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Over the past several decades numerous preclinical and clinical studies have pursued new approaches for the treatment of acute myeloid leukemia (AML). While some degree of clinical response has been demonstrated for many therapies, for the most part, fundamental changes in the treatment landscape have been lacking. Recently, the use of the BCL-2 inhibitor venetoclax has emerged as a potent therapy for a majority of newly diagnosed AML patients. Venetoclax regimens have shown broad response rates with deep and durable remissions, with a superior toxicity profile compared with traditional intensive chemotherapy agents. Numerous ongoing studies are now using venetoclax in combination with a wide range of other agents as investigators seek even more effective and well-tolerated regimens. Notably, however, while the empirical results of BCL-2 inhibition are encouraging, the mechanisms that have led to these successful clinical outcomes remain unclear. Intriguingly, the activity of venetoclax in AML patients appears to go beyond simply modulating canonical antiapoptosis mechanisms; in addition, the efficacy of venetoclax is linked to its combined use with conventional low-intensity backbone therapies. This article will evaluate the state of the field, provide a summary of key considerations, and propose directions for future studies.

Malignant stem cells have long been considered a key therapeutic target in leukemia. Therapeutic strategies designed to target the fundamental biology of leukemia stem cells while sparing normal hematopoietic cells may provide better outcomes for leukemia patients. One process in leukemia stem cell biology that has intriguing therapeutic potential is energy metabolism. In this article we discuss the metabolic properties of leukemia stem cells and how targeting energy metabolism may provide more effective therapeutic regimens for leukemia patients. In addition, we highlight the similarities and differences in energy metabolism between leukemia stem cells and malignant stem cells from solid tumors.

Widely-used response criteria, conditional upon count recovery, were developed for acute myeloid leukemia (AML) in the context of intensive chemotherapy (IC). Extending these definitions to continuously-administered venetoclax-based therapies might underestimate responses. Best practices for venetoclax-based therapies mandate interruption after an end-of-cycle 1 bone marrow biopsy shows morphologic remission with cytopenias. We analyzed 435 patients with newly-diagnosed AML and follow-up response assessments. Of the 101 who responded to venetoclax + azacitidine, overall survival for patients whose response was upgraded due to count recovery during a 14-day post-disease assessment period, from complete remission (CR) with incomplete recovery of blood counts to CR, was not different compared to patients who did not need the 14-day period for count recovery. These results were distinct from 138 IC patients. Although sample sizes for the comparison groups were small, and conclusions are exploratory and must be verified, these findings support consideration of new response criteria for venetoclax-based regimens.

Two factors known to contribute to the development of myelodysplastic syndrome (MDS) and other blood cancers are (i) somatically acquired mutations in components of the spliceosome and (ii) increased inflammation. Spliceosome genes, including SF3B1, are mutated at high frequency in MDS and other blood cancers; these mutations are thought to be neomorphic or gain-of-function mutations that drive disease pathogenesis. Likewise, increased inflammation is thought to contribute to MDS pathogenesis; inflammatory cytokines are strongly elevated in these patients, with higher levels correlating with worsened patient outcome. In the current study, we used RNAseq to analyze pre-mRNA splicing and gene expression changes present in blast cells isolated from MDS patients with or without SF3B1 mutations. We determined that SF3B1 mutations lead to enhanced proinflammatory gene expression in these cells. Thus, these studies suggest that SF3B1 mutations could contribute to MDS pathogenesis by enhancing the proinflammatory milieu in these patients.

Venetoclax with azacitidine (ven/aza) has emerged as a promising treatment regimen for acute myeloid leukemia (AML), with a high percentage of clinical remissions in newly diagnosed patients. However, approximately 30% of newly diagnosed patients and the majority of patients who have relapsed do not achieve remission with ven/aza. We previously reported that ven/aza efficacy is based on eradication of AML stem cells through a mechanism involving inhibition of amino acid metabolism, a process required in primitive AML cells to drive oxidative phosphorylation. Herein we demonstrate that resistance to ven/aza occurs via upregulation of fatty acid oxidation (FAO), which occurs either due to RAS pathway mutations or as a compensatory adaptation in relapsed disease. Utilization of FAO obviates the need for amino acid metabolism, thereby rendering ven/aza ineffective. Pharmacological inhibition of FAO restores sensitivity to ven/aza in drug-resistant AML cells. We propose inhibition of FAO as a therapeutic strategy to address ven/aza resistance. Jordan and colleagues show the role of increased fatty acid metabolism in AML stem cells, in both intrinsic and acquired resistance to combination venetoclax and azacitidine, and find that cells can be re-sensitized by inhibiting fatty acid oxidation.

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.

Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.

Purpose: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically. Experimental Design: AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized. Results: We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating ALL cells in vitro and in vivo. Conclusions: Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Background Patients with relapsed and refractory (R/R) acute myeloid leukemia (AML) have limited treatment options. Genomically-defined personalized therapies are only applicable for a minority of patients. Therapies without identifiable targets can be effective but patient selection is challenging. The sequential combination of azacitidine with high-dose lenalidomide has shown activity; we aimed to determine the efficacy of this genomically-agnostic regimen in patients with R/R AML, with the intention of applying sophisticated methods to predict responders. Methods Thirty-seven R/R AML/myelodysplastic syndrome patients were enrolled in a phase 2 study of azacitidine with lenalidomide. The primary endpoint was complete remission (CR) and CR with incomplete blood count recovery (CRi) rate. A computational biological modeling (CBM) approach was applied retrospectively to predict outcomes based on the understood mechanisms of azacitidine and lenalidomide in the setting of each patients’ disease. Findings Four of 37 patients (11%) had a CR/CRi; the study failed to meet the alternative hypothesis. Significant toxicity was observed in some cases, with three treatment-related deaths and a 30-day mortality rate of 14%. However, the CBM method predicted responses in 83% of evaluable patients, with a positive and negative predictive value of 80% and 89%, respectively. Interpretation Sequential azacitidine and high-dose lenalidomide is effective in a minority of R/R AML patients; it may be possible to predict responders at the time of diagnosis using a CBM approach. More efforts to predict responses in non-targeted therapies should be made, to spare toxicity in patients unlikely to respond and maximize treatments for those with limited options.

Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.

Introduction: Multiple myeloma (MM) is a plasma cell malignancy that remains incurable, with patients enduring multiple relapses and development of drug resistance. Immunomodulatory drugs (IMiDs) are critical anti-MM agents. IMiDs act by inducing CRBN-dependent proteasomal degradation of the transcription factors IKZF1 and IKZF3, which leads to IRF4 and MYC downregulation (collectively termed the “Ikaros axis”). Although CRBN aberrations occur, whether they are a functional mechanism of resistance in patients remains unclear. Based on the importance of CRBN in IMiD response, we hypothesized that IMiD treatment fails to downregulate the Ikaros axis in IMiD-resistant MM cells. Methods: To measure IMiD-induced Ikaros axis downregulation, we designed an intracellular flow cytometry assay that measured relative IKZF1, IKZF3, IRF4 and MYC protein levels in MM cells following ex vivo pomalidomide (Pom) treatment. We established this assay using IMiD-sensitive parental and dose-escalated Pom resistant MM1S and H929 cell lines before utilizing it in patient samples (isolated mononuclear cells). To assess the Ikaros axis in the context of MM intratumoral heterogeneity, we used mass cytometry to simultaneously characterize MM subpopulations in patient samples. Lastly, we determined ex vivo drug sensitivity in patient samples via flow cytometry. Results: Our hypothesis was supported in MM cell lines, as sensitive parental lines showed a significant decrease in all Ikaros axis protein levels following Pom treatment, while resistant lines showed no IMiD-induced decrease in any Ikaros axis protein. However, when assessed in CD38+CD138+ cells from patient samples, Pom treatment caused a significant decrease in IKZF1, IKZF3 and IRF4 regardless of IMiD sensitivity. Mass cytometry in patient samples revealed that individual Ikaros axis proteins were differentially expressed between subpopulations. When correlating this with ex vivo Pom sensitivity of MM subpopulations, we observed that low IKZF1 and IKZF3 corresponded to Pom resistance. Interestingly, most of these resistant populations still expressed MYC. We therefore assessed whether IMiD resistant MM was MYC dependent by treating Pom resistant MM cells with MYCi975. In 88% (7/8) of patient samples tested, IMiD resistant MM cells were sensitive to MYC inhibition. Conclusions: Our findings did not support our initial hypothesis, as IMiD-induced IKZF1 and IKZF3 degradation remains intact in IMiD resistant MM cells from patient samples. However, our data support a mechanism where the Ikaros axis no longer drives MYC expression in IMiD-resistant MM. Therefore, we propose that the critical mediator of IMiD resistance is conversion to a cell state where MYC expression is Ikaros axis independent. This suggests targeting MYC directly or via the as yet uncharacterized mechanism controlling MYC may be an effective strategy to eradicate IMiD resistant MM. Citation Format: Lorraine N. Davis, Zachary J. Walker, Denis Ohlstrom, Brett M. Stevens, Peter A. Forsberg, Tomer M. Mark, Craig T. Jordan, Daniel W. Sherbenou. MYC inhibition overcomes IMiD resistance in heterogeneous multiple myeloma populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3956.

Summary Isolation of leukemia stem cells presents a challenge due to the heterogeneity of the immunophenotypic markers commonly used to identify blood stem cells. Several studies have reported that relative levels of reactive oxygen species (ROS) can be used to enrich for stem cell populations, suggesting a potential alternative to surface antigen-based methods. Here, we describe a protocol to enrich for stem cells from human acute myeloid leukemia specimens using relative levels of ROS. This protocol provides consistent enrichment of leukemia stem cells. For complete details on the use and execution of this protocol, please refer to Lagadinou et al. (2013) and Pei et al. (2018)., Graphical abstract, Highlights • Low levels of reactive oxygen species are a hallmark of stem cells • Relative levels of reactive oxygen species allow for enrichment of leukemia stem cells • This protocol is an alternative to surface antigen-based methods, Isolation of leukemia stem cells presents a challenge due to the heterogeneity of the immunophenotypic markers commonly used to identify blood stem cells. Several studies have reported that relative levels of reactive oxygen species (ROS) can be used to enrich for stem cell populations, suggesting a potential alternative to surface antigen-based methods. Here, we describe a protocol to enrich for stem cells from human acute myeloid leukemia specimens using relative levels of ROS. This protocol provides consistent enrichment of leukemia stem cells.

MERTK is ectopically expressed and promotes survival in acute lymphoblastic leukemia (ALL) cells and is thus a potential therapeutic target. Here we demonstrate both direct therapeutic effects of MERTK inhibition on leukemia cells and induction of anti-leukemia immunity via suppression of the coinhibitory PD-1 axis. A MERTK-selective tyrosine kinase inhibitor, MRX-2843, mediated therapeutic anti-leukemia effects in immunocompromised mice bearing a MERTK-expressing human leukemia xenograft. In addition, inhibition of host MERTK by genetic deletion (Mertk-/- mice) or treatment with MRX-2843 significantly decreased tumor burden and prolonged survival in immune-competent mice inoculated with a MERTK-negative ALL, suggesting immune-mediated therapeutic activity. In this context, MERTK inhibition led to significant decreases in expression of the coinhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages in the leukemia microenvironment. Furthermore, although T cells do not express MERTK, inhibition of MERTK indirectly decreased PD-1 expression on CD4+ and CD8+ T cells and decreased the incidence of splenic FOXP3+ Tregs at sites of leukemic infiltration, leading to increased T cell activation. These data demonstrate direct and immune-mediated therapeutic activities in response to MERTK inhibition in ALL models and provide validation of a translational agent targeting MERTK for modulation of tumor immunity.

Purpose:The prognosis of patients with multiple myeloma who are resistant to proteasome inhibitors, immunomodulatory drugs (IMiD), and daratumumab is extremely poor. Even B-cell maturation antigen–specific chimeric antigen receptor T-cell therapies provide only a temporary benefit before patients succumb to their disease. In this article, we interrogate the unique sensitivity of multiple myeloma cells to the alternative strategy of blocking protein translation with omacetaxine.Experimental Design:We determined protein translation levels (n = 17) and sensitivity to omacetaxine (n = 51) of primary multiple myeloma patient samples. Synergy was evaluated between omacetaxine and IMiDs in vitro, ex vivo, and in vivo. Underlying mechanism was investigated via proteomic analysis.Results:Almost universally, primary patient multiple myeloma cells exhibit >2.5-fold increased rates of protein translation compared with normal marrow cells. Ex vivo treatment with omacetaxine resulted in >50% reduction in viable multiple myeloma cells. In this cohort, high levels of translation serve as a biomarker for patient multiple myeloma cell sensitivity to omacetaxine. Unexpectedly, omacetaxine demonstrated synergy with IMiDs in multiple myeloma cell lines in vitro. In addition, in an IMiD-resistant relapsed patient sample, omacetaxine/IMiD combination treatment resensitized the multiple myeloma cells to the IMiD. Proteomic analysis found that the omacetaxine/IMiD combination treatment produced a double-hit on the IRF4/c-MYC pathway, which is critical to multiple myeloma survival.Conclusions:Overall, protein translation inhibitors represent a potential new drug class for myeloma treatment and provide a rationale for conducting clinical trials with omacetaxine alone and in combination with IMiDs for patients with relapsed/refractory multiple myeloma.

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction., Germline mutations in transcription factor ETV6 cause cytoplasmic localization of the HDAC3/NCOR2 complex, substantively altering the transcriptional signature of blood cells.

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.

Due to the disseminated nature of leukemia, malignant cells are exposed to many different tissue microenvironments, including a variety of extramedullary sites. In the present study, we demonstrate that leukemic cells residing in the liver display unique biological properties and also contribute to systemic changes that influence physiologic responses to chemotherapy. Specifically, the liver microenvironment induces metabolic adaptations via upregulating expression of endothelial lipase in leukemia cells, which not only stimulates tumor cell proliferation through polyunsaturated fatty acid–mediated pathways, but also promotes survival by stabilizing antiapoptotic proteins. Additionally, hepatic infiltration and tissue damage caused by malignant cells induces release of liver-derived enzymes capable of degrading chemotherapy drugs, an event that further protects leukemia cells from conventional therapies. Together, these studies demonstrate a unique role for liver in modulating the pathogenesis of leukemic disease and suggest that the hepatic microenvironment may protect leukemia cells from chemotherapeutic challenge. Significance: The studies presented herein demonstrate that the liver provides a microenvironment in which leukemia cells acquire unique metabolic properties. The adaptations that occur in the liver confer increased resistance to chemotherapy. Therefore, we propose that therapies designed to overcome liver-specific metabolic changes will yield improved outcomes for patients with leukemia. This article is highlighted in the In This Issue feature, p. 211

Current treatment options for relapsed acute myeloid leukemia (AML) are limited and ineffective for the majority of patients. In AML, primitive leukemia stem cells (LSCs) and pre-leukemic populations are able to maintain the disease and drive relapse. Thus, therapies targeting LSC populations may increase the overall survival of AML patients. In this study, we aim to identify the drivers favoring LSC expansion following treatment and relapse and develop potential therapies for AML. The transcriptome analyses of 12 pairs of functionally defined LSC fractions at diagnosis and relapse revealed significant changes of IL-1 signaling in AML patients. We demonstrated that the protein expression levels of interleukin-1 receptor type I (IL1R1) and its complex member interleukin-1 receptor accessory protein (IL1RAP) were both up-regulated in human leukemia stem and progenitor cells (LSPCs) at diagnosis or in relapse compared to normal hematopoietic stem and progenitor cells (HSPCs). Knockdown of IL1R1 and IL1RAP suppressed the clonogenicity and engraftment growth of primary human AML cells but showed low impacts on HSPCs in the normal bone marrow. Additionally, knockout of IL1R1 in leukemia MLL-AF9 mice significantly reduced the LSC frequency and prolonged the overall survival rate. To target IL-1/TLR signaling in LSCs, we performed iterative structure-activity relationship (SAR) guided medicinal chemistry, in silico modeling and leukemia cell line reporter assays to screen and identify a novel interleukin-1 receptor-associated kinase 1/4 (IRAK1/4) inhibitor (termed UR241-2). UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB following IL-1 stimulation. UR241-2 repressed LSPC function as assessed by colony-forming unit assays in primary human AML cells at diagnosis and in relapse while minimally impacting normal HSPC function. Taken together, our findings demonstrate the important role of IL-1/TLR signaling in supporting AML LSC expansion following treatment and relapse and suggest that targeting IL-1/TLR signaling using the novel IRAK1/4 inhibitor, UR241-2, can target LSC function to improve patient outcomes in AML. Disclosures No relevant conflicts of interest to declare.

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.

Background: The mechanisms that cause the progression of myelodysplastic syndrome (MDS) are poorly understood. Little is known about major signaling networks and energy metabolism in MDS cells as patients progress from low risk (LR) to high risk (HR) disease and from high risk to secondary acute myelogenous leukemia (sAML). As many as 30% of HR MDS patients progress to sAML and a portion of LR MDS patients progress to HR. The goal of this project is preventing progression by identifying MDS-specific targets for therapy. A deeper understanding of the metabolic properties of leukemia stem cells (LSCs) in AML has shown these cells are uniquely vulnerable to venetoclax and azacitidine (Ven/Aza) (Pollyea et al, Nat. Medicine, 2018) and metabolic changes cause resistance to Ven/Aza (Stevens et al, Nat. Cancer, 2021). Little however is known about the contribution of metabolism to the pathogenesis of MDS. The contributing factors to progression including metabolic properties, transcriptional programs, and immunophenotype are examined in this study. Methods: Bone marrow specimens from MDS patients at various disease stages, including serial samples during progression, were obtained. Single cell techniques including mass cytometry, antibody based single cell RNA sequencing (CITE-Seq) and transcriptional profiling with RNA sequencing were used to elucidate novel mechanisms of progression. Selective targeting of primitive MDS cells was tested using several agents. Results: Our previous work characterizing MDS stem cells (MDSC) showed significant similarities between MDSCs and AML LSCs (Stevens et al, Nat. Communications, 2018). However, little is known about lower risk disease. In order to understand transcriptional changes and their relationship to metabolism across pathogenesis, the transcriptome of blasts from patients with LR, intermediate (INT), or HR IPSS scores was investigated. The first major transcriptional difference identified was enrichment of glycolysis pathway at LR and INT stage. In contrast, HR MDS demonstrated enrichment of oxidative phosphorylation. Furthermore, comparison of intermediate to HR MDS showed increased RNA polymerase and Ribosome pathways at the HR stage. These changes demonstrate the progressive alteration of metabolic properties during MDS pathogenesis with cells first relying on mechanisms associated with normal stem cells (i.e. glycolysis) and later transitioning to a state associated with AML stem cells (i.e. reliance on oxidative phosphorylation). Using serial specimens of patients of who progressed from LR to HR MDS we performed CITE-Seq and mass cytometry. CITE-seq in serial specimens showed up-regulation of protein translation and oxidative phosphorylation in a subset of MDS stem and progenitor cells (CD34+ at transcript and antibody level) present at LR stage and conserved at HR stage (Fig 1A-C). MDSCs also acquired surface antigens including CD99 and CD52 upon progression from LR to HR. Analysis of the mass cytometry data showed significant overlap with CITE-Seq data including increased CD123+ and MCL1 expression in MDS stem cells upon progression. In order to understand therapeutic vulnerabilities as they relate to progression, we investigated ex vivo drug response in LR and HR specimens. MDS samples were challenged with two regimens, Ven/Aza, a regimen known to inhibit OXPHOS; and omacetaxine and azacitidine (Oma/Aza), which inhibits translation. CITE-seq showed that MDSC were selectively sensitive to these agents (Fig 1D). Importantly, addition of either drug regimen caused ablation of MDSC at LR and HR stages and these changes were most profound in cells with LSC properties. Based on preclinical findings, we are investigating MDS patients treated with Ven/Aza or Oma/Aza via CITE-seq and metabolomics for correlation of clinical response with properties of MDSC. Preliminary studies show that patients that respond to Oma/Aza present with a population of MDSC with transcriptional signatures of protein translation and LSCs (Fig. 1E). Studies are underway to investigate overlapping properties of ven/aza resistance in AML to resistance in MDS specifically investigating fatty acid metabolism in MDSC. Conclusions: Analysis of MDS patient bone marrow reveals acquisition of aberrant metabolic properties at both low and high risk stages of disease. These distinct aspects of MDSC biology create unique and targetable features. Figure 1 Figure 1. Disclosures Pollyea: Genentech: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Syndax: Consultancy; Takeda: Consultancy; Daiichi Sankyo: Consultancy; Celgene/BMS: Consultancy; Amgen: Consultancy; AbbVie: Consultancy, Research Funding; Agios: Consultancy; Glycomimetics: Other.

Genes encoding spliceosome components including SF3B1, U2AF1 , and SRSF2 , are frequently somatically mutated in myelodysplastic syndromes (MDS), other hematologic malignancies, and solid tumors.[1][1] Typically these are mutually exclusive, heterozygous, missense, hotspot mutations that result in

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.

Most cancers evolve over time as patients initially responsive to therapy acquire resistance to the same drugs at relapse. Cancer stem cells have been postulated to represent a therapy-refractory reservoir for relapse, but formal proof of this model is lacking. We prospectively characterized leukemia stem cell populations (LSCs) from a well-defined cohort of patients with acute myelogenous leukemia (AML) at diagnosis and relapse to assess the effect of the disease course on these critical populations. Leukemic samples were collected from patients with newly diagnosed AML before therapy and after relapse, and LSC frequency was assessed by limiting dilution analyses. LSC populations were identified using fluorescent-labeled cell sorting and transplantation into immunodeficient NOD/SCID/interleukin 2 receptor γ chain null mice. The surface antigen expression profiles of pretherapy and postrelapse LSCs were determined for published LSC markers. We demonstrate a 9- to 90-fold increase in LSC frequency between diagnosis and relapse. LSC activity at relapse was identified in populations of leukemic blasts that did not demonstrate this activity before treatment and relapse. In addition, we describe genetic instability and exceptional phenotypic changes that accompany the evolution of these new LSC populations. This study is the first to characterize the evolution of LSCs in vivo after chemotherapy, identifying a dramatic change in the physiology of primitive AML cells when the disease progresses. Taken together, these findings provide a new frame of reference by which to evaluate candidate AML therapies in which both disease control and the induction of more advanced forms of disease should be considered.

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.

Hematopoietic stem cell (HSC) quiescence supports lifelong blood regeneration and guards against pre-leukemic clonal expansion. Acute exposure to the pro-inflammatory cytokine interleukin (IL)-1 drives myeloid cell production and HSC cell cycle entry. However, HSC return to a quiescent state suggesting the presence of a ‘braking’ mechanism that limits HSC proliferative capacity during chronic inflammation. To identify mechanism(s) regulating HSC cell cycle activity, we injected mice with IL-1 for 20 days modeling chronic inflammation in vivo. RNA-seq analysis of HSC following IL-1 exposure revealed repression of cell cycle and protein synthesis genes, suggesting the activation of a ‘growth arrest’ gene program. This gene program coincided with increased PU.1 expression, and ChIP-seq analysis identified PU.1 binding on nearly all repressed genes, suggesting PU.1 enforces HSC quiescence during chronic inflammation. Strikingly, HSC from IL-1 treated PU.1-deficient mice exhibited loss of quiescence associated with aberrant myeloid expansion in the bone marrow and spleen. Together, our results suggest PU.1 induction maintains HSC quiescence under chronic inflammatory stress. They also suggest IL-1 may confer a competitive advantage to HSC with impaired PU.1 function, triggering aberrant proliferation and myeloid expansion. Several oncogenic mutations found in acute myelogenous leukemia (AML) impair the expression and/or function of PU.1. As AML pre-dominantly affects elderly individuals and leukemogenesis is often associated with chronic inflammation, our data supports a model where chronic inflammation triggers the selective expansion of HSC harboring oncogenic mutations, leading to a pre-leukemic state. Thus, blockade of inflammation may be a tractable approach to delay and/or prevent leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Leukemia stem cells (LSCs) drive the initiation and perpetuation of AML, are quantifiably associated with worse clinical outcomes, and often persist after conventional chemotherapy resulting in relapse1–5. In this report, we show that treatment of older patients with AML with the B cell lymphoma 2 (BCL-2) inhibitor venetoclax in combination with azacitidine results in deep and durable remissions and is superior to conventional treatments. We hypothesized that these promising clinical results were due to targeting LSCs. Analysis of LSCs from patients undergoing treatment with venetoclax + azacitidine showed disruption of the tricarboxylic acid (TCA) cycle manifested by decreased α-ketoglutarate and increased succinate levels, suggesting inhibition of electron transport chain complex II. In vitro modeling confirmed inhibition of complex II via reduced glutathionylation of succinate dehydrogenase. These metabolic perturbations suppress oxidative phosphorylation (OXPHOS), which efficiently and selectively targets LSCs. Our findings show for the first time that a therapeutic intervention can eradicate LSCs in patients with AML by disrupting the metabolic machinery driving energy metabolism, resulting in promising clinical activity in a patient population with historically poor outcomes. Targeting of mitochondrial metabolism in combination with BCL-2 inhibition eradicates leukemia stem cells and induces long-lasting responses in patients with acute myeloid leukemia.

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.

AMP-Activated protein kinase (AMPK) acts as a central metabolic sensor at the interface of metabolic and signaling networks. Activated AMPK promotes multiple catabolic processes to generate ATP, such as glucose uptake, glycolysis, fatty acid uptake and oxidation, and mitochondrial biogenesis. In addition, AMPK activation suppresses the cell cycle, and anabolic processes such as the mammalian Target of Rapamycin Complex 1 (mTORC1)-dependent protein synthesis and fatty acid biosynthesis via inactivating phosphorylation of cytosolic acetyl-CoA carboxylase 1 (ACC1) and mitochondrial ACC2. The activation of AMPK under conditions of energetic stress is modulated by an allosteric mechanism where the depletion of ATP promotes AMP binding at the γ subunit of AMPK with a subsequent conformational change in the catalytic α subunit. In the context of cancer AMPK has been reported to promote tumor survival and progression under hypoxia and the maintenance of cancer stem cells (CSCs). A major challenge to interrogating the therapeutic potential of targeting AMPK in cancer is the lack of potent and selective small molecule inhibitors. Compound C has been widely used as an AMPK inhibitor, but it lacks potency and has a poor selectivity profile. The multi-kinase inhibitor, sunitinib, has demonstrated potent nanomolar inhibition of AMPK activity, but has broad-spectrum activity across the kinome. Although sunitinib targets multiple kinases, its nanomolar potency for AMPK inhibition and large scope for chemical substitution on the core oxindole ring make it an attractive lead for AMPK inhibitor development. We have used a computational model of sunitinib docked into the ATP-binding site of the α subunit of AMPK to design and synthesize several series of oxindoles to examine structural modifications to improve AMPK inhibition and selectivity. These candidate inhibitors were evaluated against the activity of the α1 and α2 isoforms of AMPK using the TR-FRET assay and cell engagement was determined by ELISA, measuring p-ACC(Ser79), in the chronic myeloid leukemia K562 cell line. Our assays identified two novel, potent oxindole-based AMPK inhibitors that hadsignificantly reduced binding affinity when compared with sunitinib against members of the receptor tyrosine kinase (RTK) family, including VEGFR1, VEGFR2 and CSF1R, as well as BTK, a member of the B cell receptor (BCR) signaling pathway. Interestingly, cellular AMPK inhibition did not impact cell viability or result in cytotoxicity in K562 cells and may only sensitize these cells to chemotherapy. AMPK inhibition may be a more effective strategy to eliminate cancer cells in hypoxic microenvironments or against certain cell types, such as CSCs, that may be more reliant on AMPK for survival. Our current studies are designed to examine our AMPK inhibitors as single agents and in combination therapy in cancer cells grown under hypoxia and in CSC populations in order to eliminate these drug-resistant cancer cell populations with an aim to prevent cancer recurrence. In summary, we have developed two novel, potent AMPK inhibitors that were designed to interact with the DFG motif in the ATP-binding site of the α subunit of AMPK that are undergoing evaluation in advanced models of leukemia. Citation Format: Christopher J Matheson, Kimberly A Casalvieri, Donald S Backos, Craig T Jordan, Philip Reigan. Substituted oxindoles as AMP-activated protein kinase (AMPK) inhibitors and their evaluation in models of leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C078. doi:10.1158/1535-7163.TARG-19-C078

A rare but well-known association between plasma cell neoplasms and neutrophilia is known to exist. Whether the neutrophilia is secondary to the plasma cell neoplasm or this convergence represents two independent clonal disorders is unclear. We reviewed five consecutive cases from a single institution over a 3-year period, applying molecular, cytogenetic and cytokine-profiling approaches to determine whether neutrophilia associated with plasma cell neoplasms represents a reactive or clonal process. We report, for the first time, the occurrence of a SETBP1 mutation in two cases, as well as changes in G-CSF and IL-6 in SETBP1 wild type vs. mutated patients that are supportive of a hypothesis that neutrophilia associated with plasma cell neoplasms may sometimes be reactive and may sometimes represent a second clonal entity. Finally, using an ex vivo drug screening platform we report the potential efficacy of the multi-kinase inhibitor dasatinib in select patients.

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is an essential part of the cellular machinery that executes "programmed" or "regulated" necrosis. Here we show that programmed necrosis is activated in response to many chemotherapeutic agents and contributes to chemotherapy-induced cell death. However, we show that RIP3 expression is often silenced in cancer cells due to genomic methylation near its transcriptional start site, thus RIP3-dependent activation of MLKL and downstream programmed necrosis during chemotherapeutic death is largely repressed. Nevertheless, treatment with hypomethylating agents restores RIP3 expression, and thereby promotes sensitivity to chemotherapeutics in a RIP3-dependent manner. RIP3 expression is reduced in tumors compared to normal tissue in 85% of breast cancer patients, suggesting that RIP3 deficiency is positively selected during tumor growth/development. Since hypomethylating agents are reasonably well-tolerated in patients, we propose that RIP3-deficient cancer patients may benefit from receiving hypomethylating agents to induce RIP3 expression prior to treatment with conventional chemotherapeutics.

Purpose: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL+ leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph+ leukemia and AML upon TK inhibition. Experimental Design: Ph+ cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined. Results: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL+ leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo. Conclusions: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL+ and FLT3ITD leukemias. Clin Cancer Res; 21(6); 1360–72. ©2014 AACR.

Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

A series of novel, heteroaryl carboxylic acid conjugates of the sesquiterpene melampomagnolide-B (MMB, 3 ) has been evaluated as antitumor agents against an NCI panel of 64 human hematopoetic and solid tumor cell lines. The indole-3-acrylic acid conjugate 7j and the indole-3-carboxylic acid conjugate 7k were found to be the most potent analogs in the series. Compounds 7j and 7k exhibited remarkable growth inhibition, with GI 50 values in the range 0.03–0.30 μM and 0.04–0.28 μM, respectively, against the cell lines in the leukemia sub-panel, and GI 50 values of 0.05–0.40 μM and 0.04–0.61 μM, respectively, against 90% of the solid tumor cell lines in the NCI panel. Compound 7a was particularly effective against the sub-panel of breast cancer cell lines with GI 50 values in the range 7j , 7a and its water soluble analog 7p also exhibited potent anticancer activity against rat 9L-SF gliosarcoma cells in culture. Compound 7j was the most potent compound in the series in the M9-ENL1 AML cell assay with a lethal dose concentration EC 50 value of 720 nM, and exhibited the greatest cytotoxicity against a collection of primary AML stem cell specimens, which included a specimen that was unresponsive to PTL, affording EC 50 values in the range 0.33–1.0 μM in three out of four specimens. The results from this study provide further evidence that analogs of the sesquiterpene MMB can be designed to afford molecules with significantly improved anticancer activity. Thus, both 7j and 7k are considered potential lead molecules in the search for new anticancer agents that can be used as treatments for both hematopoetic and solid tumors.

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus, it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression–based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide, we identified AR-42 (OSU-HDAC42), a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate, but with improved activity at submicromolar concentrations. Here, we report that AR-42 induces NF-κB inhibition, disrupts the ability of Hsp90 to stabilize its oncogenic clients, and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide, the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2–driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials, we expect that our results can be extended to the clinic. Mol Cancer Ther; 13(8); 1979–90. ©2014 AACR.

Eradicating cancer stem-like cells (CSC) may be essential to fully eradicate cancer. Metabolic changes in CSC could hold a key to their targeting. Here, we report that the dietary micronutrient selenium can trigger apoptosis of CSC derived from chronic or acute myelogenous leukemias when administered at supraphysiologic but nontoxic doses. In leukemia CSC, selenium treatment activated ATM-p53–dependent apoptosis accompanied by increased intracellular levels of reactive oxygen species. Importantly, the same treatment did not trigger apoptosis in hematopoietic stem cells. Serial transplantation studies with BCR–ABL-expressing CSC revealed that the selenium status in mice was a key determinant of CSC survival. Selenium action relied upon the endogenous production of the cyclooxygenase-derived prostaglandins Δ12-PGJ2 and 15d-PGJ2. Accordingly, nonsteroidal anti-inflammatory drugs and NADPH oxidase inhibitors abrogated the ability of selenium to trigger apoptosis in leukemia CSC. Our results reveal how selenium-dependent modulation of arachidonic acid metabolism can be directed to trigger apoptosis of primary human and murine CSC in leukemia. Cancer Res; 74(14); 3890–901. ©2014 AACR.

Acute myeloid leukemia (AML) affects approximately 15,000 persons per year in the United States and is the sixth leading cause of cancer-related deaths. The treatment of AML has advanced little in the past thirty years, in part because of the biologic heterogeneity of the disease and the difficulty in targeting AML cells while sparing normal hematopoietic cells. Advances in preventing and treating AML are likely to occur once the cellular and molecular differences between leukemia and normal hematopoietic cells are better understood. Aldehyde dehydrogenase (ALDH) activity is highly expressed in hematopoietic stem cells (HSCs), while, in contrast, a subset of AMLs are lacking this activity. This difference may be relevant to the development of AML and may also provide a better avenue for treating this disease. In this review, we summarize what is known about the ALDHs in normal HSCs and AML and propose strategies for capitalizing on these differences in the treatment of acute leukemia, and possibly other cancers as well.

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

PTPN11 mutations occur in roughly 5-10% of AML patients. There is no defined impact on prognosis in the literature, although a single center analysis from our group suggested an association with poor outcomes. The pathogenesis of activating PTPN11 mutations is hypothesized to result in increased signaling through RAS resulting in uncontrolled cellular proliferation. PU-H71 is an epichaperome inhibitor that has shown pre-clinical promise in ex-vivo and PDX models of AML (Cell Reports 2015). The efficacy of the drug is dependent on the tumor possessing epichaperomes, hyperconnected networks of chaperones and co-chaperones facilitating tumor survival (Nature 2016, Nature Medicine 2018). Prior studies have found an association between epichaperome abundance and overactivity of the transcription factor MYC. Activating PTPN11 mutations have also been shown to result in MYC overactivity through increased SHP2 signaling (protein product of PTPN11 gene) and thus we hypothesized that through increased activity of MYC, activating PTPN11 mutations in AML lead to an abundant epichaperome network, and in turn render these tumors sensitive to the epichaperome inhibitor, PU-H71. 15 PTPN11 mutated AML peripheral blood and bone marrow samples from two academic medical centers (Weill-Cornell Medical Center and University of Colorado) were collected to be analyzed for epichaperome abundance and sensitivity to the epichaperome inhibitor PU-H71. To detect epichaperome abundance, PTPN11 mutated AML samples were incubated with FITC-bound PU-H71 (labeled F2) as well as a chemically altered FITC- bound PU-H71 "control" (labeled F9) which does not bind the epichaperome (Bioorg Med Chem Lett 2011). The AML cell line MV411 was used as a positive control (given known epichaperome abundance and sensitivity to PU-H71) and intra-sample T-cells were used as negative controls. These samples were analyzed using multi-parameter flow cytometry including markers to distinguish blasts, progenitors and lymphocytes. For analysis of sensitivity to PU-H71, samples were incubated with two different concentrations of PU-H71, 1uM and 0.5uM, for 48 hours and subsequently evaluated for viability using multi-parameter flow cytometry (including markers to distinguish blasts, progenitors and lymphocytes). Leukemic blasts and T cells were gated based on immunophenotype, and the ratio of blast F2 to F9 was combined with the F2-F9 blast to T-cell ratio to calculate epichaperome abundance, using a cut-off value of 4.5 to determine medium to high or low levels of the epichaperome (medium to high, >/=4.5, low, Of the 15 samples analyzed for epichaperome abundance. Seven out of 15 (46.7%) samples showed an abundant epichaperome. Nine of these samples had complete viability data, of which, five out of nine (55.5%) showed >60% cell death after 48 hour incubation with PU-H71. For these nine samples with data on sensitivity to PU-H71, the presence or absence of epichaperome abundance was predictive of ex vivo sensitivity (5 sensitive, 4 resistant). We have established PDX-mouse models to evaluate in vivo sensitivity to PU-H71, using epichaperome abundant samples. We have confirmed the epichaperome levels in AML blast cells from the peripheral blood of the animals. Currently the treatment and analysis is ongoing. These data suggest that PTPN11 mutated AML represents a subset of AML that could be particularly sensitive to epichaperome inhibition. Additionally, the findings from our studies suggest this assay has promise as a means to accurately predict sensitivity to PU-H71, a useful precision medicine tool for a difficult to treat disease. All samples are currently being analyzed for MYC transcriptional activity and other signaling pathways. Disclosures Chiosis: Samus Therapeutics: Equity Ownership, Patents & Royalties: Intellectual rights to the PU-FITC assay. Ritchie:agios: Other: Advisory board; Pfizer: Other: Advisory board, travel support; Celgene: Other: Advisory board; Celgene, Novartis: Other: travel support; Celgene, Incyte, Novartis, Pfizer: Consultancy; Ariad, Celgene, Incyte, Novartis: Speakers Bureau; AStella, Bristol-Myers Squibb, Novartis, NS Pharma, Pfizer: Research Funding; Tolero: Other: Advisory board; Genentech: Other: Advisory board; Jazz Pharmaceuticals: Research Funding. Desai:Astellas: Honoraria; Celgene: Consultancy; Cellerant: Consultancy; Astex: Research Funding; Sanofi: Consultancy. Lee:Ai Therapeutics: Research Funding; Karyopharm Therapeutics: Consultancy; AstraZeneca Pharmaceuticals: Consultancy; Roche Molecular Systems: Consultancy; Helsinn: Consultancy; Jazz Pharmaceuticals, Inc: Consultancy. Roboz:Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: 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; Actinium: 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; Trovagene: 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; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: 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; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: 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; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: 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; Bayer: 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. Guzman:Cellectis: Research Funding; SeqRx: Consultancy; Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay.

Introduction: Acute myeloid leukemia (AML) is an aggressive disease with a dismal prognosis. This is largely due to high relapse rates, which stem from our inability to eliminate leukemia stem cells (LSCs) with conventional chemotherapy. As we have gained a deeper understanding of the biology of LSCs, new targets against these chemo-resistant cells have surfaced. One such example is treatment using venetoclax/azacititine, a therapy that has significantly improved the outcome for these patients. Notwithstanding, some patients show resistance to all treatments, and developing a larger repertoire of agents that target LSCs remains an unmet need in this disease. One key vulnerability of LSCs is their dependence on oxidative phosphorylation (OXPHOS). Although signal transducer and activator of transcription 3 (STAT3) has been classically studied as a transcription factor that regulates self-renewal and proliferation, it has also been shown to play an essential role in OXPHOS via regulation of the electron transport chain (ETC). Given that STAT3 is commonly overexpressed in AML, and LSCs are dependent on OXPHOS, we hypothesized that STAT3 may be an effective target for eradication of LSCs. Methods: We have developed a novel small molecule inhibitor of STAT3, SF25. This compound, as well as genetic knockdown of STAT3, was employed to test the functional role of STAT3 in primary AML specimens. Flow cytometry, colony-forming potential, and engraftment of primary samples in PDX mouse models were performed to assess therapeutic efficacy. RNA-seq, seahorse assays, and metabolomics experiments were also performed to determine molecular mechanisms linked to targeting STAT3. Results: Our data shows that inhibition of STAT3 in primary AML samples leads to decreased cell viability, colony-formation and engraftment potential in xenograft models, while not affecting normal hematopoietic stem cells. This effect appears to be a result of mitochondrial dysfunction in LSCs, as seen by a significant decrease in oxygen consumption rate of STAT3 depleted cells. The mitochondrial dysfunction and reduction in OXPHOS is mediated by the downregulation of several mitochondrial and nuclear encoded genes that are important for oxidative phosphorylation, including several electron transport chain complex genes. Inhibition of STAT3 also affects glutaminolysis, as noted by metabolomics analysis of leukemia stem cells treated with STAT3 inhibitor. We suspect this effect is mediated by down-regulation of Myc upon STAT3 inhibition, which blocks glutamine conversion to glutamate, and leads to further decrease in TCA cycle intermediates. Conclusions: Acute myeloid leukemia is an aggressive disease, largely due to the presence of a chemo-resistant population of leukemia stem cells. LSCs highly depend on proper mitochondrial function and OXPHOS, a process that is partly regulated by STAT3 via multiple mechanisms. We propose that inhibition of STAT3 is therefore an effective way of eliminating this population, making this a promising new target in the treatment of AML. Disclosures No relevant conflicts of interest to declare.

The combination of venetoclax with hypomethylating agents has resulted in highly promising clinical outcomes for acute myeloid leukemia (AML) patients. However, a subset of patients are refractory or develop resistance to venetoclax based regimens, resulting in disease recurrence. The goal of this project was to determine a mechanism to re-sensitize resistant leukemia stem cells (LSCs) to venetoclax with azacitidine (ven/aza) treatment. LSCs are the population of leukemia cells that initiate disease and are not fully eradicated by conventional treatments resulting in disease recurrence. We have previously reported that ven/aza targets LSCs in de novo AML patients by perturbing amino acid uptake resulting in decreased oxidative phosphorylation (OXPHOS). To investigate how some AML patients, develop resistance to ven/aza, we first determined if ven/aza reduced amino acid uptake in primary human AML ven/aza resistant LSCs by stable isotope labeled metabolic flux and mass spectroscopy analysis. Amino acid uptake was significantly reduced in both ven/aza sensitive and resistant LSCs upon ven/aza treatment, indicating that ven/aza is still biologically active in resistant LSCs. Next, we performed gene expression analysis from LSCs isolated from AML patients who were treated with ven/aza, responded, and then either remained in remission or progressed on ven/aza therapy. Gene set enrichment analysis revealed that fatty acid transport was enriched in LSCs isolated from patients who eventually progressed on ven/aza therapy (FDR = 0.0088) (Figure A). We then determined differences in overall fatty acid levels by lipidomics mass spectroscopy analysis in ven/aza sensitive and resistant LSCs. We observed a significant increase in abundance of 20% (6/29) of fatty acids detected in resistant LSCs. To determine if targeting fatty acid transport could re-sensitize resistant LSCs to ven/aza we knocked down genes involved in fatty acid transport including CD36, CPT1A and CPT1C in 4 ven/aza resistant AML specimens and then measured viability and colony-forming potential upon ven/aza treatment (Figure B and C). Knockdown of CD36, CPT1A, or CPT1C in combination with ven/aza treatment significantly decreased both viability and colony forming ability in each of the AML specimens. In addition, knockdown of CPT1A or CPT1C in combination with ven/aza reduced OXPHOS, a known metabolic requirement of LSCs. To perturb fatty acid transport in a therapeutically relevant manner, we treated LSCs isolated from ven/aza resistant patient specimens with a CPT1 inhibitor, etomoxir, as a single agent or in combination with ven/aza, and then measured viability and OXPHOS. The combination but not single agents reduced viability and OXPHOS, consistent with our genetic studies. To determine if ven/aza with etomoxir targeted functional LSCs we treated a primary AML specimen with etomoxir, ven/aza or the combination and measured engraftment into immune deficient mice. Combination therapy significantly reduced engraftment potential compared to ven/aza or etomoxir alone indicating that the combination of ven/aza with etomoxir decreased LSC function (Figure D). To determine if ven/aza with etomoxir could target AML cells in vivo, we treated a primary patient derived xenograft model with ven/aza, etomoxir, or the combination for 2 weeks and measured leukemic burden in the bone marrow (Figure E). The combination reduced leukemic burden more significantly than ven/aza or etomoxir alone. Finally, we measured the consequences of ven/aza, etomoxir, or the combination on normal hematopoietic stem and progenitor cells. Neither single agents nor combination therapy decreased CD34+ cell viability or colony forming ability, indicating that there may be a therapeutic window to targeting these metabolic pathways in AML without harming normal stem cells. Gene expression analysis revealed that CD36, CPT1A, and CPT1C are expressed at significantly higher levels in AML compared to HSCs, which may contribute to this therapeutic window. In conclusion, these data indicate that ven/aza resistance can be overcome by targeting fatty acid transport in LSCs. Furthermore, combining ven/aza with a CPT1 inhibitor such as etomoxir may be a clinically relevant strategy to overcoming ven/aza resistance. Figure Disclosures Pollyea: Gilead: 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; 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; Celgene: 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; Forty-Seven: 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; Takeda: 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.

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.

Myelodysplastic syndrome (MDS) is a group of heterogeneous disorders caused by ineffective hematopoiesis and characterized by bone marrow dysplasia and cytopenia. Current treatment options for MDS are limited to supportive care, hypomethylating agents, and stem cell transplant. Most patients eventually succumb to the disease or progress to leukemia. Previously, we found that CD123 can be used to delineate MDS stem cells in patients at high risk for MDS and that the CD123-positive population is biologically distinct from normal hematopoietic stem cells. Furthermore, selective targeting of MDS stem cells can dramatically reduce tumor burden in preclinical models. On the basis of these findings, we propose CD123 as a candidate target for chimeric antigen receptor (CAR) T-cell therapy in high-risk MDS patients. To test this concept, we employed a CAR lentiviral vector containing a CD123-specific single-chain variable fragment in combination with the CD28 costimulatory domain, CD3ζ signaling domain, and truncated estimated glomerular filtration rate. Utilizing this system, we illustrate that CD123 CAR can be expressed on both healthy donor and MDS patient-derived T lymphocytes with high efficiency, leading to the successful elimination of MDS stem cells both in vitro and in patient-derived xenografts. These results provide the concept for the use of CD123-targeted CAR T cells as a therapeutic option for patients with MDS.

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

A phase I study utilizing decitabine (DAC) followed by the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, in patients with relapsed/refractory adult AML was undertaken to assess safety and feasibility. Patients received DAC 20mg/m(2) intravenously daily for 5 days followed by rapamycin from day 6 to day 25 at doses of 2 mg, 4 mg, and 6 mg/day in a standard 3+3 dose escalation design. Twelve patients completed treatment for safety evaluation. Maximum tolerated dose (MTD) was not reached, and except for grade 3 mucositis in 4 patients, no other significant unexpected non-hematologic toxicities have occurred indicating safety of this regimen. This trial is registered at clinical trials.gov as NCT00861874.

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

SummaryWe used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.

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.

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.