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Chronic myelomonocytic leukemia (CMML) is an aggressive myeloid neoplasm of older individuals characterized by persistent monocytosis. Somatic mutations in CMML are heterogeneous and only partially explain the variability in clinical outcomes. Recent data suggest that cardiovascular morbidity is increased in CMML and contributes to reduced survival. Clonal hematopoiesis of indeterminate potential (CHIP), the presence of mutated blood cells in hematologically normal individuals, is a precursor of age-related myeloid neoplasms and associated with increased cardiovascular risk. To isolate CMML-specific alterations from those related to aging, we performed RNA sequencing and DNA methylation profiling on purified monocytes from CMML patients and from age-matched (old) and young healthy controls. We found that the transcriptional signature of CMML monocytes is highly proinflammatory, with upregulation of multiple inflammatory pathways, including tumor necrosis factor and interleukin (IL)-6 and -17 signaling, whereas age per se does not significantly contribute to this pattern. We observed no consistent correlations between aberrant gene expression and CpG island methylation, suggesting that proinflammatory signaling in CMML monocytes is governed by multiple and complex regulatory mechanisms. We propose that proinflammatory monocytes contribute to cardiovascular morbidity in CMML patients and promote progression by selection of mutated cell clones. Our data raise questions of whether asymptomatic patients with CMML benefit from monocyte-depleting or anti-inflammatory therapies.

Background Chronic phase chronic myeloid leukemia (CP-CML) is characterized by overproduction of differentiating myeloid cells, while blast phase CML (BP-CML) cells exhibit a differentiation block. Tyrosine kinase inhibitors (TKIs) are effective in CP-CML, but resistance is common in BP-CML and can occur without explanatory BCR-ABL1 kinase mutations (BCR-ABL1-independent resistance). Similarly, CML stem/progenitor cells (LSPCs) are insensitive to TKIs, and residual leukemia persists in the majority of CML patients on TKI therapy. We previously reported overlap between the transcriptomes of CD34 + cells from BP-CML and TKI-naïve CP-CML patients with primary TKI resistance, pointing to commonalities between LSPC quiescence, BCR-ABL1-independent TKI resistance, and BP-CML. Results To identify common mechanisms, we performed a meta-analysis of published CML transcriptomes. We identified a small set of genes with consistently low expression in LSPC quiescence, BCR-ABL1-independent TKI resistance, and BP-CML, including Membrane Spanning 4-Domains A3 (MS4A3), a signaling protein previously reported to inhibit hematopoietic cell cycle progression. Low MS4A3 in CD34 + cells from TKI-naïve CP-CML patients was associated with shorter survival on subsequent TKI therapy, suggesting that MS4A3 governs TKI response. To understand the function of MS4A3, we lentivirally introduced MS4A3 shRNA or an MS4A3 expression vector into CML CD34 + LSPCs. MS4A3 knockdown increased clonogenicity and imatinib resistance, while ectopic MS4A3 expression had opposite effects. MS4A3 KD also increased LSPC persistence ex vivo in LTC-IC assays, and in vivo in NSG mice xenografts, while modulating MS4A3 expression had no effect on normal CD34 + cells. We next generated Ms4a3+/+│-/-; Scl-tTA+; TRE-BCR-ABL1+ compound transgenic mice. Upon BCR-ABL1 induction, Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ mice developed leukocytosis comparable to Ms4a3+/+ controls. However, BM of Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ mice showed increased short-term HSCs and multipotent progenitor cells, and reduced granulocyte-macrophage progenitors. When Lin - BM cells from leukemic mice were transplanted into irradiated recipients, Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ cells showed increased engraftment and myeloid leukocytosis, validating our observations in human cells. To determine how MS4A3 is downregulated in CML, we expressed BCR-ABL1in 32D-cl3 cells. p210 BCR-ABL1 drastically reduced Ms4a3 expression, while kinase-inactive p210 BCR-ABL1-K271R had no effect. Moreover, we found that suppression of C/EBPε by MECOM reduces MS4A3, consistent with previous reports of MECOM as a driver of TKI resistance and progression to BP. Treatment of CML CD34 + cells with a library of epigenetic pathway inhibitors revealed that MS4A3 is suppressed by both DNA methylation and PRC2/EZH2-mediated H3K27 trimethylation, which was confirmed by patch-PCR and ChIPseq. These data indicate that multi-levelled mechanisms cooperate in the suppression of MS4A3 in CML. To determine how MS4A3 regulates clonogenicity and TKI response, we expressed MS4A3-EGFP fusion protein in LAMA-84 CML cells. We found that MS4A3 resides on the plasma membrane and in endosomes. Surface protein biotin labelling and tandem mass spectrometry ± MS4A3 KD showed that MS4A3 controls endocytosis of membrane proteins, including common β chain (βc) cytokine receptors. Specifically, MS4A3 promotes endocytosis of βc cytokine receptors upon GM-CSF/IL-3 stimulation of primary LSPCs and enhances downstream signaling and differentiation, suggesting that restoring MS4A3 expression has therapeutic efficacy. To test this, we manufactured a prototype MS4A3 protein-loaded liposomal nanoparticle (NP) using coating with the CD34 CD62L for targeted delivery to CD34 + cells. Compared to MS4A3-free NPs, MS4A3 NPs increased CD34 +CD38 + and CD34 -CD38 + at the expense of CD34 +CD38 - cells, reduced clonogenicity, and increased sensitivity to TKIs, mimicking ectopic MS4A3 expression. Conclusion MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of primitive LSPCs and BP-CML cells. We posit that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation to maintain a primitive, TKI-insensitive state. MS4A3 re-expression or delivery of ectopic MS4A3 may eliminate LSPCs. Figure 1 Figure 1. Disclosures Druker: Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; EnLiven: Consultancy, Research Funding; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aileron: Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Nemucore Medical Innovations, Inc.: Consultancy; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Research Funding; Recludix Pharma, Inc.: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Agios: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Constellation: Research Funding; Schrodinger: Research Funding. Oehler: BMS: Consultancy; OncLive: Honoraria; Pfizer: Research Funding; Takeda: Consultancy; Blueprint Medicines: Consultancy. Radich: BMS: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Deininger: Sangamo: 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, Other: Part of a Study Management Committee, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Novartis: Consultancy, Research Funding; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis. FLT3 internal tandem duplications (ITDs) are found in ~30% of AML patients and are associated with inferior survival. Addition of FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin improve survival of AML treated with cytotoxic chemotherapy. However, relapse rates remain unacceptably high. Protection by stroma-derived survival signals is thought to enable survival of leukemia initiating cells (LICs) in the presence of FLT3 TKIs, providing a reservoir for subsequent overt relapse. We previously reported that HS-5 conditioned medium (CM) rescued FLT3-ITD positive AML cell lines and primary cells from the effects of the 2nd generation FLT3 TKI AC220 (quizartinib) as compared to cells cultured in regular medium (RM). Earlier work from our lab and others has demonstrated that HS-5 CM increases pSTAT3Y705 in chronic myeloid leukemia cells treated with BCR-ABL1 TKIs, leading to TKI resistance, while pSTAT5Y694 levels remain under the control of the BCR-ABL1 kinase (Eiring et al, Leukemia, 2015). Similar to BCR-ABL1, FLT3 signaling results in potent pSTAT5Y694 activation. We hypothesized that STAT3 and/or STAT5 activation contributes to stroma-mediated protection of FLT3-ITD+ AML cells upon FLT3 inhibition with AC220. We found that FLT3-ITD+ cell lines and primary cells grown in HS-5 CM exhibited strong upregulation of pSTAT3Y705 and pSTAT5Y694 that was unaffected by treatment with 10nM AC220. In RM, pSTAT3Y705 was undetectable irrespective of AC220 dose and pSTAT5Y694 was abolished by 10nM AC220. shRNA knockdown experiments of STAT3 and STAT5 in FLT3-mutated cell lines grown in CM and treated with AC220 showed that STAT5 knockdown alone consistently resulted in greater inhibition of cell proliferation than STAT3 knockdown alone. This suggests that in FLT3-ITD+ AML cells cultured in CM, upregulation of pSTAT5Y694 by stroma-derived soluble factors is the main contributor to AC220 resistance. We decided to use a candidate approach to identify the upstream kinases leading to pSTAT5Y694, focusing on inhibitors of pathways previously implicated in STAT5 activation in AML. We used phosphoflow to quantify pSTAT5Y694 in MOLM-13 cells grown for 24 hours in CM in the presence of 10nM AC220 combined with ruxolitinib, dasatinib, ibrutinib, PD173074 (FGFR1i) or PRT062607 (SYKi). Results from this screen indicated that the combination of dasatinib and AC220 most effectively decreased pSTAT5Y694 in MOLM-13 cells cultured in CM (minimum dasatinib dose =100nM). To further validate dasatinib as a candidate for combination drug treatment, we performed cell proliferation experiments in MOLM-13 cells cultured in CM for 72h with graded concentrations of AC220 and the candidate inhibitors at a minimum candidate drug dose of 100nM. Of the four inhibitors tested, the combination of dasatinib and AC220 most significantly decreased the IC50 of AC220 in MOLM-13 cells grown in CM (IC50 AC220 alone: 11.09nM; IC50 AC220 + 100nM dasatinib: 0.41nM; p It has recently been reported that FLT3-ITD AML cells harbor a highly glycolytic phenotype that can be partially suppressed by FLT3 TKIs (Huntly et al, Blood, 2018). Our preliminary Seahorse experiments show that the combination of dasatinib and AC220 more effectively inhibits glycolysis in MOLM-13 cells than AC220 alone, with differential modulation in CM versus RM. These results suggest that dasatinib inhibits a metabolic pathway critical to maintaining glycolysis in MOLM-13 cells challenged by AC220-based FLT3 inhibition. Moreover, these data indicate that extrinsic signals contribute to metabolic reprogramming of FLT3+ AML cells, enabling survival despite FLT3 inhibition. Experiments are underway to delineate how CM contributes to metabolic resistance and how dasatinib intercepts this survival signal. Our results support the concept of combining AC220 with dasatinib to reduce residual leukemia in FLT3+ AML and prolong survival. Further investigation is ongoing and will be reported at the time of abstract presentation. Disclosures Deininger: Blueprint: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Chronic myelomonocytic leukemia (CMML) is a genetically heterogeneous hematopoietic stem cell disorder that combines features of a myelodysplastic syndrome and a myeloproliferative neoplasm and exhibits a strong bias towards older age. The prognosis of CMML is poor, with overall survival of less than 3 years in most studies, however recurrent somatic mutations explain only 15-24% of the clinical heterogeneity of CMML (Elena C. et al. Blood 128:1408-17, 2016). The extreme skewing of the CMML age distribution suggests that CMML reflects the malignant conversion of the myelomonocytic-biased differentiation characteristic of an aged hematopoietic system. We hypothesized that separating the contribution of the normal aging process from bona fide CMML-specific alterations will improve the molecular characterization and biological understanding of CMML. We decided to focus on monocytes as the phenotypic minimal common denominator of genetically heterogeneous diseases. CD14+ monocytes were sorted from the blood of untreated CMML patients (N=12, median age 77 years, range 61-90), age-matched healthy controls (old controls: N=12, median age 68 years, range 62-74) and young healthy controls (young controls: N=16, median age 29 years, range 24-44) and subjected to RNA sequencing and DNA methylation profiling. Differentially expressed genes in CMML monocytes compared to healthy controls were identified with DESeq2 using a 1% false discovery rate (FDR) and a fold-change cutoff set at >│2│ (Figure 1A). We identified the 2480 CMML-specific genes by subtracting all genes with significant differences in the young controls vs. old controls comparison from the CMML vs. old controls comparison. The top-25 most significantly upregulated genes (Figure 1B) included transcription factors, TNFα signaling genes, genes that regulate genomic stability, and genes involved in apoptosis. The most significantly downregulated transcripts were genes involved in response to DNA damage, RNA binding, monocyte differentiation and mediators of inflammatory process. To link these observations to function, we imputed the 2480 CMML-specific differentially expressed genes into the ingenuity pathway analysis (IPA) application. This analysis uncovered significant enrichment of pathways involved in: mitotic roles of Polo-like kinase, G2/M DNA damage checkpoint regulation, lymphotoxin β receptor signaling, IL-6 signaling and ATM signaling (Figure 1C). DNA methylation profiling revealed 909 differentially methylated regions (DMRs) between CMML and age-matched controls, with most regions being hypermethylated in CMML monocytes. Of these, 37% of the DMRs were intronic, 22% were exonic, 14 % were in the promoter region (Figure 1D), 10% were downstream, 10% were upstream, the remainder were 3' and 5'-overlaps. We also performed integrated analysis using the promoter DMRs and the gene expression profile to identify CMML-associated genes that are likely to be regulated by specific changes in methylation. We observed concomitant changes in CMML-specific mRNA transcripts and DNA methylation promoter regions in the CMML vs. old controls contrast for 10 genes (Figure 1E). AOAH, SERINC5, TAF3 and AHCYL1 were downregulated and hypermethylated; MS4A3, TNF, VCAM1, and IFT80, were upregulated and hypermethylated; TUBA1B was upregulated and hypomethylated and PITPNA was downregulated and hypomethylated. Our study is the first to combine transcriptional and methylation profiling for molecular characterization of CMML monocytes. Conclusions: (i) age-related gene expression changes contribute significantly to the CMML transcriptome; (ii) the CMML-specific transcriptome is characterized by differential regulation of transcription factors, inflammatory response genes and anti-apoptotic pathway genes; (iii) differences in promoter methylation represent only a small proportion of overall differences in methylation, suggesting that intragenic or intronic methylation is a major contributor to the leukemic phenotype; (iv) age-related changes may be necessary, but are not sufficient to realize the CMML phenotype. Figure 1. Figure 1. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm with five-year overall survival rates To identify survival-critical AML genes irrespective of mutational status, we performed an shRNA screen on patient samples (N=12) using a barcoded lentiviral shRNA library. Transduced cells were cultured for 9 days on HS-5 stromal cells (to mimic the BM microenvironment) and subjected to NGS for barcode quantification. Sirtuin 5 (SIRT5), the only known enzyme with lysine desuccinylase, demalonylase, or deglutarylase activity, was amongst the top candidates. SIRT5 is implicated in regulating metabolic pathways, including energy metabolism. We first confirmed growth inhibition upon SIRT5 knockdown (KD) in cryopreserved AML cells from the original screen. Next, we transduced a panel of 25 AML cell lines with doxycycline (dox) - inducible shSIRT5 (dox-shSIRT5). SIRT5 KD strongly inhibited growth, reduced colony formation and increased apoptosis in 18/25 lines (SIRT5-dependent cell lines). Seven cell lines showed 80% reduction of SIRT5 protein and were considered SIRT5-independent. SIRT5 dependence was neither correlated with specific mutations nor basal SIRT5 expression. To control for off-target effects we tested three unique shSIRT5s targeting different sequences in three SIRT5-dependent and three SIRT5-independent cell lines, and confirmed the differential sensitivity for all lines and all constructs. We next transduced CD34+ cells from AML patients (n=15) or cord blood (CB, n=5) with dox-shSIRT5 and plated cells in colony assays. SIRT5 KD (~40% in AML and CB) reduced AML colonies by ~50%, with no effect on CB. Colony formation by SIRT5 null mouse bone marrow infected with FLT3-ITD or MLL-AF9 retrovirus was reduced by 50-60% compared to wild type controls. Metabolic profiling showed that SIRT5 KD induced a profound reduction of oxidative phosphorylation and glycolysis in SIRT5-dependent, but not SIRT5-independent cell lines. Mitochondrial reactive oxygen species (ROS) in SIRT5-dependent cell lines were strongly increased upon SIRT5 KD, and this increase preceded apoptosis. Ectopic expression of superoxide dismutase 2 (SOD2, mitochondrial) abrogated the increase in ROS and rescued cells from apoptosis. Metabolomics and RNAseq suggest that SIRT5-dependent cells exhibit profound metabolic disruption, including reduced TCA cycle activity, and recurrent transcriptional changes, while SIRT5 KD in SIRT5-independent cells is inconsequential. We next injected mice with CMK-1 cells (SIRT5-dependent) expressing dox-shSIRT5 and luciferase. Mice were randomized to dox-supplemented (dox-water) or control water 18 hours post injection, and monitored by luminescence imaging. Control mice showed abundant luminescence at week 3, and died before week 5, while mice receiving dox-water survived throughout the 13-week experiment without evidence of leukemia. When mice with established leukemia were switched to dox-water following week 3, luminescence rapidly decreased and the mice survived without evidence of leukemia until termination of the experiment (week 13). Dox-water had no effect on mice engrafted with OCI-AML3 cells (SIRT5-independent), all of whom died with extensive leukemic involvement despite downregulation of SIRT5 in leukemia cells. We are currently testing the requirement of SIRT5 in MLL-AF9-mediated AML using a retroviral BM transplant mouse model. Preliminary data suggest that absence of SIRT5 may prolong survival. Our data suggest that SIRT5 KD preferentially targets AML cells over normal cells. As SIRT5 null mice are viable with only minor metabolic abnormalities at steady state, these data implicate SIRT5 as a potential therapy target in AML and support the development of clinical SIRT5 inhibitors. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.