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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.

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.

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival and continually adapt to fluctuations in nutrient and oxygen availability in the bone marrow (BM) microenvironment. The first-in-class OxPhos inhibitor IACS-010759 (Molina, Nat. Medicine 2018) is currently in Phase 1 clinical trial in AML. To identify biomarkers of sensitivity or resistance to OxPhos inhibition, we performed Cap Analysis of Gene Expression analysis (CAGE) in primary AML samples (11 sensitive and 3 resistant to IACS-010759) and 6 AML cell lines (OCI-AML3, MOLM13, THP-1, U937, MV4;11, HL60). CAGE identified 41 gene transcripts that were significantly higher in IACS-010759 resistant primary AML samples than in sensitive samples (fold change >3.0, FDR We next studied OxPhos inhibition under conditions mimicking BM microenvironment, by co-culturing AML cells with healthy BM-derived mesenchymal stem cells (MSC). Anti-proliferative effects of IACS-010759 were reduced by co-culture with MSC (IC50 0.01nM vs 2.1nM OCI-AML3, 65.4nM vs >10µM MOLM13). MSC co-culture increased OCR and glycoPER, and IACS-010759 decreased OCR / increased glycoPER in both cell lines. To determine the mechanism of resistance to IACS-010759 under MSC co-culture conditions, we studied the mitochondrial exchange between AML cells and MSC. We observed the bidirectional mitochondrial transfer between MSCs and AML cells. Using AML and MSC cells stably infected with mitochondria-targeted PDHA1 (GFP in AML, dsRed in MSC) to visualize the mitochondria, we found that IACS-010759 treatment facilitated transfer of AML-derived mitochondria into the adhered MSCs (24h), and MSC-derived mitochondria transferred to floating AML cells (48-72h) along with formation of tunneling nanotubes (TNTs) of AML cells, in sensitive OCI-AML3 cells (PDHA1-GFP/DsRed double positive %, control vs IACS; 9.0% vs 23.3 %). Mitochondria transfer from MSCs to AML cells was minimally observed in MOLM13 cells with elevated basal OxPhos (4.0% vs 5.0%). We next investigated IACS-010759-induced metabolic changes in AML cells interacting with bone marrow (BM) stromal cells. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) metabolite analysis further detected that IACS-010759 suppressed multiple anaplerotic amino acids in OCI-AML3 cells including glutamine and glutamic acid, which was reversed by MSC co-culture along with increase in intra-autophagosomal LC3-II in OCI-AML3 cells. These findings were not observed in MOLM13. Microenvironment-induced activation of glutamine metabolism and autophagy-associated amino acid metabolism could be additional compensatory mechanisms in response to OxPhos inhibition by IACS-010759 in BM microenvironment. Taken together, basal metabolic energetic capacity including elevated OxPhos and high compensatory glycolysis confer the AML-intrinsic resistance to IACS-010759, which can be reversed by simultaneous glycolysis inhibition. BM microenvironment facilitates secondary resistance to OxPhos inhibition through modulation of amino acid metabolism and direct mitochondrial transfer. Inhibition of these processes could enhance the anti-AML efficacy of OxPhos inhibition. Disclosures Andreeff: AstraZeneca: Research Funding. Konopleva:Stemline Therapeutics: Research Funding.