ALBERT

Description: 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

Description: 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

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

Description: 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