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Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis.Significance:Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

Supplementary Figures S1-S5 supporting Main Figures 1-5.

Supplementary Tables S1-S6 describing patient characteristics, gene lists, LSC signatures, and reagents.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. Significance: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

Chronic myeloid leukemia (CML) is a heterogeneous disease, initiated by reciprocal translocation of chromosome 9 and 22, resulting in the generation of a BCR-ABL fusion protein and constitutive activation of the ABL kinase. ABL tyrosine kinase inhibitors (TKIs) have been very successful in suppressing CML disease. However, TKIs may not eliminate leukemia stem cells (LSCs), as evidenced by the frequent re-emergence of the disease upon TKI discontinuation. Moreover, blast phase CML (bpCML) remains a formidable challenge in disease management. Recent clinical evidence suggests that the BCL2 inhibitor venetoclax (Ven) in combination with ABL-targeting tyrosine kinase inhibitors (TKI) can eradicate bpCML LSCs. However, the exact mechanism by which this combination may targets LSCs is not known. In this report, we confirm the efficacy and LSC-targeting capacity of Ven/TKI combination in preclinical models of bpCML and we further identify that inhibition of free fatty acid (FFA) mobilization pathways may provide enhanced efficacy against LSCs. To establish the efficacy of Ven/TKI combination, we treated bpCML samples with Ven+Dasatinib (Das) combination for 24h, this resulted in a significant decrease in the viability of bulk and primitive populations (CD34+, CD38+). Patient-derived xenografts of bpCML samples in NSGS-mice, were treated with Ven/Das as well as single agents. The result showed a significant decrease in leukemia burden in the combination treated group, compared to either drug alone, albeit, some resistant cells survived in the combo treated group. Furthermore, using a syngeneic mouse model of bpCML, co-expressing Bcr-Abl and Nup98-HOXA9 translocations, the mouse leukemic cells treated with Ven/Dasatinib combination demonstrated a significant loss of viability of the bulk as well as phenotypically defined LSCs (Lin-/Sca1+). Treatment of leukemic mice with Ven/Das had a significant survival benefit and remained disease free at 80 days post treatment. We also showed significant survival benefits of Ven/ponatinib in NSGS-mice harboring syngeneic bpCML cells with the T315I gatekeeper mutation. Treatment of normal mice with Ven/Das combo did not affect the colony forming ability of LSK cells from the bone marrow, indicating a leukemia-specific response. Based on these results, we conclude that Ven/TKI combination effects were due to direct targeting of the LSC population. To investigate the potential mechanism of Ven/TKI activity in LSC targeting, we performed gene expression studies using RNA-seq based methods after short term treatment. Our findings indicated that the LSC population from Ven/TKI-treated mice showed enrichment of a gene signature associated with lysosome biology. Pre-treatment of mouse leukemia cells with bafilomycin, an inhibitor of lysosome function, resulted in increased sensitivity to Ven/TKI combo. Intriguingly, we also found significant induction of lysosomal acid lipase (LIPA), an enzyme involved in the generation of free fatty acids for energy needs. Metabolomic analysis of LSCs isolated after short term treatment with Ven/TKI, showed that a number of fatty acids were up-regulated in the Ven/Das treated group compared to control. Knocking down Lipa using CRISPR technology resulted in increased sensitivity to Ven/TKI combination, whereas overexpression of Lipa resulted in decreased sensitivity to the Ven/TKI combination, implicating Lipa upregulation and a resultant increase in free fatty acids as a protective response to Ven/TKI treatment. Furthermore, knocking down CPT1A, an important free fatty acid mitochondrial transporter, resulted in increased sensitivity to Ven/TKI combination both in mouse and primary human leukemic cells, leading to the hypothesis that activation of fatty acid processing through enhanced Lipa activity may represent a compensatory response to venetoclax based therapies in bpCML. In summary, we demonstrate the preclinical efficacy of Ven/TKI combination therapies for targeting of bpCML LSCs. Furthermore, our data suggest that blocking upregulation of free fatty acids through mechanisms such as inhibition of LIPA activity, might synergize with Ven/TKI combinations to eradicate LSCs, allowing for more durable response. Our findings provide a therapeutic rationale for blocking pathways involved in free fatty acids generation, as a potential strategy for increasing remission duration. Disclosures Pollyea: Amgen: Consultancy; Janssen: Consultancy; Genentech: Consultancy; AbbVie: Consultancy, Research Funding; Syndax: Consultancy; Daiichi Sankyo: Consultancy; Takeda: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Glycomimetics: Other. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding.

In this study we interrogated the metabolome of human acute myeloid leukemia (AML) stem cells to elucidate properties relevant to therapeutic intervention. We demonstrate that amino acid uptake, steady-state levels, and catabolism are all elevated in the leukemia stem cell (LSC) population. Furthermore, LSCs isolated from de novo AML patients are uniquely reliant on amino acid metabolism for oxidative phosphorylation and survival. Pharmacological inhibition of amino acid metabolism reduces oxidative phosphorylation and induces cell death. In contrast, LSCs obtained from relapsed AML patients are not reliant on amino acid metabolism due to their ability to compensate through increased fatty acid metabolism. These findings indicate that clinically relevant eradication of LSCs can be achieved with drugs that target LSC metabolic vulnerabilities.

Although multidrug approaches to cancer therapy are common, few strategies are based on rigorous scientific principles. Rather, drug combinations are largely dictated by empirical or clinical parameters. In the present study we developed a strategy for rational design of a regimen that selectively targets human acute myelogenous leukemia (AML) stem cells. As a starting point, we used parthenolide, an agent shown to target critical mechanisms of redox balance in primary AML cells. Next, using proteomic, genomic, and metabolomic methods, we determined that treatment with parthenolide leads to induction of compensatory mechanisms that include up-regulated NADPH production via the pentose phosphate pathway as well as activation of the Nrf2-mediated oxidative stress response pathway. Using this knowledge we identified 2-deoxyglucose and temsirolimus as agents that can be added to a parthenolide regimen as a means to inhibit such compensatory events and thereby further enhance eradication of AML cells. We demonstrate that the parthenolide, 2-deoxyglucose, temsirolimus (termed PDT) regimen is a potent means of targeting AML stem cells but has little to no effect on normal stem cells. Taken together our findings illustrate a comprehensive approach to designing combination anticancer drug regimens.

Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs) and may promote tumor development. Here, we show that a subpopulation of leukemic stem cells (LSCs) can utilize gonadal adipose tissue (GAT) as a niche to support their metabolism and evade chemotherapy. In a mouse model of blast crisis chronic myeloid leukemia (CML), adipose-resident LSCs exhibit a pro-inflammatory phenotype and induce lipolysis in GAT. GAT lipolysis fuels fatty acid oxidation in LSCs, especially within a subpopulation expressing the fatty acid transporter CD36. CD36(+) LSCs have unique metabolic properties, are strikingly enriched in AT, and are protected from chemotherapy by the GAT microenvironment. CD36 also marks a fraction of human blast crisis CML and acute myeloid leukemia (AML) cells with similar biological properties. These findings suggest striking interplay between leukemic cells and AT to create a unique microenvironment that supports the metabolic demands and survival of a distinct LSC subpopulation.

We have previously demonstrated that oxidative phosphorylation is required for the survival of human leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML). More recently, we demonstrated that LSCs in patients with de novo AML rely on amino acid metabolism to drive oxidative phosphorylation. Notably, although overall levels of amino acids contribute to LSC energy metabolism, our current findings suggest that cysteine may be of particular importance for LSC survival. We demonstrate that exogenous cysteine is metabolized exclusively to glutathione. Upon cysteine depletion, glutathione synthesis is impaired, leading to reduced glutathionylation of succinate dehydrogenase A (SDHA), a key component of electron transport chain complex (ETC) II. Loss of SDHA glutathionylation impairs ETC II activity, thereby inhibiting oxidative phosphorylation, reducing production of ATP, and leading to LSC death. Given the role of cysteine in driving LSC energy production, we tested cysteine depletion as a potential therapeutic strategy. Using a novel cysteine-degrading enzyme, we demonstrate selective eradication of LSCs, with no detectable effect on normal hematopoietic stem/progenitor cells. Together, these findings indicate that LSCs are aberrantly reliant on cysteine to sustain energy metabolism, and that targeting this axis may represent a useful therapeutic strategy.

Myelodysplastic syndrome (MDS) is a chronic hematologic disorder that frequently evolves to more aggressive stages and in some cases leads to acute myeloid leukemia (AML). MDS arises from mutations in hematopoietic stem cells (HSCs). Thus, to define optimal therapies, it is essential to understand molecular events driving HSC pathogenesis. In this study, we report that during evolution of MDS, malignant HSCs activate distinct cellular programs that render such cells susceptible to therapeutic intervention. Specifically, metabolic analyses of the MDS stem cell compartment show a profound activation of protein synthesis machinery and increased oxidative phosphorylation. Pharmacological targeting of protein synthesis and oxidative phosphorylation demonstrated potent and selective eradication of MDS stem cells in primary human patient specimens. Taken together, our findings indicate that MDS stem cells are reliant on specific metabolic events and that such properties can be targeted prior to the onset of clinically significant AML, during antecedent MDS., Myelodysplastic syndrome (MDS) arises from mutations in hematopoietic stem cells (HSCs). Here, the authors demonstrate that HSCs in higher-risk MDS express the surface marker CD123 and are characterized by activation of protein synthesis machinery and increased oxidative phosphorylation.

Leukemia stem cells (LSCs) are thought to drive the genesis of acute myeloid leukemia (AML) as well as relapse following chemotherapy. Because of their unique biology, developing effective methods to eradicate LSCs has been a significant challenge. In the present study, we demonstrate that intrinsic overexpression of the mitochondrial dynamics regulator FIS1 mediates mitophagy activity that is essential for primitive AML cells. Depletion of FIS1 attenuates mitophagy and leads to inactivation of GSK3, myeloid differentiation, cell cycle arrest, and a profound loss of LSC self-renewal potential. Further, we report that the central metabolic stress regulator AMPK is also intrinsically activated in LSC populations and is upstream of FIS1. Inhibition of AMPK signaling recapitulates the biological effect of FIS1 loss. These data suggest a model in which LSCs co-opt AMPK/FIS1-mediated mitophagy as a means to maintain stem cell properties that may be otherwise compromised by the stresses induced by oncogenic transformation.

Recent clinical trials have reported that in combination with hypomethylating agents, the BCL-2 inhibitor venetoclax can induce responses in over 70% of older previously untreated AML patients who are unfit for conventional chemotherapy. These findings led to the recent United States Food and Drug Administration approval of this regimen for this population, and it is now considered to be the standard care. However, a significant minority of patients do not achieve a remission and are refractory. In addition, the majority of patients who do achieve a remission ultimately relapse. It is therefore critical to identify AML patients who are likely to be resistant to venetoclax-based therapy. To initially address this question, we retrospectively reviewed 75 newly diagnosed AML patients who received venetoclax + azacitidine (VEN+AZA) at our institution and analyzed several baseline clinical features to determine the ability of each to predict disease that was refractory to treatment (defined as a lack of complete remission [CR], CR with incomplete recovery of peripheral blood counts [CRi], partial remission, or morphological leukemia free state [MLFS]). Both univariate and multivariate analyses revealed the presence of FAB-M5 to be associated with disease that was refractory to VEN+AZA (Table 1). Given that FAB-M5 represents AML with monocytic differentiation, these findings indicate a strong correlation between myeloid differentiation status and resistance to venetoclax. Using multicolor flow cytometry, we show bone marrow specimens of typical FAB-M5 patients who were refractory to VEN+AZA presented dominant monocytic disease that has an immunophenotype of CD45-bright/SSC-high/CD117-/CD11b+/CD68+ (Figure A). In contrast, bone marrow specimens of typical FAB-M0/M1/M2 patients who achieved CR with VEN+AZA presented as a single dominant disease population that is CD45-med/SSC-low/CD117+/CD11b-/CD68- (Figure B). In a subset of AML patients, we observed the co-existence of both phenotypically primitive and monocytic populations, which we term "MPM" AML (for Mixed Primitive/Monocytic). We observe that after attaining CR with VEN+AZA treatment and subsequent relapse, MPM-AML showed almost complete loss of the primitive subpopulation, and evolved to a dominant monocytic disease (Figure C). These data indicate that VEN+AZA treatment induces strong selection of the monocytic phenotype. Importantly, when we compared the immunophenotype of six pairs of diagnostic/relapse specimens from AML patients treated with conventional intensive induction chemotherapy, we observed selection of a more primitive phenotype, suggesting the drive toward a monocytic phenotype observed at relapse appears to be a unique consequence of VEN+AZA therapy. To our knowledge, selection of a monocytic phenotype at relapse has never been previously observed in AML, suggesting the relapse after VEN+AZA may represent a new clinical entity. Mechanistically, using RNA-seq we show the global transcriptome of monocytic AMLs are distinct from primitive AMLs, suggesting they represent two broad classes of AML with likely differential responses to therapy. Indeed, we demonstrate that AML with a primitive immunophenotype is dependent on BCL-2 activity as a means to drive oxidative phosphorylation, a critical requirement for survival of leukemia stem cells. Conversely, AML with a more differentiated monocytic phenotype is no longer dependent on BCL-2, but rather switches to MCL-1 as a mediator of oxidative phosphorylation. Using colony-forming and xenograft assays, we show the stem and progenitor potential of monocytic AMLs are selectively more sensitive to MCL-1 inhibition comparing to BCL-2 inhibition. Together, our study suggests a significantly higher refractory/relapse risk for monocytic AML patients treated with VEN+AZA (Figure D). Further, for those AML patients who do respond to initial VEN+AZA treatment, the therapy drives a powerful selective process resulting in emergence of more differentiated monocytic disease in some patients. Based on these findings, we propose that AML exists on a developmental spectrum that is inherently fluid, where with appropriate selective pressure the disease can acquire characteristics of a more differentiated state. Further, our data indicate that optimal AML therapy will require strategies designed to target both primitive and myeloid phenotypes. Disclosures Pollyea: Forty-Seven: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Diachii Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Savona:TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Selvita: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Patents & Royalties; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Fabrication of superior and cost-effective cathodic materials is vital in manufacturing sustainable microbial electrolysis cells (MECs) for biofuels production. In the present study, a novel manganese dioxide (MnO2) coated felt cathode (Mn/CF) has been developed for MECs using electrodeposition method via potentiostat. MnO2 is considered to encourage exogenous electron exchange and, in this way, improves the reduction of carbon dioxide (CO2). MnO2, as a cathodic catalyst, enhances the rate of biofuel production, electron transfer, and significantly reduces the cost of MECs. A maximum stabilized current density of 3.70 ± 0.5 mA/m2 was obtained in case of MnO2-coated Mn/CF based MEC, which was more than double the non-coated carbon felt (CF) cathode (1.70 ± 0.5 mA/m2). The dual chamber Mn/CF-MEC achieved the highest production rate of acetic acid (37.9 mmol/L) that was significantly higher (43.0%) in comparison to the non-coated CF-MEC. The cyclic voltammograms further verified the substantial enhancement in the electron transfer between the MnO2 coated cathode and microbes. The obtained results demonstrate that MnO2 interacted electrochemically with microbial cells and enhanced the extracellular electron transfer, therefore validating its potential role in biofuel production. The MnO2 coated CF further offered higher electrode surface area and better electron transfer efficiency, suggesting its applicability in the large-scale MECs.

SUMMARY The NADPH-dependent oxidase NOX2 is an important effector of immune cell function, and its activity has been linked to oncogenic signaling. Here, we describe a role for NOX2 in leukemia-initiating stem cell populations (LSCs). In a murine model of leukemia, suppression of NOX2 impaired core metabolism, attenuated disease development, and depleted functionally defined LSCs. Transcriptional analysis of purified LSCs revealed that deficiency of NOX2 collapses the self-renewal program and activates inflammatory and myeloid-differentiation-associated programs. Downstream of NOX2, we identified the forkhead transcription factor FOXC1 as a mediator of the phenotype. Notably, suppression of NOX2 or FOXC1 led to marked differentiation of leukemic blasts. In xenotransplantation models of primary human myeloid leukemia, suppression of either NOX2 or FOXC1 significantly attenuated disease development. Collectively, these findings position NOX2 as a critical regulator of malignant hematopoiesis and highlight the clinical potential of inhibiting NOX2 as a means to target LSCs., Graphical Abstract, In Brief The NADPH-dependent oxidase NOX2 is important for normal myeloid cell function. Adane et al. show that NOX2 is expressed in leukemic stem cells, where it regulates the balance of myeloid differentiation and self-renewal. Deficiency of NOX2 altered core metabolism, exacerbated inflammatory signaling, and limited in vivo disease development.

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is high in hematopoietic stem cells and functions in part to protect stem cells from reactive aldehydes and other toxic compounds. In contrast, we found that approximately 25% of all acute myeloid leukemias expressed low or undetectable levels of ALDH1A1 and that this ALDH1A1- subset of leukemias correlates with good prognosis cytogenetics. ALDH1A1- cell lines as well as primary leukemia cells were found to be sensitive to treatment with compounds that directly and indirectly generate toxic ALDH substrates including 4-hydroxynonenal and the clinically relevant compounds arsenic trioxide and 4-hydroperoxycyclophosphamide. In contrast, normal hematopoietic stem cells were relatively resistant to these compounds. Using a murine xenotransplant model to emulate a clinical treatment strategy, established ALDH1A1- leukemias were also sensitive to in vivo treatment with cyclophosphamide combined with arsenic trioxide. These results demonstrate that targeting ALDH1A1- leukemic cells with toxic ALDH1A1 substrates such as arsenic and cyclophosphamide may be a novel targeted therapeutic strategy for this subset of acute myeloid leukemias.

A novel process is described to produce biopesticides of Trichoderma harzianum Rifai , Pochonia chlamydosporia Zare and Gams, Bacillus subtilis Cohn amend . Prazmowski and Pseudomonas fluorescens (Threvesan) Migula by taking 1 part of stock culture (sawdust:soil:5% molasses, 15:5:1) of the biocontrol agents and 20 parts carrier (flyash:soil:5% molasses mixture, 5:3:1) (w/w). Greatest CFU counts of the microorganisms were recorded at 25 °C or room temperature during 2–12 weeks of 32 weeks long shelf life test. Seed treatment with the biopesticides @ 5 g/kg seeds carried 10 3−6 CFU/g seed of chickpea and pigeonpea. The treatments with T. harzianum and P. chlamydosporia effectively controlled the wilt ( Fusarium oxysporum f. sp. ciceri Padwick, Fusarium udum Butler) and root knot ( Meloidogyne incognita Kofoid and white, Chitwood) on chickpea ( Cicer arietinum L.) and pigeonpea ( Cajanus cajan L.) and greatly reduced the soil population of the pathogens. The biocontrol agents established in the soil and their CFU increased significantly ( P ⩽ 0.05), being greater in pathogen infested soils ( P ⩽ 0.05) than non infested soil during 4 months period.

The goal of this project was to identify and target metabolic vulnerabilities of leukemia stem cells (LSCs) to improve therapeutic outcomes for patients with AML. We have previously shown that primary human LSCs reside in a unique metabolic condition characterized by a relatively low oxidative state (termed "ROS-low") and increased levels of glutathione (Lagadinou et al. Cell Stem Cell, 2013). Cells in this condition are highly dependent on oxidative phosphorylation for survival, in striking contrast to many tumor cells which often rely on glycolysis; indicating that LSCs are governed by distinct metabolic properties. To further elucidate key metabolic properties of LSCs, we measured differences in the global metabolome of ROS-Low LSCs in comparison to ROS-high AML blasts. Our preliminary data demonstrated that ROS-low LSCs have higher levels of amino acids and require amino acid catabolism for survival. We hypothesized that certain individual amino acids may be more important for LSC survival. If true, then targeting specific amino acids may be an avenue towards improved AML therapy. To determine if any individual amino acid is essential for LSC survival, we analyzed AML cells from five patients that were systematically cultured in media lacking one of the twenty amino acids. Cysteine depletion was consistently the most cytotoxic, showing decreased cell viability and colony forming potential of LSCs. We next determined the effect of an engineered human enzyme that selectively degrades cysteine and cystine (AEB3103, Aeglea BioTherapeutics, Inc.) on LSC viability and colony forming potential. We found that AEB3103 treatment decreased viability of LSCs in all AML specimens tested and significantly decreased colony formation (p To determine how AEB3103 decreased LSC viability we investigated the metabolic changes that occur upon AEB3103 treatment by mass spectroscopy. We found that AEB3103 treatment decreased the abundance of metabolites involved in glutathione synthesis (cysteine, cystine, glutathione, taurine, and Cys-Gly) in ROS-low LSCs. Tracing of cysteine13C315N in LSCs demonstrated that all detectable heavy cysteine was metabolized to cystine and glutathione. Furthermore, pretreatment with cell permeable glutathione rescued cell viability and colony-forming potential upon AEB3103 treatment. These data suggest that modulation of glutathione is central to the mechanism by which AEB3103 kills LSCs. Glutathione is a well-characterized antioxidant, therefore, we measured ROS levels and the expression of genes known to be expressed upon ROS induction in LSCs. Surprisingly, we did not observe changes in ROS induction or the expression of ROS induced genes upon AEB3103 treatment. Next, we interrogated cellular functions of glutathione that are independent from the role of glutathione as an antioxidant. Glutathione has previously been shown to mediate electron transport chain complex II function via posttranslational glutathione modification, glutathionylation. Therefore, we hypothesized that decreased glutathione levels upon AEB3103 treatment could result in decreased complex II activity and therefore decreased levels of OXPHOS and ATP production. To test this hypothesis, we measured complex II activity, complex II glutathionylation, oxidative phosphorylation, and ATP levels upon AEB3103 treatment. We observed AEB3103 treatment significantly decreased glutathionylation, complex II activity, oxidative phosphorylation, and ATP levels in AML cells (p Disclosures Pollyea: AbbVie: Consultancy, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Curis: Membership on an entity's Board of Directors or advisory committees.

Epidemiological studies indicate that obese populations have an increased risk for leukemia. However, the mechanism underlying this phenomenon remains unclear. In the current study, utilizing both murine AML models and human AML samples, we observe that AML pathogenesis leads to aberrancies in adipose tissue, pancreatic function, and gut/microbiome, all of which contribute to an insulin resistant phenotype. We demonstrate that through induction of insulin resistance, leukemic disease alters systemic metabolism and thereby redirects systemic glucose to be preferentially available to malignant cells. We found that insulin effect on leukemic mice was significantly impaired as shown by insulin tolerance tests (ITT) (Fig.1) as well as by reduced glucose utilization in both adipose and muscle tissues. Interestingly, glucose utilization in leukemia cells was not affected by insulin. Several mechanisms were found to underlie this insulin resistant phenotype. First, leukemia induced a high-level production of IGFBP1 from adipose tissue and led to a 100-fold increase in circulating IGFBP1, which impaired insulin sensitivity (Fig.1). Blocking IGFBP1 partially restored insulin sensitivity and reduced leukemic burden, whereas pretreating with IGFBP1 facilitated leukemic progression. Second, a 95% reduction in the gut-derived circulating serotonin led to a significant decrease in insulin secretion from pancreas in leukemic mice (Fig.2). Serotonin supplementation partially restored serum insulin levels and decreased leukemic burden by 50% (Fig.2). Finally, the profile of gut microbiota in leukemic mice was distinct from normal mice. Leukemia-associated microbiota functionally contributed to the insulin resistant phenotype and therefore promoted disease progression. Mechanistically, decreased productions of microbiota-derived short chain fatty acids (SCFAs) i.e. butyrate and propionate, were found in leukemic fecal materials and butyrate supplementation reduced leukemic burden by 50% (Fig.2). Of note, there was interplay between these mechanisms. For example, agents increasing insulin levels resulted in a decreased IGFBP1 production in leukemic mice. Further, leukemia-associated microbiota also contributed to the elevated IGFBP1 level and the reduced insulin level in leukemic mice. To test the therapeutic relevance of our findings, we combined serotonin supplementation with butyrate, and found this combination (hereafter termed 'Ser-Bu') reduced leukemic burden by 80% (Fig.3) and provided survival benefits. More importantly, multiple assays including PET-CT scanning (Fig.3), 3H-2-DG labeling, and 13C-NMR labeling were employed to demonstrate that Ser-Bu treatment increased glucose uptake/utilization in adipose and muscle tissues by 200% and 50% respectively and reduced glucose uptake/utilization in leukemia cells by 70% (Fig.3). Therefore, leukemia progression can be significantly attenuated solely by modulation of systemic glucose metabolism. To examine the human relevance of our findings, we analyzed serum samples from normal controls, MDS and AML patients and found that a 6-fold and a 14-fold increase of IGFBP1 in MDS and AML serum respectively compared to normal controls (Fig.4). Additionally, other insulin resistance indicators such as serum free fatty acids, inflammatory cytokines and serum Leptin and Resistin levels were all elevated in AML serum. An insulin resistant phenotype and elevated serum IGFBP1 were also observed in a primary human leukemia specimen xenograft model. Further, a 65% reduction in serotonin was found in AML serum compared to normal controls (Fig.4). Impressively, analyses of paired diagnostic, remission and relapsed BM specimens showed that IGFBP1 was decreased in remission samples and rebounded in the relapsed state, whereas serotonin level showed the opposite pattern (Fig.5). Together, these data support that an insulin resistant phenotype is also evident in AML patients. Collectively, our studies suggest that leukemic tumors gain a competitive advantage by co-opting multiple mechanisms to induce a diabetes-like physiological condition and thereby subvert systemic glucose metabolism to facilitate disease progression. Our studies demonstrate that restoration of normal glucose regulation may be a feasible strategy to suppress systemic growth of malignant cell types. Disclosures Pollyea: Gilead: Consultancy; Curis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Outcomes for AML patients remain poor because of the inability to fully eliminate leukemia stem cells (LSCs). We have previously shown that primary human LSCs reside in a unique metabolic condition characterized by a relatively low oxidative state (termed "ROS-low") and increased levels of glutathione (Lagadinou et al. Cell Stem Cell, 2013). Cells in this condition are highly dependent on oxidative phosphorylation (OXPHOS) for survival, in striking contrast to many tumor cells which often rely on glycolysis, suggesting that LSCs are governed by distinct metabolic properties. Therefore, the goal of this project was to identify and target metabolic vulnerabilities of LSCs. To achieve this objective, we used mass spectroscopy to interrogate the metabolome of leukemia stem cells (LSCs) isolated from primary human AML specimens. We observed significant increases in the levels, uptake, and metabolism of amino acids in LSCs compared to bulk AML cells. These data suggest that LSCs may preferentially rely on amino acids for survival. To investigate this hypothesis, we cultured LSCs and bulk leukemia cells isolated from primary leukemia specimens in media lacking amino acids and measured cell viability and colony forming potential. We found that LSCs were uniquely sensitive to amino acid loss. In addition, LSCs formed significantly fewer colonies upon amino acid depletion compared to LSCs cultured in media containing amino acids. To confirm that amino acid depletion was targeting functionally-defined LSCs, we employed engraftment assays in immune incompetent mice. Culturing primary AML cells without amino acids for 24 hours resulted in significantly reduced levels of leukemia cell engraftment. Next, we interrogated whether amino acid depletion impaired normal HSC survival and function by culturing mobilized peripheral blood without amino acids and measuring the frequency of CD34+ cells, colony forming ability, and engraftment into immune deficient mice. HSC frequency, colony forming ability, and engraftment potential were not changed by amino acid depletion. Altogether, these data demonstrate the LSCs are selectively dependent on amino acids for survival. We next determined how amino acids modulate LSC biology by measuring the consequences of amino acid loss on LSC metabolism. We observed that amino acid depletion decreased OXPHOS specifically in LSCs and not in bulk leukemia cells. We have previously shown that BCL-2 inhibition decreases OXPHOS in LSCs (Lagadinou et al. Cell Stem Cell, 2013). Importantly, recent studies have shown that inhibition of BCL-2 using the BCL-2 inhibitor venetoclax in combination with azacitidine has resulted in superior outcomes for AML patients (Dinardo et al. Lancet Oncology, 2018). Furthermore, our preliminary data demonstrates that venetoclax with azacitidine targets LSCs in AML patients. Therefore, we hypothesized that venetoclax with azacitidine may be targeting LSCs by modulating OXPHOS via amino acid metabolism. To test this hypothesis, we isolated LSCs from AML patients undergoing treatment with venetoclax and azacitidine. LSC specimens obtained pre and 24 hours after initiation of therapy were analyzed for changes in OXPHOS, gene expression, and the metabolome. We observed that venetoclax with azacitidine treatment decreased OXPHOS and reduced amino acid levels. In addition, expression of amino acid transporters was down-regulated. Finally, we sought to determine if culturing LSCs in high levels of amino acids before venetoclax and azacitidine treatment could rescue LSC viability and OXHPOS. We found that culturing LSCs with increased levels of amino acids rescued LSCs survival and OXPHOS, demonstrating that venetoclax with azacitidine targets LSCs by decreasing amino acid levels. Taken together, our data indicate that LSCs are selectively reliant on amino acid metabolism to fuel OXPHOS. Furthermore, amino acid metabolism can be targeted in AML patients by venetoclax with azacitidine treatment. These studies are the first to characterize metabolic targeting of LSCs in AML patients. Disclosures Nemkov: Omix Technologies inc: Equity Ownership. Pollyea:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Curis: Membership on an entity's Board of Directors or advisory committees.

Summary From an organismal perspective, cancer cell populations can be considered analogous to parasites that compete with the host for essential systemic resources such as glucose. Here, we employed leukemia models and human leukemia samples to document a form of adaptive homeostasis, where malignant cells alter systemic physiology through impairment of both host insulin sensitivity and insulin secretion to provide tumors with increased glucose. Mechanistically, tumor cells induce high-level production of IGFBP1 from adipose tissue to mediate insulin sensitivity. Further, leukemia-induced gut dysbiosis, serotonin loss, and incretin inactivation combine to suppress insulin secretion. Importantly, attenuated disease progression and prolonged survival are achieved through disruption of the leukemia-induced adaptive homeostasis. Our studies provide a paradigm for systemic management of leukemic disease.

Formation of cell surface protrusions has long been associated with tumor cell migration, metastasis & bone marrow homing & retention. It was shown that increased expression of endogenous or exogenous hyaluronic acid synthesizing enzyme 3 (HAS3) induces growth of microvillus-like cell surface protrusions. Some of the biological signals triggered by HA are dependent on its recognition by the cell surface receptor CD44. In our studies we observe that overexpression of the eukaryotic translation initiation factor 4E (eIF4E) increases formation of cell surface protrusions resembling those produced by HAS3 upregulation. eIF4E regulates mRNA export & translation through binding to the m7G cap of mRNA in the nucleus & the cytoplasm, respectively. Strikingly, analysis of our two complimentary screens (immunoprecipitation of mRNAs bound to eIF4E in the nucleus, and mRNA export assay with ribavirin, an established inhibitor of eIF4E) that we carried out to identify candidate eIF4E mRNA export targets, showed that nearly all the enzymes involved in HA synthesis as well as CD44. Here, we hypothesize that eIF4E regulates HA synthesis & CD44 overexpression to reshape the surface of cancer cells producing HA-rich microvillus-like pseudopods. Following validation of the positive hits, we used immunofluorescence staining with biotinylated HA binding protein (HABP) to determine the effect of eIF4E expression on protrusion formation. Our data revealed that eIF4E overexpression increased formation of HA-rich cell surface protrusions & was correlated with increased invasiveness of eIF4E overexpressing cells in in vitro matrigel assay, compared to respective controls. Colocalization studies using confocal microscopy indicated that protrusions positively stained for CD44. Further, pharmacological inhibition with ribavirin and genetic knockdown of eIF4E (or HAS3/CD44 in the context of eIF4E overexpression) abrogated protrusion formation and decreased the invasion capacity of eIF4E overexpressing cells. Together, these data indicate that eIF4E modulates protrusion formation & tumor invasion through coordinately regulating the export of RNAs in the CD44-HA regulon. To establish the clinical relevance of our findings in AML, we examined primary AML specimens with high eIF4E levels for expression of HA synthesizing enzymes, CD44 & we directly measured HA using confocal methods. This comparison showed that these pathways are dysregulated in AML patients & that their inhibition correlates with response to eIF4E inhibition by ribavirin. While HA has long been considered as a component of the tumor microenvironment required for the migration of cancer cells though interaction with surface CD44, our results provide a novel role for eIF4E in the motility & bone marrow homing. These findings offer a rationale for potentially inhibiting tumor cell metastasis through the combinatorial inhibition of eIF4E and CD44 or HAS3. Citation Format: Hiba Zahreddine, Biljana Kraljacic-Culjkovic, Valbona Cali, Mark Lauer, Lucy Skrabanek, Nabilah Khan, Ronald Midura, Leandro Cerchietti, Vincent Hascall, Craig Jordan. eIF4E reshapes the surface of migrating AML cells through regulating Hyaluronic Acid synthesis & CD44 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4898. doi:10.1158/1538-7445.AM2017-4898

We recently reported that adipose tissue functions as a reservoir for leukemia stem cells (LSCs) using a murine model of leukemia (Ye et al., Cell Stem Cell, 2016). Intriguingly, the presence of leukemic cells in adipose tissue induces increased lipolysis and the release of free fatty acids (FFA) which in turn fuels the growth of LSCs. Further, adipose tissue protects resident LSCs from chemotherapy. These findings indicate that the unique characteristics of adipose tissue may provide key insights on the growth and survival of leukemic cells. Thus, in the present study we focus on the endocrine function of adipose tissue and explore its role in leukemia development. First, to evaluate secreted factors, we applied adipokine arrays which detect 38 adipokines to leukemia serum collected at different stages of leukemia pathogenesis. Interestingly, we observed a significantly elevated level of serum IGFBP1 as soon as leukemic disease became evident at low levels (~0.5%) in marrow. IGFBP1 increased to levels approximate 200X normal at peak stages of disease burden. ELISA analyses further confirmed these observations. IGFBP1 is normally considered as a liver-derived protein. However, we did not find any changes of IGFBP1 expression in leukemic liver. Rather, we observed a significant increase in the expression of IGFBP1 in adipose tissue. IGFBP1 in conditioned medium (CM) from leukemic gonadal adipose tissue (GAT) is approximate 100X higher than naive GAT. Together, these findings suggest adipose tissue-derived IGFBP1 contributes to the increased serum IGFBP1 we detected in leukemic animals. We next examined the role of IGFBP1 in leukemia development using a neutralizing antibody. Treatment of experimental animals with anti-IGFBP1 antibody significantly decreased leukemic burden in GAT (~40% IgG treated VS. 20% Anti-IGFBP1 treated) while bone marrow (BM) and spleen leukemic engraftment remained unchanged (~40%). Further, less atrophy of adipose tissue as well as less body weight loss was seen in the IGFBP1 neutralizing antibody treated group. Consistent with this observation, serum FFA level was also reduced, suggesting less lipolysis in the IGFBP1 antagonized group. Together, these results indicate that IGFBP1 is involved in the regulation of leukemic cells homing to adipose tissue and consequently leukemia-induced lipolysis. Next we explored the mechanisms for the increased expression of IGFBP1 in adipose tissue. Studies have shown that both FGF21 and hypoxia induce IGFBP1 expression. We did not find any changes of FGF21 expression in adipose tissue or in liver, suggesting FGF21 was not involved in IGFBP1 regulation in our system. In contrast, we observed a five-fold elevation in IGFBP1 mRNA in primary adipose tissue cultured under hypoxic conditions. Studies have shown that both BM and spleen in leukemia mice are already hypoxic in early stages of leukemic development (Benito et al., Plos One, 2011). Thus, we hypothesize that tissue hypoxia may at least partially regulate IGFBP1 in leukemia. Ongoing experiments are testing this hypothesis. Additionally, inflammatory cytokines have been reported to increase IGFBP1 expression. We previously reported significantly increased levels of inflammatory cytokines in the adipose tissue of leukemic mice including TNF-α, IL1 and CSF2 (1.5X normal adipose tissue). Therefore, we hypothesize the local inflammatory cytokine production may also contribute to increased expression of IGFBP1, a theory that is also currently under investigation. IGFBP1 has recently been reported to activate osteoclasts and thus promotes bone metabolism (Wang et al., Cell Metabolism, 2015). Studies have found an increased number of osteoclasts and thus bone loss in our leukemic model (Frisch et al., Blood, 2012). We hypothesize that adipose tissue-derived IGFBP1 contributes to bone loss in leukemic mice. Ongoing experiments are testing whether antagonization of IGFBP1 in leukemic mice will rescue bone loss. Collectively, these findings suggest that adipose tissue-derived IGFBP1 facilitates progression of leukemia through regulation of adipose tissue lipolysis and may promote bone marrow colonization by leukemia cells through activation of osteoclasts. Disclosures No relevant conflicts of interest to declare.

NADPH dependent oxidase 2 (NOX2) is the founding member of a family of multimeric, oxido-reductase enzymes that catalyze the production of superoxides by transferring a single electron from the cofactor NADPH to molecular oxygen. It is primarily utilized in neutrophils and macrophages to generate copious amount of reactive oxygen species (ROS) to facilitate the neutralization of engulfed particulates during phagocytosis. In sharp contrast to this specialized function however, recent evidence implies a non-phagocytic role for NADPH oxidases in which physiologic levels of ROS generated by these enzymes modulate key signaling proteins and transcription factors to exert profound biological effects. Based on this information we decided to investigate the potential role of NOX2 in normal and leukemic stem cells. Using transgenic NOX2 knock out mice, genetically defined murine models of myeloid leukemia and primary human acute myeloid leukemia (AML) specimens, we show that NOX2 is critical for the proper function of normal and malignant hematopoietic stem cells. In silico analysis using published transcriptional profiles of hematopoietic populations revealed that multiple subunits of the NOX2 complex are expressed at low levels in hematopoietic stem cells (HSCs) and at relatively higher levels in multipotent progenitors (MPPs). Next, we characterized the different hematopoietic compartments from age and sex matched wild type (WT) and transgenic NOX2 knock out (KO) mice. Our studies revealed that in the bone marrow of KO mice, a subset of multipotent progenitor populations (MPP2 & MPP3), which often have biased myelo-erythroid output are markedly expanded relative to their wild type counterparts. Consistently, we found increased levels of granulocytes and monocytes in the peripheral circulation of NOX2 KO mice. To test whether NOX2 has a functional, biological role in the self-renewal of HSCs, we performed competitive transplantation assays using equal numbers of whole BM cells from WT and KO mice to co-repopulate lethally irradiated hosts. Analysis of engrafted mice showed that the contribution from NOX2 KO HSCs was severely compromised in all lineages and developmental stages of hematopoiesis examined. Collectively, these results suggest a critical biological role for NOX2 in maintaining the quiescence and long term self-renewal of HSCs. Similar to normal hematopoiesis, we found out that NOX2 is also widely expressed by functionally defined leukemic stem cells in a murine model of myeloid leukemia generated by expressing the oncogenic translocations BCR-ABL and NUP98-HOXA9. To evaluate the role of NOX2 in leukemogenesis, we established the BCR-ABL/NUP98-HOXA9 model using primitive cells derived from either WT or KO. Intriguingly, NOX2 KO leukemic cells generated a much less aggressive disease upon transplantation into primary and subsequently into secondary recipients. Furthermore, leukemic cells in which NOX2 is suppressed displayed aberrant mitotic activity and altered developmental potential marked by loss of quiescence, enhanced entry into cycle and terminal differentiation. To gain mechanistic insight into the observed phenotype, we isolated leukemic stem cells and performed whole genome expression analysis. The data showed that deficiency of NOX2 leads to downregulation of the cell cycle inhibitor CDKN2C (p18) and robust activation of the granulocyte fate determining transcription factor CEBPε. Thus we conclude that loss of NOX2 impacts leukemogenesis through rewiring of the cell cycle machinery and developmental programs in leukemic stem cells. Finally, we found that in CD34+ primary human AML cells, NOX2 and the other subunits of the complex are abundantly expressed. Furthermore, pharmacologic inhibition of NOX2 with VAS2870, a selective NADPH oxidase inhibitor, reduced the level of ROS and limited the in vitro proliferation and survival of leukemic cells. Next we genetically suppressed the expression of NOX2 in primary human AML cells using sh-RNAs and transplanted these cells into immune compromised mice. Consistent with the murine leukemia, NOX2 knocked down AML cells failed to engraft and expand in vivo. Taken together, our results firmly establish a hitherto unrecognized, prominent regulatory role for NOX2 in the biology of normal and malignant hematopoietic stem cells and imply a potential therapeutic opportunity that can get exploited to treat AML. Disclosures Pollyea: Celgene: Other: advisory board, Research Funding; Ariad: Other: advisory board; Pfizer: Other: advisory board, Research Funding; Glycomimetics: Other: DSMB member; Alexion: Other: advisory board.

This project is focused on characterizing the malignant stem cells that drive the pathogenesis of MDS, with the goal of developing improved therapeutic strategies. To this end, we have established methods by which MDS stem cells can be identified, isolated and characterized in xenograft models. These approaches have been employed to permit global metabolomic and transcriptomic analyses, which have subsequently led to modeling novel therapeutic regimens. Initial studies exploited previous work in acute myelogenous leukemia to identify candidate phenotypic markers of stem cell malignancy. These efforts demonstrated that up-regulation of CD123 in the hematopoietic stem cell compartment identifies MDS stem cells as they transition from low risk to high risk stages of pathogenesis. Thus, using cell sorting, we are able to isolate early (CD123-) vs. late (CD123+) stage stem cells from MDS patient specimens and subject them to the experimental analyses outlined below. We first performed a transcriptomic study that demonstrated a massive increase in cellular protein translation machinery through significant increases in ribosomal proteins as stem cells progress to advanced stages of MDS. To functionally validate these findings, MDS stem cell populations were cultured with the fluorescent protein substrate OP-puro, which detects newly synthesized polypeptide chains. CD123+ stem cells strongly increase protein synthesis levels (~13-fold increase). Given the established role of protein translation homeostasis in hematopoietic stem cells [Signer et al., 2014], this finding indicates a major change in cellular metabolism as well as a potential therapeutic entry point. To further characterize cellular changes occurring as MDS stem cells evolve, we performed global mass spectrometry-based metabolomic analyses which further demonstrates increased protein biosynthesis as well as altered glutathione metabolism (elevated oxidized glutathione). Taken together, these findings indicate major metabolic changes in MDS stem cells as they acquire increasing malignant phenotypes. Next, we investigated signaling related to increased ribosomal protein production. Specifically, we examined the hypothesis that ribosomal subunit binding of MDM2, may play a role in pathogenesis. Intriguingly, our data show that the specific subunits known to bind MDM2 are increased in CD123+ MDS stem cells. Consistent with this observation, MDM2 is elevated as well. This finding indicates that increased ribosomal protein levels may function to inhibit p53 activity, thereby enhancing pathogenic outgrowth of MDS stem cells. Using the mechanistic insights outlined above, we explored novel therapeutic strategies. First, we examined drugs known to selectively target metabolism through altering protein synthesis and targeting ribosomal proteins (e.g. homoharringtonine, HHT)). In addition, given the prevalent role of hypomethylating agents in current MDS treatment regimens, we also examined how these inhibitors interact with 5-azacytidine (5-aza). In vitro studies indicate that multiple protein synthesis inhibitors selectively eradicate MDS stem cells (CD123+). In addition, combination with 5-aza yielded additive to synergistic eradication of MDS stem cells. We have demonstrated that high risk MDS specimens effectively engraft the NSG-S strain of immune deficient mice, when T cell depletion and a busulfan conditioning regimen are employed. Using this model, we transplanted primary MDS specimens and challenged using the regimens above. Treatment with HHT demonstrated selective eradication of MDS stem cells, with a significant differential toxicity observed in multiple samples (50-60% selective ablation). Finally, analysis of protein synthesis inhibitors in combination with the hypomethylating agent 5-aza demonstrated potent efficacy in targeting the MDS stem cell population with greater then additive toxicity when compared to single agent treatment(70-80% selective ablation). Taken together, these data show that changes in metabolic properties represent a critical inflection point in the pathogenesis and progression of the MDS. Focusing on such changes, we have identified novel pharmacological approaches that may effectively target the MDS stem cell population. Importantly, these approaches function well in conjunction with commonly used agents used in the treatment of MDS. Disclosures Pollyea: Alexion: Other: advisory board; Celgene: Other: advisory board, Research Funding; Glycomimetics: Other: DSMB member; Ariad: Other: advisory board; Pfizer: Other: advisory board, Research Funding.

Aberrant function of adipose tissue (AT) is seen in several diseases including cancer. Studies show that AT facilitates the progression of tumors through paracrine signaling of adipokines as well as regulation of cancer cell metabolism. However, the mechanism by which cancer cells corrupt the normal function of AT to gain proliferative and survival advantages is unknown. Using a murine model of blast crisis CML, we have shown enrichment of phenotypically primitive leukemia cells (Sca+/lin- leukemia cells, termed "PLCs") in the gonadal AT (GAT) as well as a fatty acid oxidation (FAO) regulatory role of AT. In this study, we evaluated the functional alteration of GAT in leukemic mice. We hypothesized that resident leukemia cells change the characteristics of GAT to obtain metabolic benefits. To test this hypothesis, we first examined whether PLCs in GAT differed from PLCs in other tissues including bone marrow, spleen and peripheral blood. To this end, we utilized RNA-seq to obtain a genome-wide transcriptional profile of PLCs in different tissues. We found PLCs in GAT had a distinct gene expression pattern with enrichment of inflammatory response genes. Specifically, pro-inflammatory cytokines and chemokines were highly expressed by PLCs in GAT (Figure 1). Furthermore, the expression of those genes was also increased in the stromal vascular fraction (SVF) of GAT, indicating resident leukemia cells induced inflammation in GAT. Collectively, these results suggest that leukemia cells found in GAT are distinct from leukemia cells in other tissues and may alter the function of GAT. Another characteristic observed in our model was atrophy of GAT (Figure 2) as well as loss of body weight during leukemia progression, indicating the presence of cancer cachexia. Loss of GAT was also found prior to loss of body weight, suggesting the presence of a pre-cachexia stage. We speculated that atrophy of GAT was due to lipolysis induced by inflammation. Indeed, leukemic GAT released more free fatty acid (FFA) and had a lipolytic pattern of adipokines compared to normal GAT. Elevated FFA and lipolytic adipokines were also detected in leukemic serum. Together, these observations demonstrate that GAT in leukemic mice is lipolytic. To gain insights into mechanisms involved in lipolysis of leukemic GAT, we examined expression of lipolysis-related genes (Figure 3). We found that leukemic GAT had increased expression of the adipose triglyceride lipase (Atgl), which is a rate-limiting lipase controlling lipolysis, and reduced expression of lipoprotein lipase (Lpl), whose expression correlates with the influx of fatty acids into adipocytes. Additionally, decreased expression of the cell death activator CIDE-A (Cidea), which is a lipid droplet (LD) associated protein that shields LDs from lipases and inhibits lipolysis, was found in leukemic GAT. Together, these findings suggest that regulation of lipid metabolism is disrupted in leukemic GAT, leading to lipolysis. To test whether resident leukemia cells contribute to the atrophy of GAT, we examined the lipolytic effect of the pro-inflammatory cytokines and chemokines that were highly expressed by PLCs in GAT. We found that IL-1β and CSF2 induced lipolysis and engendered similar gene expression patterns of lipolysis-related genes in 3T3-L1 adipocytes. Notably, palmitate induced the expression of IL-1β in leukemia cells while it had an opposite effect in naive hematopoietic cells, implying a positive feedback loop where inflammation induces lipolysis which induces IL-1β which in turn augments inflammation. Additionally, an increased amount of IL-1β was observed in leukemic serum. Taken together, these data suggest that resident leukemia cells contribute to the atrophy of GAT through paracrine signaling of pro-inflammatory agents. This phenomenon appears to benefit leukemia cells by fostering FAO and metabolic properties that enhance leukemia cell survival. Thus, targeting pathways that mediate inflammation and/or lipolysis may create a microenvironment that is less favorable to leukemia cells. Disclosures No relevant conflicts of interest to declare.

Adipose tissue (AT) serves as a storage site for lipids as well as an endocrine organ. In the context of cancer, AT can function to facilitate the progression of tumors. Interestingly, recent studies have shown that AT acts as an extramedullary reservoir of hematopoietic stem cells (HSCs), suggesting the presence of a HSC niche in AT. The fact that leukemia stem cells (LSCs) co-opt the HSC marrow and the supportive effect of AT on cancer cells led us to hypothesize that AT functions as a reservoir for LSCs. To test this hypothesis, we first examined whether there were LSCs residing in AT. Using a murine model of primary blast crisis CML, we found enrichment of phenotypically primitive leukemia cells (PLCs) in the gonadal adipose tissue (GAT) relative to bone marrow, spleen and peripheral blood (Figure 1). The high percentage of PLCs in GAT led us to postulate that PLCs are preferentially reliant on fatty acids as an energy source since GAT was found to be lipolytic in leukemic mice. Indeed, we observed that PLCs had a higher rate of fatty acid oxidation (FAO) compared to lineage+ leukemia cells and their non-leukemic counterparts. Additionally, conditioned medium from adipocytes selectively increased the FAO rate in PLCs suggesting a FAO regulatory role of AT. Further, we showed that CD36, a fatty acid transporter, was highly expressed by a subset of PLCs and selectively regulated FAO in PLCs. Interestingly, CD36+ PLCs were strikingly enriched in GAT. Together, these results suggested that GAT functions as a reservoir for CD36+ PLCs and facilitates use of FAO for energy metabolism. Next we examined the characteristics of CD36+ PLCs. CD36+ PLCs differed metabolically from CD36- PLCs with regard to fatty acid metabolism. Specifically, we found CD36+ PLCs had a higher FAO rate and were more dependent on the transportation function of CD36 for FAO (Figure 2). We also compared the cell cycle status between these two populations and found CD36+ PLCs were more quiescent. Interestingly, both CD36+ and CD36- PLCs contained LSCs and were able to reconstitute the whole leukemic BM when transplanted into recipients. Collectively, these findings indicated that at least two metabolically distinct types of leukemia-initiating cells exist in our blast crisis model, and that the major forms of energy metabolism can differ as a function of anatomical location and expression of CD36. Since front line agents commonly used in cancer generally target actively cycling cells, we speculated that CD36+ PLCs might be more drug resistant due to their increased quiescence. Indeed, we found enrichment of CD36+ PLCs in BM after applying a 5-day chemotherapy regimen to leukemic mice, while CD36- PLCs were not protected. More importantly, we observed that CD36+ PLCs were highly enriched in GAT after chemotherapy suggesting GAT conferred chemo-resistance to resident CD36+ PLCs (Figure 3). Taken together, our observations imply that the heterogeneity found in PLCs is translated into drug sensitivities in different leukemic sub-fractions. Specifically, CD36+ PLCs represent a relatively drug resistant subpopulation and GAT serves as a reservoir for resident CD36+ PLCs. Lastly, we investigated whether these findings in the murine model could be recapitulated in primary human bcCML cells. We found that within the CD34+ subpopulation of primary human bcCML cells, CD36+ subset had a high FAO rate compared to CD36- subset. Furthermore, this CD36+ subset was more quiescent and drug resistant. These data suggest that a similar heterogeneity can be found in primary human bcCML cells based on the expression of CD36. Collectively, our findings suggested that GAT in leukemia mice functions as a reservoir for LSCs and confers chemo-resistance to resident leukemia cells, implying a potential role of GAT in the pathogenesis of leukemia and relative efficacy of therapeutic challenge. Furthermore, our data indicate metabolic heterogeneity within LSC populations, where pathways controlling energy consumption can differ. We propose that metabolic heterogeneity in LSCs may contribute to the challenge in effectively eradicating such cells. Disclosures No relevant conflicts of interest to declare.

Studies from normal cell biology have demonstrated that mitochondria can switch between fission-active and fusion-active states to dramatically change their morphology during important developmental events such as lineage differentiation and cell division. Further, very recent research has revealed intriguing interactions between mitochondrial morphology regulation and oncogenic signals in cancer. To date however, whether mitochondrial morphology regulation plays a role in cancer stem cell function remains largely unknown. We report, in acute myelogenous leukemia (AML), leukemia stem cells (LSCs), characterized by a low level of reactive oxygen species (ROS), have hyper-active mitochondrial fission regulators as evidenced by higher levels of mitochondrial fission 1 (FIS1) and activating phosphorylation of dynamin-related protein 1 (DRP1), compared to non-LSCs. To directly compare the mitochondrial morphology in LSCs versus non-LSCs, we imaged mitochondrial morphology using transmission electron microscopy. Quantification of the imaging results showed that the LSCs contain an increased number of smaller and globular-shaped mitochondria (see Figure), indicating that they reside in a fission-active state relative to non-LSCs. Inhibition of mitochondrial fission through genetic knock-down of FIS1 (FIS1-KD) induces mitochondrial fusion and dramatically diminishes both colony-forming ability and serial engraftment potential of primary AML cells, suggesting a fission-active state of mitochondrial morphology is critical for LSC function. To dissect the mechanism by which FIS1 regulates primitive leukemia cells, we performed detailed analysis of molecular events following FIS1-KD. We found that AML cells lacking the function of FIS1 up-regulated the expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins, suggesting an increased OXPHOS activity. We then observed through metabolomic studies that knock-down of FIS1 altered the level of metabolites involved in glycolysis and the citric acid cycle (TCA), suggesting that the FIS1-KD-induced change in OXPHOS activity affected other aspects of global cellular energy metabolism as well. Among the metabolic changes seen was an increase in Acetyl-CoA, which plays an important role in epigenetic regulation by serving as the substrate for histone acetylation. Consistently, we observed an increase in H3K27-acetylation in AML cells following FIS-KD, and our transcriptomic analysis using RNA-seq showed that AML cells lacking FIS1 function exhibited global up-regulation of a hematopoietic lineage differentiation gene signature including CD11B, CD14, CD36, and CD68, indicating an induction of differentiation. In addition to affecting energy metabolism and epigenetic landscape, FIS1-KD also increased cellular ROS level in AML cells. This may happen as a consequence of induced OXPHOS activity, since ROS is a major byproduct of oxygen metabolism in OXPHOS machineries. Taken together, our functional studies raise the hypothesis that mitochondrial morphology critically regulates LSC function. Our subsequent molecular studies begin to reveal two major potential mechanisms by which mitochondrial morphology regulators can affect LSC potential. First, FIS1-regulated mitochondrial morphology may sequentially affect mitochondrial OXPHOS activity, global energy metabolism, homeostasis of metabolites such as Acetyl-CoA that can link metabolism to epigenetics, and regulation of the epigenome that is required for sustaining LSCs. Second, FIS1 can also affect cellular ROS, a critical determinant of self-renewal ability of stem cells, to effect LSC function. Overall, our current study elucidates a previously unknown link between the regulation of mitochondrial morphology and LSC function, targeting of which represents a novel therapeutic means to eradicate LSCs. Figure 1. Figure 1. Disclosures Pollyea: Ariad: Consultancy; GlycoMimetics: Other: Member of data safety monitoring board; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy; Karyopharm: Consultancy.