV-825 against T-ALL cell lines, including GSI-resistant lines. Since microenvironmental signals are critical for the survival of T-ALL, we specifically tested the impact of BRD4 degradation on CD44/CD44v, which integrates cell-extrinsic microenvironmental signals and is part of cysteine transporter that maintains low intra-cellular reactive oxygen species (ROS), necessary for T-ALL survival and the persistence of disease. We also examined the anti-leukemic effect of ARV-825 in a T-ALL patient-derived xenograft (PDX) mouse model of disseminated leukemia with a constitutively active NOTCH1 mutation. Results: The IC50s for all tested T-ALL cell lines at 72 hours were in the low nanomolar range (< 50 nM). ARV-825 leads to sustained degradation of BRD4 and down-regulation of its transcriptional targets MYC, Bcl-2 and Bcl-XL and inhibits cell proliferation and induces apoptosis in GSI-sensitive (HPB-ALL, KOPT1) and GSI-resistant (MOLT4, SUPT1) cell lines. Mass cytometry based proteomic analysis (CyTOF) and immunoblotting showed that ARV-825 down-regulated cell intrinsic oncogenic molecules: transcription factors Myc and NFkB, cell cycle regulator CDK6, activated PI3K/Akt, and anti-apoptotic Bcl2 family proteins. In addition ARV-825 down regulated two key molecules involved in leukemia-stroma interaction; CD44 (Fig. 1), and CD98, a component of amino acid transporters xCT, LAT1 and 2, both essential in regulation of oxidative stress. Quantitative PCR and immunoblotting analysis confirmed the transcriptional down regulation of total CD44 and CD44 variants 8-10 (2-fold change treated vs . untreated). As a functional correlate of down-regulation of CD98/CD44/CD44v, flow cytometry confirmed increased intracellular ROS generation (Fig. 2). Finally, in a PDX mouse model of human T-ALL, ARV-825 treatment resulted in lower leukemia burden (confirmed by flow cytometry for human CD45+ cells in bone marrow) and better survival compared to vehicle-treated control mice (p=0.002) (Fig.3). Reference: 1. Marks DI, Rowntree C. Management of adults with T-cell lymphoblastic leukemia. Blood 2017; 129(9): 1134-1142. 2. Litzow MR, Ferrando AA. How I treat T-cell acute lymphoblastic leukemia in adults. Blood 2015; 126(7): 833-41. 3. Sanchez-Martin M, Ferrando A. The NOTCH1-MYC highway toward T-cell acute lymphoblastic leukemia. Blood 2017; 129(9): 1124-1133. 4. Demarest RM, Ratti F, Capobianco AJ. It's T-ALL about Notch. Oncogene 2008; 27(38): 5082-91. 5. Girardi T, Vicente C, Cools J, De Keersmaecker K. The genetics and molecular biology of T-ALL. Blood 2017; 129(9): 1113-1123. 6. Joshi I, Minter LM, Telfer J, Demarest RM, Capobianco AJ, Aster JC et al. Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 2009; 113(8): 1689-98. 7. Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 2013; 153(2): 320-34. Disclosures Qian: 4Arvinas, LLC. New Haven, CT: Employment. Raina: 4Arvinas, LLC. New Haven, CT: Employment. McKay: 6 ImmunoGen, Inc.Waltham, MA: Employment. Kantarjian: Novartis: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding. Andreeff: Daiichi Sankyo: Consultancy.