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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.

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.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress., Journal of Experimental Medicine, 218 (6), ISSN:0022-1007, ISSN:1540-0069, ISSN:1540-9538

Contains fulltext : 229479.pdf (Publisher’s version ) (Open Access) Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.

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.

Background: Venetoclax-based therapy regimens are now FDA-approved for treatment of acute myeloid leukemia (AML) in older patients or those unfit to tolerate intensive chemotherapy (IC). Remission rates are high at 60-70% but relapses do frequently occur. Outcomes for newly-diagnosed patients who receive venetoclax-based therapies and proceed to a potentially curative allogeneic stem cell transplant (SCT) have largely been unreported. In the current study we compare outcomes of patients who received SCT following either IC or venetoclax + azacitidine (ven/aza) at the University of Colorado Hospital. Methods: Patients 18 years or older who received SCT in first remission of AML between 2010-2020 were included in the analysis. Patients were stratified into the IC arm if they initially received a backbone of cytarabine and an anthracycline; some patients in this cohort received IC in combination with other targeted agents. Patients who received ven/aza as first-line therapy followed by SCT were grouped in a separate cohort. Demographic and clinical information - including flow cytometry-based (MCF) MRD - was extracted from the electronic medical record. Comparisons of demographic and clinical variables between IC and ven/aza groups were made with t-test, Chi-squared, or Fisher's exact test depending on the nature of the variable. Relapse-free (RFS) and overall (OS) survival were calculated from the day of SCT to the respective endpoint or last documented follow-up using log-rank statistics. Finally, a Cox proportional hazards model was used to assess the interplay between variables pre- and post-SCT. P-values Results: We identified 179 patients who received SCT for AML in first remission. Of these patients, 151 received IC and 28 received ven/aza prior to SCT. Patients in the ven/aza group had higher median age than those in the IC group, as well as a higher proportion with adverse ELN genetic risk scoring. Patients in the ven/aza group received less intensive conditioning regimens. Sex, rates of MCF MRD negativity pre-BMT, incidence of severe acute or chronic GVHD, and causes of death were not significantly different between the two groups. There was no difference between the two groups in post-transplant RFS or OS (Figure 1). In a multivariate Cox model of pre-transplant variables predicting OS, the only factor that achieved significance was pre-SCT MCF MRD; the induction regimen was not a multivariate factor. Negative MCF MRD going into SCT was associated with decreased likelihood of relapse, GVHD, and death, respectively. Conclusions: In our cohort of AML patients receiving SCT, we found that ven/aza as a pre-transplant therapy yielded equivalent post-transplant outcomes compared to IC, in a population of older age and with higher ELN genetic risk. MCF MRD pre-SCT was confirmed as a key prognostic factor for post-SCT outcome. These findings support ongoing use of ven/aza as a first line therapy for elderly patients with AML as well as its exploration as a candidate therapy for younger patients. Figure 1 Figure 1. Disclosures Pollyea: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other; Aprea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kiadis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Syndax: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Agios: Other, Research Funding; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva: Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Curis, Servier: Other; Pfizer: Research Funding; Syros: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Other: advisory board; Foghorn: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Other: advisory board; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: advisory board.

Background Myelodysplastic syndrome (MDS) arises from and is perpetuated by malignant stem cells. Curative therapy requires eradication of this population, but this is not achieved with currently available medical therapies. MDS stem cells possess unique molecular properties that can be therapeutically exploited, but in general these strategies have not been explored clinically. Our recent in vitro studies showed that combination treatment with the protein synthesis inhibitor omacetaxine (oma) and the hypomethylating agent (HMA) azacitidine (aza) efficiently targeted MDS stem cells with minimal impact on normal stem cells. Based on these findings we conducted a Phase 1 clinical trial for MDS patients with concomitant oma + aza (NCT03564873). Methods Patients were eligible if they had newly diagnosed MDS with ≥5% blasts. Aza was given at 75 mg/m 2 IV days 1-7 of a 28-day cycle; oma was given subcutaneously twice daily on days 1-7 of a 28-day cycle with 4 dose cohorts (0.75, 1.0 and 1.25 mg/m 2, and a de-escalation cohort of 0.5 mg/m 2). Dose escalation was based on a 3+3 design. Bone marrow biopsies occurred at baseline and day 8 and 28 of cycle 1, and at the conclusion of odd-numbered cycles. The primary endpoint was the maximum tolerated dose (MTD) of oma with aza. Responses were assessed according to 2006 IWG criteria with hematologic improvement (HI). Using pre- and post-treatment patient samples, advanced single cell techniques including mass cytometry and antibody-based single-cell RNA sequencing (CITE-Seq) were used to elucidate mechanisms of sensitivity and resistance. High sensitivity DNA sequencing techniques were used to understand clonal evolution and detect measurable residual disease (MRD). Results Nine patients were required to complete phase 1. See Table 1 for baseline characteristics. Two patients in cohort 1 experienced dose limiting toxicity (DLT) (grade 3 hypoxia and grade 3 respiratory failure). De-escalation to cohort 0 resulted in one DLT event (grade 4 GI bleed) among the six patients treated in this cohort. The MTD was established as 0.5 mg/m 2 oma days 1-7. Adverse events are detailed in Table 2. Median number of cycles was 2 (1-3). 8/9 patients achieved a marrow CR; 5 had HI. 6 patients proceeded to transplantation and none have relapsed with median time from transplant of 772 (183-1158) days. Median response duration is 587 days (29-938). 5 remain alive. MRD negativity, as measured with droplet digital PCR (sensitivity up to 0.1%), was achieved in 2 out of 4 patients measured. Both MRD negative patients had complete clearance of mutated splice factor genes; we previously showed no ability to clear these mutations (0/13 patients) using venetoclax+aza in AML (Nature Medicine (2018) 24:1859-1866). Baseline analysis of patient specimens by single cell transcriptomics demonstrates a distinct population of MDS stem cells (Figure 1A). This subset has overlapping transcriptional properties with known AML stem cell profiles as highlighted by increased geneset expression. Within the stem cell compartment, we observed up-regulation of protein synthesis pathways (Figure 1B) (e.g. KEGG ribosome pathway in MDS stem cells), indicating that oma/aza treatment may specifically target the malignant stem cell population. Subsequent studies are investigating the use of CITEseq as a means to evaluate drug response, which will provide very high resolution analyses of transcriptional signatures in MDS patients. By specifically monitoring eradication of MDS stem cells during the course of treatment, it should be possible to predict therapeutic efficacy and response duration while elucidating genes that correspond to oma/aza sensitivity and resistance. Conclusions Phase 2 of this study is ongoing; 23 newly-diagnosed high risk MDS patients will be enrolled with a primary endpoint to determine the overall response rate. In addition a ten-patient HMA failure expansion cohort will be enrolled to determine preliminary toxicity and efficacy assessments. Correlative studies will seek to determine the impact of this regimen on the MDS stem cell population and the mechanism of this impact. Figure 1 Figure 1. Disclosures McMahon: Takeda: Membership on an entity's Board of Directors or advisory committees. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding. Pollyea: Foghorn: Honoraria; Gilead: Consultancy, Honoraria; Celgene: Honoraria; Bristol Myers Squibb: Honoraria; Genentech: Consultancy, Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Amgen: Honoraria; Astellas: Honoraria; Aprea: Honoraria; Karyopharm: Consultancy, Honoraria; Kiadis: Honoraria; Novartis: Consultancy, Honoraria; Syndax: Honoraria; Syros: Consultancy, Honoraria; Takeda: Honoraria; Teva: Research Funding. OffLabel Disclosure: Omacetaxine for MDS

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.

For more than 50 years, investigators have considered a malignant stem cell as the potential origin of and a key therapeutic target for acute myeloid leukemia (AML) and other forms of cancer.1-4 The nature and existence of tumor-initiating cells for leukemia and other malignancies have long been the subject of intense and rigorous study; indeed, the promise of the potential to eradicate such cells is clear. However, until recently, deficiencies in our understanding of the nature of these cell populations, coupled with a limited ability to therapeutically exploit their weaknesses, have been limiting factors in realizing the goal of targeting leukemic stem cells (LSCs). Exciting new insights into the fundamental underpinnings of LSCs are now being made in an era in which drug development pipelines offer the potential to specifically target pathways of significance. Therefore, the focus in this new era, characterized by the confluence of understanding LSCs and the ability to target them, is shifting from “if it can be done” to “how it will be done.” Moving from a theoretical stage to this hopeful era of possibilities, new challenges expectedly arise, and our focus now must shift to determining the best strategy by which to target LSCs, with their well-documented heterogeneity and readily evident intra- and interpatient variability. The purpose of this review is therefore both to summarize the key scientific findings pertinent to AML LSC targeting and to consider methods of clinical evaluation that will be most effective for identifying successful LSC-directed therapies.

Relapse is the most common cause of mortality in patients undergoing allogeneic stem cell transplantation (ASCT) for acute myeloid leukemia (AML). The presence of measurable residual disease (MRD) at the time of transplant is associated with very high relapse rates, and novel strategies to address relapse risk are needed for these patients. Venetoclax has demonstrated single agent activity in AML, and venetoclax combined with azacitidine, decitabine, or low dose cytarabine is FDA approved to treat newly diagnosed patients >75yrs or older and those unfit for induction. Given the ability of venetoclax to target leukemic stem cells combined with its favorable toxicity profile and ease of administration, we have initiated off label post-transplant venetoclax maintenance for AML patients with MRD at the time of transplant. We report outcomes of our experience to date. Venetoclax therapy is planned to be initiated approximately 40-80 days post-ASCT upon count recovery (ANC>1000 109/L and platelets>100 109/L) and recovery from early ASCT toxicity. Dose is initiated at 100 mg daily for one week and titrated up by 100 mg weekly to a final maintenance dose of 400 mg daily. Therapy is planned until one year post-ASCT. Since February 2019, we have administered venetoclax to 23 post-ASCT patients (22 AML (6 with prior diagnosis of MDS) and 1 MDS), median age 65 (range 19-73). Venetoclax therapy was initiated beginning median 67 days (range 36-146) after ASCT. 16 patients underwent transplant following one line of therapy (twelve of whom received venetoclax azacitadine), 5 patients received 2 lines of therapy, and 2 patients received 3 lines of therapy. Three patients were second transplants. Donor sources were cord (n=14), haplo cord (n=5), and matched related donor (n=4). 7 patients underwent myeloablative, 14 reduced-intensity, and 2 non-myeloablative conditioning. All patients had MRD at time of transplant (15 by cytogenetics/FISH, 10 by flow cytometry, and 6 by digital droplet PCR). At time of transplant, 1 patient was aplastic, 6 were in morphologic leukemia free state (MLFS), one was in complete remission with incomplete count recovery (CRi), 15 were in complete remission (CR), and 1 had MDS with ringed sideroblasts with multilineage dysplasia. Patient data is summarized in Table 1. Median follow up among survivors is 219 days (range 92-439). Details of individual patients' courses on venetoclax are summarized in Figure 1. In total, 11 patients were still on venetoclax at the time of this analysis, and 4 had completed the planned 1 year of treatment. 3/23 (11%) had the drug permanently discontinued due to potential adverse effects in the setting of additional post-ASCT complications. Four patients relapsed 191, 288, 325, and 367 days post-transplant and all died, and 3 patients experienced transplant related mortality 146, 164, and 307 days post-ASCT. 6 month overall survival (OS) and relapse free survival (RFS) were both 87%. Venetoclax was temporarily held or dose-reduced in 11/23 (47.8%) patients due to adverse events (AEs). 14/23 (61%) patients experienced at least one AE that could be potentially attributed to venetoclax. Most common AEs were cytopenias (7/23, 30%) and diarrhea (7/23, 30%). 12 patients experienced grade 2-4 acute GVHD (3 grade 3). 10 of these patients developed GVHD symptoms prior to starting venetoclax. 5/7 patients who held venetoclax due to diarrhea had acute GVHD prior to initiation of ventoclax, and whether the diarrhea was GVHD or venetoclax related was unclear. Three patients have developed chronic GVHD (2 mild and 1 severe). Our preliminary data suggests venetoclax is tolerable in the post-ASCT maintenance setting without unexpected side effects. In this small cohort, GVHD rates are comparable to historical controls. Ongoing follow-up will continue to examine the safety and efficacy of this approach. Disclosures Pollyea: Novartis: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Agios: Consultancy; 47: Consultancy, Research Funding; Genentech: Consultancy; Glycomimetics: Other; Celgene/BMS: Consultancy; Syndax: Consultancy; Syros: Consultancy; Abbvie: Consultancy, Research Funding; Daiichi Sankyo: Consultancy. OffLabel Disclosure: off label venetoclax post-transplant maintenance

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

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.

Venetoclax and azacitidine (ven/aza) is our institutional preference for initial treatment of non-core binding factor AML patients age ≥60. Given potential durability of response in these patients, the role of transplant (SCT) in patients who achieve response to ven/aza is uncertain. To examine the potential contribution of SCT, we identified 119 patients age ≥60 who received ven/aza as initial AML therapy at our center between 1/2015 and 1/2020. We then compared outcomes for these patients based on whether they were deemed eligible or ineligible for SCT; for those deemed eligible, we compared outcomes between patients who received SCT and those who did not due to personal preference. SCT consults were generally conducted within 2 cycles of initiation of ven/aza but were delayed in some patients achieving CR until evidence of persistent or recurrent measurable residual disease (MRD). Among the 119 patients, 61 (median age 76 (61-90)) were not SCT candidates due to age or comorbidities (n=44) or early death due to primary refractory disease/acute toxicity (n=17). 60 patients were potential SCT candidates. 21 patients median age 65 (60-73) underwent SCT. 2 of these patients had MDS prior to developing AML and underwent SCT consult before developing AML and initiating ven/aza. For the remaining 19 patients, consult occurred median of 50 days (20-287) from diagnosis. 2 patients relapsed after initiating ven/aza prior to transplant, underwent induction, and then went on to SCT. SCT occurred median 176 (69-643) days from diagnosis and median 133 (49-615) days from SCT consult. 37 patients median age 72 (64-78) had SCT consults but did not undergo SCT. 5 of these patients had refractory disease and never achieved SCT eligibility, one patient was deemed not an SCT candidate after consult due to social issues, and one patient was lost to follow up. The remaining 30 patients median age 72 (64-78) achieved adequate response for SCT but elected to defer. 2 of these 30 patients had MDS prior to developing AML and underwent SCT consult before developing AML and initiating ven/aza. Among the 28 patients who had SCT consults after initiating ven/aza and deferred SCT, SCT consult occurred median of 64 days (14-222) from diagnosis. Details of ELN risk status and disease status at time of SCT consult, time of SCT, and best disease response for non-SCT patients are provided in Table 1. MRD positivity included disease detected by cytogenetics, FISH, flow cytometry, or digital droplet PCR (ddPCR). Donor sources were cord (n=11), haplocord (n=3), and matched related donor (n=7). Conditioning regimens included 7 non-myeloablative, 12 reduced-intensity, and 2 myeloablative. Overall survival (OS) was significantly greater among SCT patients (median survival not reached) versus patients undergoing SCT consult and deferring SCT (median 588 days) (p=0.01) versus patients not considered SCT candidates due to age or comorbidities (median 291 days) (p= For SCT eligible patients, SCT appears to improve outcomes among patients who respond to ven/aza compared to patients who defer SCT. Preliminary data suggests that outcomes may be improved for patients going to transplant with MRD following initial therapy with ven/aza as compared to traditional chemotherapy induction. Ongoing data to further refine the significance of MRD, molecular and cytogenetic prognostic factors, and optimal timing of SCT, as well as standardized tools to assess MRD, are necessary. Disclosures Pollyea: Syndax: Consultancy; Glycomimetics: Other; Syros: Consultancy; Abbvie: Consultancy, Research Funding; Karyopharm: Consultancy; Takeda: Consultancy; Daiichi Sankyo: Consultancy; Agios: Consultancy; Celgene/BMS: Consultancy; Pfizer: Consultancy; 47: Consultancy, Research Funding; Janssen: Consultancy; Amgen: Consultancy; Genentech: Consultancy; Novartis: Consultancy.

Background: Venetoclax (ven)+azacitidine (aza) is the standard of care for untreated acute myeloid leukemia (AML) patients (pts) ≥75 years or unfit for intensive induction chemotherapy (IC) due to comorbidities. While the standard of care for younger newly diagnosed pts is IC, long term outcomes for all but favorable risk pts are suboptimal. Specifically, pts with adverse risk disease, per the European Leukemia Network (ELN), are more likely to be refractory to IC and have low rates of long term survival. Ven based regimens have a ~70% response rate in the upfront setting; in contrast to IC, those with adverse risk do not have lower response rates. This study explores the feasibility and outcomes of ven+aza for newly diagnosed younger AML pts who are not necessarily unfit for IC, but carry adverse risk features that make them less likely to respond to this therapy. Methods: This is an interim analysis of an ongoing phase 2 study (NCT03573024) of a planned 36 untreated adverse risk AML pts 18-59. Pts have adequate organ function and white blood cell counts Results: 8 have enrolled between November 2018 and July 2020; median age is 33 (22-52). All have adverse risk disease (Table 1). There were 179 adverse events (AEs); 35 related. The ≥grade 3 events were: fatigue (N=1), leukopenia (N=2), neutropenia (N=2), anemia (N=2), thrombocytopenia (N=1). There were no deaths in the first 60 days. In cycle 1 pts spent a median 10 days (4-19) inpatient and required a median of 2 (0-10) red blood cell transfusions and 3 (0-15) platelet transfusions. Six of 8 (75%) pts responded; all 6 were CRs. 4/6 responses were cytogenetic remissions, 5/6 were MRD negative by MFC and 1/6 was MRD negative by ddPCR. Two pts relapsed, 35 and 84 days after remission; one was successfully salvaged with IC and proceeded to transplant while the other was refractory to IC and died from AML. Two pts were refractory to ven+aza; both were successfully salvaged with IC and have proceeded to transplant. 7/8 proceeded to transplant; 4 required only ven+aza for this outcome and proceeded to transplant a median of 89 days (56-93) after diagnosis. No pts who have proceeded to transplant have relapsed, a median of 340 days (136-444) after transplant. 7/8 remain alive, 345 days (91-586) after enrolling. Conclusions: At the stage 1 interim analysis, the CR rate for ven+aza remains >60% in adverse risk younger patients. With small numbers, up-front treatment with ven+aza allows some younger pts with adverse risk disease a non IC opportunity to achieve a response and proceed to transplant; most of those who relapsed or were refractory were salvaged with IC. Disclosures Pollyea: Syndax: Consultancy; Syros: Consultancy; Abbvie: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; Karyopharm: Consultancy; Novartis: Consultancy; Genentech: Consultancy; Amgen: Consultancy; 47: Consultancy, Research Funding; Janssen: Consultancy; Agios: Consultancy; Glycomimetics: Other; Celgene/BMS: Consultancy; Pfizer: Consultancy; Takeda: Consultancy. OffLabel Disclosure: Venetoclax for younger patients fit for induction chemotherapy

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.

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.

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: The prognosis of patients with multiple myeloma (MM) who are resistant to proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) is extremely poor. Even daratumumab and BCMA CAR-T cell therapies appear to provide only temporary benefit before patients eventually succumb to their disease. We show that protein translation is upregulated in MM cells. We also demonstrate that translational inhibition with omacetaxine (Oma) is specifically cytotoxic in a manner that is preserved in MM cells from PI/IMiD resistant patients. Furthermore, protein translation inhibition is synergistic with IMiDs, even in IMiD refractory patients. We hypothesize these findings stem from the origin of MM cells as professional antibody secretory cells, with highly active protein translation fundamental to their biology. Here, we show these findings and provide support to test the translation inhibitor omacetaxine with a clinical trial in multiple myeloma patients. Methods: Primary MM cells in mononuclear cell (MNC) cultures were assayed ex vivo for Oma effect at 48-hrs by high-throughput flow cytometry using our myeloma drug sensitivity testing (My-DST) platform. Protein synthesis rates were measured via flow cytometry using the Cayman OP-puromycin assay. Initial drug combination matrices were performed in MM cell lines via MTS assay after 96-hr incubation. Cell line xenograft study of the Oma and IMiD combination in vivo were performed using an orthometastatic MM model with MM1.S luciferase-expressing cells injected IV in NSG mice. Vehicle control, Oma, pomalidomide (Pom) and the combination were dosed 5 days/week by IP injection beginning on day 30. Disease burden was monitored with imaging of luciferase bioluminescence. Results: Oma had specific cytotoxicity across 5 MM cell lines (EC50 = 15-35 nM, data not shown). Oma also showed MM cell specific cytotoxicity in patient samples with EC50 ranging from 25-100 nM (Fig 1A). Ex vivo treatment with 50 nM Oma for 48 hrs caused >50% reduction in viable MM cells in 39/50 (78%) patient samples. Importantly, 50 nM Oma retained activity in PI/IMiD-refractory patients compared to sensitive patients (64.2% vs 71.6% mean decrease in MM cell viability, respectively, P = 0.44, Fig 1B). Primary MM cells exhibited significantly higher levels of baseline protein translation than other bone marrow MNCs with mean 3.9-fold increase (n = 14, P = Conclusion: Protein translation inhibitors represent a potential new drug class for clinical myeloma treatment. We have shown that the protein synthesis inhibitor omacetaxine possesses a potent and specific killing effect against MM cells. Importantly, this anti-myeloma cytotoxicity was equally active in biopsy samples obtained from patients at diagnosis and patients with disease advanced to the late stage of PI/IMiD resistance. The pretreatment level of MM cell protein translation may serve as a biomarker for clinical response. In further exploration of the optimized treatment approach for clinical trials, we found synergy between omacetaxine and pomalidomide in vitro, in vivo and using patient samples ex vivo. A phase I clinical trial of omacetaxine single agent and in combination with pomalidomide is currently in development at the University of Colorado. Disclosures Mark: Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Celgene: Honoraria.

Background: Venetoclax (ven) was approved for older untreated acute myeloid leukemia (AML) patients due to high response rates and durable remissions. As a participating site in the dose escalation study, we observed deeper/more durable responses in some who received >400mg ven. We also noted 16/33 discontinued azacitidine (aza) after achieving a response; 9 relapsed and 7 remained in long term remission on ven only. Based on these observations, we designed a study that hypothesized: A)Higher initial doses of ven would allow deeper/more durable responses, and B)Multi modality high sensitivity measurable residual disease (MRD) testing could identify patients able to discontinue aza and remain on maintenance ven. Methods: This is an ongoing phase 2 study (NCT03466294) of 42 untreated AML patients ≥60 who decline/are ineligible for induction. Patients have adequate organ function and white blood cell counts Results: 30 patients enrolled between May 2018 and July 2019; median age is 71 (60-88), 10% evolved from MDS and 10% and 73% had intermediate and unfavorable risk disease by ELN, respectively (Table 1). 732 adverse events (AEs) occurred; 46 (6%) were serious, the most common were neutropenic fever (37%) and pneumonia (13%). The most common >grade 2 related AEs were leukopenia (53%), thrombocytopenia (44%) and neutropenia (35%); there were no related grade 5 AEs. The overall response rate was 70% (21/30; CR=19, MLFS=2). Median number of cycles to achieve best response was 1. Significant blast reductions were seen on day 8; of the 28 with interpretable day 8 BMBXs, 10 achieved MLFS on day 8. 4 completed ≥1 cycle and were refractory. An additional 4 did not complete cycle 1: 1 died of disease and 3 elected to come off therapy (all subsequently died of disease). Four (19%) responders relapsed, after a median 180 days (27-279). With median follow up of 214 days, median response duration has not been reached. 10 patients died, after a median 65 days (29-256); 1/30 died within 30 days. Median overall survival has not been reached. Of the 26 who completed ≥1 cycle, 19 were MRD- by MPFC, including 18/19 who achieved CR. Of these 26, 3 were not monitored by ddPCR: for 2 patients this was due to the absence of detectable baseline mutations and for 1 patient it was due to refractory disease. The remaining 23 had ddPCR monitoring; 3 became MRD- by this modality (Fig 2). All 3 were also MRD- by MPFC and per protocol discontinued aza and initiated ven maintenance (Fig 1). MRD negativity by both parameters occurred after cycles 1, 2 and 3, respectively. One MRD- patient relapsed after 216 days; two remain in remission after 301 and 124 days. An additional 4 who achieved MRD+ responses discontinued aza at their insistence (and in violation of the protocol); 1 relapsed after 279 days, and 3 remain in ongoing remission. Univariate predictors of refractory disease were FAB M0/M1 (OR 0.070, p=0.02) and RAS pathway mutations (OR 14.25, p=0.02). Conclusions: Higher initial doses of ven are tolerated in this population. Blast reduction occurs quickly in many patients (day 8), for this low intensity regimen. Response rates are consistent with lower doses of ven. Very deep responses, as measured by highly sensitive MRD methods (MPFC and ddPCR are capable of sensitivity up to 0.02%), are attainable. Longer follow up time will determine if higher ven doses and MRD-driven decisions related to continuation of aza result in more durable responses. Increased maturation of blasts and RAS pathway mutations are predictors for refractory disease. Disclosures Lyle: Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo Incyte: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Pollyea:Agios: 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; Astellas: 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; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celyad: 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; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forty-Seven: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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.

Hematopoietic stem cell (HSC) quiescence is crucial for maintaining lifelong blood production and preventing potentially toxic overproduction of blood cells. HSC are capable of re-entering quiescence following exposure to pro-inflammatory stimuli such as interleukin (IL)-1, implying the existence of one or more 'braking' mechanisms that limit and/or overcome the mitogenic properties of these signals to limit HSC cell cycle entry. However, mechanism(s) regulating HSC quiescence during chronic inflammation have yet to be fully elucidated. In the present study, we find that the master myeloid transcription factor PU.1 represses Myc-regulated cell cycle and protein synthesis pathways in HSC during chronic inflammation, thereby guarding HSC quiescence in this context. To gain insight into HSC cell cycle regulation in the context of chronic inflammatory signaling, we performed cell cycle and RNA-seq analysis on purified HSC from mice injected daily for 20 days with IL-1β. Strikingly, HSC from IL-1-treated mice remained in a quiescent state, and our RNA-seq analyses uncovered a significant decrease in mRNA transcripts from pathways related to cellular proliferation, translation, and ribosome biogenesis. Also, flow cytometry analyses revealed that IL-1 significantly reduced Myc protein expression and ribosomal protein S6 phosphorylation in HSC. Altogether, these data identify an inflammation-induced cell cycle restriction gene program that limits HSC proliferation in response to inflammation. Strikingly, ingenuity pathway analysis (IPA) predicted PU.1as a possible driver of the cell cycle restriction gene program. Indeed, PU.1 mRNA and protein levels in HSC increased significantly in PU.1-eYFP knockin reporter mice treated with IL-1. Furthermore, fractionating HSC based on reporter levels showed that IL-1-exposed HSC expressing high PU.1 levels activated the cell cycle restriction program more robustly than HSC with lower PU.1 levels or from untreated control mice. Along these lines, cell cycle analyses showed that PU.1-high HSC were quiescent following IL-1 exposure. Surprisingly, functional assays revealed that the PU.1-high fraction became enriched for functional HSC following IL-1 exposure, in contrast to a primarily PU.1-low phenotype under control conditions. Collectively, these data show that low Myc levels, a quiescent cell cycle state and functional HSC identity are all associated with high PU.1 levels during chronic inflammation. To address the requirement for PU.1, we compared HSC following IL-1 exposure from wild-type (WT) and PU.1-knockin (KI) mice, which express ~30% of normal PU.1 levels due to a point mutation in the PU.1 upstream regulatory element (URE). Strikingly, IL-1 induced aberrant HSC expansion in the BM of PU.1 KI mice, underwritten by IL-1-induced quiescence loss in PU.1 KI HSC. Excess cell cycle activity in HSC from IL-1-treated PU.1 KI mice was associated with Myc overexpression, hyperinduction of cycle and protein synthesis genes, and an increased protein synthesis rate. Interestingly, IL-1-treated PU.1 KI mice also exhibited exuberant platelet production and aberrant activation of megakaryocyte (Mk) lineage programs in HSC. Taken together, these data show PU.1 is required to limit HSC proliferation and cell cycle activity, and that failure to upregulate PU.1 during chronic inflammation leads to aberrant HSC expansion and dysregulated Mk lineage output. Altogether, our data identify PU.1 as a key enforcer of HSC dormancy, and as a regulator of HSC proliferation and proteostasis during chronic inflammation. As PU.1 has been shown to slow cell cycle entry to promote myeloid differentiation in actively proliferating cells, our results suggest a mechanistic conservation in which inflammation induces PU.1 expression and engages a similar cell cycle restriction program that preserves HSC quiescence. Such a mechanism can prevent excessive Mk lineage output leading to thrombosis, and/or HSC exhaustion. Importantly, these findings re-cast inflammation-induced PU.1 expression as a guardian of blood system function, rather than a pathogenic by-product. As PU.1 serves a tumor suppressor role, these findings also may provide insight into the link between inflammation and leukemogenesis, particularly in contexts where PU.1 function is impaired in leukemic stem cells (LSC) due to mutation or indirect mechanism(s). Disclosures No relevant conflicts of interest to declare.

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.

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.

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.

Acute myeloid leukaemia (AML) is a disorder characterized by a clonal proliferation derived from primitive haematopoietic stem cells or progenitor cells. Abnormal differentiation of myeloid cells results in a high level of immature malignant cells and fewer differentiated red blood cells, platelets and white blood cells. The disease occurs at all ages, but predominantly occurs in older people (>60 years of age). AML typically presents with a rapid onset of symptoms that are attributable to bone marrow failure and may be fatal within weeks or months when left untreated. The genomic landscape of AML has been determined and genetic instability is infrequent with a relatively small number of driver mutations. Mutations in genes involved in epigenetic regulation are common and are early events in leukaemogenesis. The subclassification of AML has been dependent on the morphology and cytogenetics of blood and bone marrow cells, but specific mutational analysis is now being incorporated. Improvements in treatment in younger patients over the past 35 years has largely been due to dose escalation and better supportive care. Allogeneic haematopoietic stem cell transplantation may be used to consolidate remission in those patients who are deemed to be at high risk of relapse. A plethora of new agents - including those targeted at specific biochemical pathways and immunotherapeutic approaches - are now in trial based on improved understanding of disease pathophysiology. These advances provide good grounds for optimism, although mortality remains high especially in older patients.

FMS-like tyrosine kinase 3–targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD–mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML.

Psoriasis is a common, immune-mediated genetic disorder of the skin and is associated with arthritis in approximately 30% of cases. Previously, we localized PSORS2 (psoriasis susceptibility locus 2) to chromosomal region 17q25.3-qter after a genome-wide linkage scan in a family of European ancestry with multiple cases of psoriasis and psoriatic arthritis. Linkage to PSORS2 was also observed in a Taiwanese family with multiple psoriasis-affected members. In caspase recruitment domain family, member 14 (CARD14), we identified unique gain-of-function mutations that segregated with psoriasis by using genomic capture and DNA sequencing. The mutations c.349G>A (p.Gly117Ser) (in the family of European descent) and c.349+5G>A (in the Taiwanese family) altered splicing between CARD14 exons 3 and 4. A de novo CARD14 mutation, c.413A>C (p.Glu138Ala), was detected in a child with sporadic, early-onset, generalized pustular psoriasis. CARD14 activates nuclear factor kappa B (NF-kB), and compared with wild-type CARD14, the p.Gly117Ser and p.Glu138Ala substitutions were shown to lead to enhanced NF-kB activation and upregulation of a subset of psoriasis-associated genes in keratinocytes. These genes included chemokine (C-C motif) ligand 20 (CCL20) and interleukin 8 (IL8). CARD14 is localized mainly in the basal and suprabasal layers of healthy skin epidermis, whereas in lesional psoriatic skin, it is reduced in the basal layer and more diffusely upregulated in the suprabasal layers of the epidermis. We propose that, after a triggering event that can include epidermal injury, rare gain-of-function mutations in CARD14 initiate a process that includes inflammatory cell recruitment by keratinocytes. This perpetuates a vicious cycle of epidermal inflammation and regeneration, a cycle which is the hallmark of psoriasis.

Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Abstract 243 Disruption of normal hematopoietic development is a major problem in acute myeloid leukemia (AML). Osteoblastic cells have been shown to support hematopoiesis. We hypothesized that myeloid leukemia inhibits osteoblastic cells contributing to the loss of normal hematopoiesis. To define effects of leukemia on osteoblastic cells, we employed a previously described syngeneic murine model of AML where transplant of leukemic cells does not require irradiation of recipients (Neering et al. Blood, 2007). Leukemic mice had decreased serum levels of the bone formation marker osteocalcin (OC) (92.6 ± 11.6 vs 25.9 ± 4.4 ng/ml Normal (N) vs Leukemic (L) p Disclosures: No relevant conflicts of interest to declare.

Myeloid leukemias are clonal disorders originating in a primitive multipotential hematopoietic cell and characterized by aberrant proliferation, differentiation and maturation of leukemic progenitors and precursor cells. These diseases are the result of multiple genetic and epigenetic events, although the nature and number of events vary widely among patients. For over four decades, studies have identified sub-populations of leukemic cells possessing different functional capabilities. Investigators have struggled to understand the origin and significance of this heterogeneity. The stem cell model for myeloid malignancies has offered one potential explanation. Since 1994, experimental data supporting the presence of leukemia stem cells has been reported and validated in numerous studies. We will review the history of the model, data from the past decade supporting the stem cell model for myeloid malignancies, more recent data regarding patient specific variability in leukemic stem cell surface antigen phenotype and the impact the stem cell model has on the care of patients with myeloid malignancies.

We have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However, despite the activity of this agent, PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus, we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal, we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment, exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together, our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells.

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

Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) remains one of the only curative therapies for acute myeloid leukemia (AML) in adult patients. However, a high proportion of patients will relapse post-HSCT. Measurable residual disease (MRD) has emerged as highly predictive of relapse after chemotherapy and multiple studies have demonstrated inferior outcomes after HSCT for patients with pre-HSCT MRD. Fewer studies have evaluated post-HSCT MRD, but those have suggested that MRD is a powerful predictive tool in this setting as well. We have previously utilized droplet digital PCR (ddPCR) as an MRD platform to track 33 unique AML-associated mutations in a cohort of patients receiving either cytotoxic chemotherapy or targeted agents, and our preliminary data suggest that persistence or recurrence of these mutations during therapy is predictive of relapse and diminished overall survival. In the present study we sought to evaluate the utility of ddPCR specifically for post-HSCT MRD monitoring. Methods: Patients 18 years of age or older with AML who had received allogeneic HSCT were identified via retrospective chart review. Thirty-seven patients were identified who had sufficient diagnostic testing to identify AML-associated point mutations and had available DNA from serial bone marrow samples. Thirty-six of these patients had at least one pre-HSCT sample analyzed; post-HSCT day 28, day 80, and day 365 samples were available for 26, 25, and 11 patients, respectively. DdPCR was utilized to track 21 different AML-associated mutations in this patient cohort. MRD negativity was defined as variant allelic frequency (VAF) below the limit of detection for all assayed mutations (1-4 mutations per patient). Relapse-free (RFS) and overall (OS) survival were defined as time from date of transplant to relapse or death. Survival statistics were analyzed as Kaplan-Meyer curves, with p-values determined by the log-rank (Mantel-Cox) test. P-values less than 0.05 were considered statistically significant. Outcome data for all patients were censored July 6, 2018. Results: Median follow-up from time of HSCT was 13.3 months. Of the 37 patients included in this cohort, the majority (73%) received regimens containing cytotoxic chemotherapy prior to HSCT; the remainder (27%) received targeted therapies +/- low-intensity chemotherapy prior to HSCT. Only 6 of 36 patients (17%) were MRD negative pre-HSCT, all of whom had received cytotoxic chemotherapy. All 37 patients had one or more post-HSCT samples available for MRD assessment. Of these, 30 achieved MRD negativity after HSCT (81%). Post-HSCT MRD status was predictive of both RFS and OS (Figure 1). This replicates previously published data with respect to ddPCR-based MRD, but is based on evaluation of both more patients and more mutations. In agreement with previously published data showing high predictive value of the 1-month post-HSCT MRD, we found this timepoint specifically to be predictive of RFS and OS in the 26 patients for whom a sample was assayable (Figure 2). Neither outcomes nor MRD status by ddPCR varied based on conditioning regimen (myeloablative versus reduced-intensity) in our cohort (data not shown). We also compared ddPCR MRD status with whole bone marrow chimerism measured by short tandem repeat (STR) analysis. Concordance between MRD negativity and >95% donor chimerism was 69% at 1 month post-HSCT and 93% at 80 days post-HSCT. Of 8 discordant samples at the 1 month post-HSCT time point, 3 had donor chimerism Conclusions: ddPCR-based molecular MRD assessment is predictive of relapse-free and overall survival post-HSCT in patients with AML, and may be a powerful adjunct to surveillance measures already in clinical use, such as chimerism. Presence of molecular MRD specifically at 1 month post-transplant showed significant correlation with relapse and with overall mortality. Detection of AML-associated mutations at this and later time points in a prospective manner could in many cases allow salvage of patients prior to clinical relapse, thus improving survival for this high-risk population. Disclosures Pollyea: Gilead: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Curis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Celyad: 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; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Most AML patients who receive intensive chemotherapy achieve a significant clinical response; however, the majority will relapse and succumb to their disease, indicating that leukemia stem cells (LSCs) are not effectively targeted. Further, it has recently been shown that LSC frequency and phenotypic diversity are increased at relapse (Ho et al. Blood, 2016), thereby creating an even more challenging clinical scenario. Thus, novel therapies specifically designed to target LSCs in relapsed AML patients are urgently needed. Previously, we have shown that LSCs can be targeted by perturbing energy metabolism (Lagadinou et al. Cell Stem Cell, 2013). Therefore, the goal of the current study was to identify and target metabolic dependencies of relapsed LSCs, with the hope that this would allow improved efficacy for AML patients with relapsed disease. To achieve this objective we first measured metabolic differences in LSCs isolated from de novo and relapsed patients. This analysis revealed that relapsed LSCs have significantly increased levels of nicotinamide compared to de novo LSCs (Figure 1A). Nicotinamide is a precursor of NAD+, an essential coenzyme in energy metabolism. We hypothesized that relapsed LSCs are dependent on nicotinamide metabolism to maintain energy metabolism. To test this hypothesis, we targeted nicotinamide metabolism with the small molecule APO866, an inhibitor of Nampt, the rate-limiting enzyme for conversion of nicotinamide to NAD+. This resulted in a significant decrease in NAD+ in LSCs isolated from both de novo and relapsed AML specimens (data not shown). However, strikingly, inhibition of nicotinamide metabolism only decreased viability and colony-forming ability of LSCs isolated from relapsed AML patients, not LSCs from untreated patients (Figure 1B). To verify that inhibition of Nampt was targeting functional LSCs, we treated a relapsed AML patient specimen with APO866 for 24 hours and measured the ability of the leukemia cells to engraft into immune deficient mice. We observed a significant reduction in leukemia engraftment upon APO866 treatment (data not shown). Importantly, inhibition of nicotinamide metabolism did not affect normal hematopoietic stem cell frequency or colony forming ability (data not shown). Altogether, these data suggest that inhibition of nicotinamide metabolism specifically targets relapsed LSCs. We next sought to understand the mechanism by which inhibiting nicotinamide metabolism targets relapsed LSCs. To this end we measured changes in the major energy metabolism pathways (oxidative phosphorylation [OXPHOS] and glycolysis) in LSCs isolated de novo and relapsed AML patient specimens. Upon APO866 treatment, we observed a significant decrease in OXPHOS and OXPHOS capacity in relapsed LSCs but not de novo LSCs (Figure 1C). Furthermore, no change in glycolysis was observed (data not shown). These data demonstrate that inhibition of nicotinamide metabolism targets OXPHOS specifically in relapsed LSCs. To determine how APO866 reduced OXPHOS, we measured stable isotope metabolic flux of amino acids, the fatty acid palmitate, and glucose into the TCA cycle after APO866 treatment. We observed an increased accumulation of citrate, malate, and α-ketoglutarate from amino acids and palmitate, consistent with decreased activity of the NAD+ dependent enzymes isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase (data not shown). Through direct measurement of enzyme activity, we confirmed that isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activity were each significantly decreased upon APO866 treatment (Figure 1D). Consistent with our previous findings we did not observe any changes in glycolysis or glucose contribution to the TCA cycle (data not shown). Overall, these data suggest that inhibition of nicotinamide metabolism through Nampt inhibition results in decreased OXPHOS through decreased TCA cycle activity. In conclusion, we have shown that relapsed LSCs have distinct metabolic properties including increased levels of nicotinamide, which can be selectively targeted to eradicate relapsed LSCs. We propose that therapeutic strategies designed to target nicotinamide metabolism may be useful for relapsed AML patients and may allow for broad efficacy such as that observed when LSCs are targeted in the up-front treatment setting. Disclosures Nemkov: Omix Technologies inc: Equity Ownership. Pollyea:Curis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees.

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

Rheumatoid arthritis (RA) is a debilitating autoimmune disease resulting from autoantibodies that cause damage to synovial joints. Joint damage causes increased systemic inflammatory cytokines which may lead to aberrant hematopoiesis. Indeed, RA is accompanied by many hematological complications including anemia, cytopenias, and suppressed bone marrow function. Hematopoietic stem cells (HSC) at root of the blood system can respond to inflammatory signals by activating the cell cycle and preferentially generating myeloid cells. However, chronic inflammation can also lead to HSC dysfunction. Previous studies using genetic mouse models of RA have identified myeloid overproduction in this context; however, HSC long-term reconstitution activity was maintained. To better understand hematopoietic alterations in RA, our group used the collagen induced arthritis (CIA) mouse model, which is inducible in adult mice and recapitulates many immunological features of the human disease, including elevated inflammatory cytokine levels in the bone marrow (BM) and peripheral blood (PB). Confirming prior reports, we found increased numbers of myeloid lineage cells in the PB and BM of CIA mice. We also found reduced erythroid and lymphoid lineage progenitor cell numbers in the BM, consistent with anemia and immunosenescence phenotypes in RA patients. Interestingly, these features were accompanied by a significant increase in the number of myeloid-biased multipotent progenitor-3 (MPP3) cells, suggesting increased activation of myeloid differentiation pathways. However, we found no changes to the number of activated (MPP1), short-term HSC (ST-HSC), or long-term HSC (LT-HSC) in CIA mice. Likewise, and in line with previous reports, long-term HSC potential was not reduced in CIA mice, as assessed by transplantation of either purified HSC or with unfractionated BM into irradiated recipient mice. While HSC from control and CIA donor mice displayed similar blood chimerism and lineage distribution over a 16-week period, we did observe increased proportions of donor-derived MPP3 in CIA recipient animals, further supporting an activation of myeloid HSC differentiation pathways. Overall, these results reveal underlying changes in the BM driving aberrant hematopoiesis, with the HSC pool remaining intact despite activation of a myeloid differentiation pathway. To better understand how HSC are impacted by arthritic inflammation, we assessed the molecular state of control and CIA HSC using RNA-seq. We found 292 genes significantly upregulated and 237 genes significantly downregulated in CIA HSC. Analysis of these genes using Ingenuity, GSEA, and DAVID tools revealed broad downregulation of inflammatory and proliferation signaling pathways including IL-1β, NFκB, MYC, and ERK. Genes for G1/S cell cycle transition, transcription, protein translation, and proliferation pathways were also significantly downregulated in CIA HSC. On the other hand, genes corresponding to cell cycle arrest and negative regulation of transcription were significantly upregulated. Notably, we find that IL-1β, which is produced in the BM of CIA mice, is sufficient to induce this molecular program. We find that HSC in CIA mice have a global downregulation of transcripts required for activation and proliferation, and a global upregulation of transcripts that would promote quiescence. Hence, HSC are forced back into a quiescent state by broad downregulation of cell growth and proliferation genes even during chronic inflammation caused by RA. Altogether, our data show that a mouse model of rheumatoid arthritis causes hematopoietic lineage skewing towards the myeloid lineage with simultaneous loss of lymphoid and erythroid lineage potential. Interestingly, in this chronic inflammatory setting HSC downregulate pathways involved in cytokine signaling, cell cycle activation, and translation. This mechanism can be triggered by chronic exposure to pro-inflammatory cytokines, and may serve to limit HSC proliferation and potential for damage in disease settings. These results may explain the relative rarity of outright bone marrow failure in autoimmune disease patients, while providing insight into mechanisms driving aberrant hematopoiesis in these individuals. Lastly, they provide functional evidence for cytokine blockade to normalize HSC function in the setting of RA and other chronic inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

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

Previous studies have demonstrated the importance of energy metabolism as it relates to numerous aspects of leukemia stem cell (LSC) biology. Specifically, in acute myelogenous leukemia (AML), it has been shown that LSCs have a unique reliance on oxidative phosphorylation (OXPHOS), and that inhibition of B-cell lymphoma 2 (BCL-2) acts to down-regulate OXPHOS and eradicate LSCs in pre-clinical models. In the clinical setting, when BCL-2 inhibitor venetoclax is combined with azacitidine, high response rates (~80%) in elderly de novo AML patients have been observed (PMID: 29339097). Nonetheless, a significant portion of these patients ultimately experience disease progression. The mechanism of resistance and characteristics of these patients is poorly understood. Our preliminary data shows that the venetoclax + azacitidine (ven/aza) regimen targets LSCs through alteration of energy metabolism. Specifically, the regimen disrupts the TCA cycle leading to decreases in ATP production and inhibition of OXPHOS. This metabolic targeting is central to ven/aza efficacy and we hypothesize that resistance and progression of AML patients is due to compensatory mechanisms that restore sufficient levels of OXPHOS (PMID:3333149). Investigation of such mechanisms led us to explore the potential activity of MCL1. Previous studies have shown MCL1 can influence venetoclax resistance, however little is known about MCL1's role in metabolism, although a report in breast cancer cells suggests MCL1 modulates OXPHOS (PMID: 28978427). In leukemia, MCL1 expression has been shown to be partially upregulated through mutations in protein tyrosine phosphatase non- receptor type 11 (PTPN11), and PTPN11 mutations have been shown to increase LSC frequency. Thus, we hypothesized that PTPN11 mutations may confer resistance to venetoclax-based regimens at least partially by up-regulation of MCL1. To test this hypothesis, we investigated the relationship between PTPN11 mutations, MCL1, and the metabolic phenotype. In comparison to specimens with a wild type allele, LSCs isolated from PTPN11 mutant patient specimens showed increased levels of OXPHOS as well as glycolysis, amino acids, and fatty acids, suggesting an ability to utilize multiple energy sources for survival. PTPN11 mutant specimens also show decreased sensitivity to venetoclax, suggesting OXPHOS is not affected by venetoclax to the same degree as PTPN11 wild type specimens (fig. 1). Furthermore, when we introduced a mutated allele of PTPN11 into a primary AML specimen we observed increased oxidative phosphorylation and glycolysis, which correlated with decreased in vitro sensitivity to venetoclax (fig. 2). To test the potential role of MCL1 in PTPN11 mutant specimens, we employed a small molecule MCL-1 inhibitor. Metabolic analysis of specimens treated with the MCL-1 inhibitor showed decreased OXPHOS in PTPN11 mutant specimens (fig 3). Further, PTPN11 mutant specimens exhibit increased sensitivity to the MCL-1 inhibitor (fig. 4). To investigate a potential mechanistic link to clinical observations, we next examined 45 older, previously untreated AML patients from our institution who received ven/aza, both in the context of the multi-institutional study NCT02203773 (N=33) and with off-label use (N=12). Of 12 variables examined, only the presence of PTPN11 predicted shorter response duration (table 1). In addition, of the 9 patients who progressed ven/aza, 2 (22%) acquired PTPN11 mutations upon progression, further suggesting PTPN11 may represent a resistance mechanism to this regimen. Notably, PTPN11 is not preferentially detected in patients who progress after regimens other than ven/aza. In conclusion, AML containing PTPN11 mutations exhibit a unique energy metabolism profile. These specimens also appear to have increased sensitivity to MCL-1 inhibitors. The presence of PTPN11 mutations represents both a novel method for predicting response to ven/aza and a potential strategy for targeting patients who progress. We propose that addition of an MCL-1 inhibitor for treatment of AML patients bearing PTPN11 or related mutations may increase therapeutic responses. Disclosures Savona: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Consultancy. Fesik:Boehringer Ingelheim: Consultancy. Pollyea:Celyad: 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; Curis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy.