ALBERT

Description: Extensive studies in prostate cancer and other malignancies have revealed thatl-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of seruml-Met, either via partial dietary restriction or with bacteriall-Met–degrading enzymes exerts potent antitumor effects. However, administration of bacteriall-Met–degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encodel-Met–degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation ofl-Met. At therapeutically relevant dosing, hMGL-4.0 reduces seruml-Met levels to 〉75% for 〉72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.

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: Recent studies indicate that acute myeloid leukemia (AML) cells, including leukemia-initiating cells, are highly dependent on oxidative phosphorylation (OXPHOS) for survival, while normal hematopoietic stem cells predominantly utilize glycolysis for energy homeostasis. We have reported development of a series of novel, highly potent mitochondrial complex I inhibitors, which in vitro inhibit complex I with IC50 values 20% or for signs of morbidity. Right, bioluminescence imaging before (D0) and 10 days after 1 st dose; left survival. Figure 1. Treatment with IACS-1131 prolongs survival in a OCI-AML3 xenograft model. Luciferase-expressing OCI-AML3 cells were injected in the tail vein of NSG mice. On day 16 after injection, engraftment was confirmed and mice were randomized on the basis of IVIS-based imaging of luciferase activity after luciferin injection. For the next 58 days, mice received either vehicle or 60 mg/kg/day of IACS-1131 via oral gavage. Mice were sacrificed when body weight was reduced by 〉20% or for signs of morbidity. Right, bioluminescence imaging before (D0) and 10 days after 1 st dose; left survival. Disclosures No relevant conflicts of interest to declare.

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: Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions. First, we performed metabolomic analysis of HL-60 cells co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids (Phe, kynurenine, Trp, Leu, Ile, Met, Tyr, Val, Thr, Ala, Gln, Asn, and His), possibly reflecting inhibition of global protein synthesis. CB-839 suppressed cysteine consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis (Fig. 1A). CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, under conditions mimicking the BM microenvironment (Fig. 1B). Stable isotope-resolved metabolomics (SIRM) analysis with 13C5, 15N2-Gln in HL-60 cells indicated that treatment with CB-839 severely hindered Gln anaplerosis to similar extent under normoxic or hypoxic conditions. Moreover, Gln is predominantly used to carry out oxidative metabolism. The enriched fraction of aspartate in treated cells dropped dramatically (to approximately 20% or less of the pool), suggesting that leukemia cells require Krebs cycle-derived oxaloacetate transamination for the generation of aspartate (Fig. 1C). Limiting Gln supply using CB-839 caused reduction in the concentration of alpha-ketoglutarate (α-KG) and the oncometabolite 2-hydroxyglutarate (2-HG), known to play a role in the pathogenesis of AML. We have previously shown that the leukemic BM microenvironment is highly hypoxic (Benito PLoS One 2011), andhypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG measured by LC-MS/MS in HL-60 (6.2 fold) and OCI-AML3 cells (2.9 fold) with wt-IDH. This increase in L-2HG was potently inhibited by CB-839, implicating Gln as a source for L-2HG production by AML cells under hypoxia. HL-60 and OCI-AML3 AML cells produced very limited amounts of the D-enantiomer of 2HG (D-2HG), and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies. Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1µM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and in primary AML cells (n=3) (Fig. 1D). To test the efficacy of both compounds in vivo, we injected NSG-S mice with genetically engineered MV4;11/Luc cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the CB-839 plus 5-AZA co-treated mice. CB-839 and 5-AZA co-treatment resulted in significant extension of survival compared with 5-AZA single agent, 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: Despite recent approval of hypomethylating agent/venetoclax (HMA+VEN) therapy for older patients (pt) with AML unfit for induction chemotherapy, their outcomes remain suboptimal. Such pts have a median overall survival of only 14 months and only approximately 35% of pts enjoying long-term survival (DiNardo, EHA 2020). Approximately 20-40% of newly diagnosed older pts with AML do not respond to the HMA+VEN regimens, with higher rates of refractory disease as well as early relapse in high-risk pts. Metabolic reprogramming and dependence on mitochondrial oxidative phosphorylation (OxPhos) is a core feature of AML leukemia stem cells (LSC). Recent reports have shown that upregulation of OxPhos confers intrinsic and acquired resistance to VEN in AML, multiple myeloma and lymphoid malignancies, which can be reversed by disrupting OxPhos; mutated TP53 has been shown to confer intrinsic resistance to venetoclax through increased OxPhos (Nechiporuk T, et al. Cancer Discovery 2019). We hypothesized that combined blockade of mitochondrial fitness by OxPhos inhibitors and of BCL-2 with VEN/azacitidine (AZA) will perform synergistically in pre-clinical AML models. To test this hypothesis, we utilized a novel complex I inhibitor IACS-010759 that effectively inhibits cell respiration and leukemia progression in the in vitro and in vivo AML pre-clinical models (Molina, Nat Med 2018). Priming of MOLM-13 cells with 20nM VEN for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while single agents reduced OCR by 70%) of viable cell numbers in several AML cell lines tested (OCI-AML2, MV-4-11, and MOLM-13) (Figure 1). By co-immunoprecipitation VEN disrupted interaction of BCL-2 with the mitochondrial protein VDAC, known to regulate ADP/ATP exchange during electron transport across mitochondria membrane; this resulted in dramatic reduction of the intracellular ATP and CTP levels measured by MS-based metabolomics. Further, VEN increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of VEN and IACS-010759, possibly because of RNA degradation. In primary AML samples (n=3) and AML PDX cells (n=4) cultured ex vivo, combined VEN and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. We next tested the efficacy of the "triple" combination of VEN, AZA and IACS-010759 in the in vivo AML PDX models. We injected AML PDX cells 3871344 (with no mutations identified by targeted sequencing) and 4404778 (harboring IDH1, NPM1, FLT3-ITD mutations) into NRG mice and upon engraftment, randomized mice into 4 groups to receive 2 cycles of treatment with 3 weeks interruption between cycles: vehicle, VEN (50mg/kg daily, 5 days on/2 days off, day 1-21) with AZA (2.5 mg/kg daily, day 1-7), IACS-010759 (1mg/kg ?daily, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. In the less aggressive model 3871344, all therapies reduced circulating leukemia burden, and the triple treatment achieved best efficacy, with average circulating tumor burden at 10 weeks after cycle 2 of 90.8%, 38.7%, 68.8% and 7.4% in vehicle, VEN+AZA, IACS-010759, and triple-therapy cohorts, respectively. In the aggressive model 4404778, the treatments were less effective, but the combination offered highest activity, with average circulating tumor burden of 71.0%, 52.3%, 88.7% and 39.9% in vehicle, IACS-010759, VEN+AZA and triple-therapy cohorts, respectively (Figure 1). Analysis of survival and additional PDX models are ongoing and will be reported. In summary, our study demonstrates that BCL-2 modulates mitochondrial respiration and mitochondrial ATP generation in addition to its established anti-apoptotic role. VEN disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitro and in vivo. Disclosures Konopleva: Ascentage: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Ablynx: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Kisoji: Consultancy; AstraZeneca: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding.

Description: Acute myeloid leukemia (AML) cells are highly dependent on mitochondrial function for survival 1. We have recently reported a novel oxidative phosphorylation (OXPHOS) inhibitor IACS-010759 that potently inhibits mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells in vitro and in vivo2. In this study, we aimed to identify chemotherapeutic agents that synergistically deplete AML cells when administered in combination with IACS-010759. We performed a high-throughput screening of a drug library (289 anti-cancer compounds) administered either individually or in combination with IACS-010759 on two leukemia cell lines (OCI-AML3, MOLM-13) and three bone marrow stromal cell lines (HS-5, HS-27A, MSC) in both hypoxia (1% O2) and normoxia conditions. Based on the cell viability datasets, we selected top candidates for combinations based on the following criteria: either bliss index 〉 0.1 (synergy of the combination treatment; red in Fig. 1A&B), or high cytotoxicity to leukemia cells (relative cell viability 〈 0.5, blue in Fig. 1A&B), as well as low toxicity against normal cells (relative cell viability in normal cells 〉 0.8, yellow in Fig. 1A&B). Twenty-four compounds satisfied the selection criteria above, either in normoxia or hypoxia, or both. Out of the 24 compounds, 5 agents (Fig. 1C) are known FLT3 (FMS-like tyrosine kinase 3) inhibitors, including AC220 (quizartinib), dovitinib, nintedanib, SGI-1776, and rebastinib, pointing to a molecular target of great potential interest in the design of synergistic drug combinations with IACS-010759. Thus, we investigated more in-depth the synergism between IACS-010759 (10nM) and 13 FLT3 inhibitors, all currently in clinical trials (AC220, sorafenib, gilteritinib, sunitinib, ponatinib, midostaurin, ibrutinib, TP-0903, crenolanib, tandutinib, FF-10101, lestaurtinib, and KW-2449; 0.0128:5x:5000nM), in AML cell lines (FLT3-wt KG-1, U937, OCI-AML2, OCI-AML3; and FLT3-mutant MOLM-13 and MOLM-14). Among the 13 FLT3 inhibitors, only AC220 combined with IACS-010759 showed concentration windows with bliss index higher than 0.1 across different lines. Next, we further characterized the synergism between AC220 and IACS-010759 in AML cell lines (U937 and OCI-AML3) under hypoxic conditions using metabolic flux analysis (MFA) to trace the incorporation of 13C5,15N2-glutamine and 1,2-13C2-glucose and study the metabolic modulation associated with the synergy. Leukemia cells were incubated with unlabeled/labeled medium for 24h and concurrently treated with 5nM IACS-010759 and/or 500nM AC220. While both individual agents modulate glutamine consumption and TCA cycle dynamics, by far the most dramatic metabolic effects on TCA cycle intermediates are observed following administration of the combined treatment. Severe drops in the levels of TCA cycle metabolites, (Fig. 1D) point to a reduced mitochondrial activity following the combined treatment, which is also validated by the increased ratio of oxidized/reduced forms of nicotinamide adenine dinucleotide (NAD/NADH). Interestingly, the total pool of the oncometabolite 2-hydroxyglutarate, while increasing following the individual treatments, significantly dropped to very low levels in response to the combined treatment. The significantly reduced metabolite levels as well as the glucose-derived enrichment fractions of glucose 6-phosphate, fructose bisphosphate, phosphoenolpyruvate and ribose 5-phosphate in the AC220-containing treatment groups (significantly more pronounced in the combined treatment) point to impaired glycolysis /pentose phosphate pathway (Fig. 1E). In turn this results in lower de novo nucleotide biosynthesis (based on the decreased glutamine and glucose incorporation). Similar results were observed in OCI-AML3 cells. Overall, the combinatorial treatment with IACS-010759 and AC220 impaired AML cell metabolism tremendously and to a much greater extent than any of the individual treatments alone. Influx inhibition of both the two main carbon sources, glucose and glutamine, was observed leading to impairment of the TCA cycle and glycolysis for energy production, as well as pentose phosphate pathway and de novo nucleotide biosynthesis. In conclusion, we identified a novel drug combination AC220 and IACS-010759 which synergistically inhibits AML cell growth regardless of FLT3 mutation at least by metabolism disruption. Disclosures Konopleva: Kisoji: Consultancy; Agios: Research Funding; Amgen: Consultancy; Cellectis: Research Funding; Eli Lilly: Research Funding; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Sanofi: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; AbbVie: Consultancy, Research Funding; AstraZeneca: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding.