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Mutations in the gene Additional Sex-Combs Like 1 (ASXL1) are recurrent in myeloid malignancies as well as the pre-malignant condition clonal hematopoiesis, where they are universally associated with poor prognosis. However, the role of ASXL1 in myeloid lineage maturation is incompletely described. To define the role of ASXL1 in myelopoiesis, we employed single cell RNA sequencing and a murine model of hematopoietic-specific Asxl1 deletion. In granulocyte progenitors, Asxl1 deletion leads to hyperactivation of MYC and a quantitative decrease in neutrophil production. This loss of granulocyte production was not accompanied by significant changes in the landscape of covalent histone modifications. However, Asxl1 deletion results in a decrease in RNAPII promoter-proximal pausing in granulocyte progenitors, indicative of a global increase in productive transcription. These results suggest that ASXL1 inhibits productive transcription in granulocyte progenitors, identifying a new role for this epigenetic regulator in myeloid development.

Mutations in SET binding protein 1 (SETBP1) are associated with poor outcomes in myeloid leukemias. In the Ras-driven leukemia, juvenile myelomonocytic leukemia, SETBP1 mutations are enriched in relapsed disease. While some mechanisms for SETBP1-driven oncogenesis have been established, it remains unclear how SETBP1 specifically modulates the biology of Ras-driven leukemias. In this study, we found that when co-expressed with Ras pathway mutations, SETBP1 promoted oncogenic transformation of murine bone marrow in vitro and aggressive myeloid leukemia in vivo. We demonstrate that SETBP1 enhances the NRAS gene expression signature, driving upregulation of mitogen-activated protein kinase (MAPK) signaling and downregulation of differentiation pathways. SETBP1 also enhances NRAS-driven phosphorylation of MAPK proteins. Cells expressing NRAS and SETBP1 are sensitive to inhibitors of the MAPK pathway, and treatment with the MEK inhibitor trametinib conferred a survival benefit in a mouse model of NRAS/SETBP1-mutant disease. Our data demonstrate that despite driving enhanced MAPK signaling, SETBP1-mutant cells remain susceptible to trametinib in vitro and in vivo, providing encouraging pre-clinical data for the use of trametinib in SETBP1-mutant disease.

Mutations in the gene Additional Sex-Combs Like 1 (ASXL1) are recurrent in myeloid malignancies as well as the pre-malignant condition clonal hematopoiesis, where they are universally associated with poor prognosis. An epigenetic regulator, ASXL1 canonically directs the deposition of H3K27me3 via the polycomb repressive complex 2. However, its precise role in myeloid lineage maturation is incompletely described. We utilized single cell RNA sequencing (scRNA-seq) on a murine model of hematopoietic-specific ASXL1 deletion and identified a specific role for ASXL1 in terminal granulocyte maturation. Terminal maturation is accompanied by down regulation of Myc expression and cell cycle exit. ASXL1 deletion leads to hyperactivation of Myc in granulocyte precursors and a quantitative decrease in neutrophil production. This failure of normal developmentallyassociated Myc suppression is not accompanied by significant changes in the landscape of covalent histone modifications including H3K27me3. Examining the genome-wide localization of ASXL1 in myeloid progenitors revealed strong co-localization with RNA Polymerase II (RNAPII) at the promoters and spread across the gene bodies of transcriptionally active genes. ASXL1 deletion results in a decrease in RNAPII promoter-proximal pausing in granulocyte progenitors, indicative of a global increase in productive transcription, consistent with the known role of ASXL1 as a mediator of RNAPII pause release. These results suggest that ASXL1 inhibits productive transcription in granulocyte progenitors, identifying a new role for this epigenetic regulator and highlighting a novel potential oncogenic mechanism for ASXL1 mutations in myeloid malignancies.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset—accessible through the Beat AML data viewer (Vizome)—that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML. Analyses of samples from patients with acute myeloid leukaemia reveal that drug response is associated with mutational status and gene expression; the generated dataset provides a basis for future clinical and functional studies of this disease.

Internal Tandem Duplication mutations of Fms Related Receptor Tyrosine Kinase 3 (FLT3), known as FLT3-ITD mutations, are associated with poor prognosis in Acute Myeloid Leukemia (AML). The clinical efficacy of inhibiting FLT3 in AML is limited by the rapid development of drug resistance and relapse, underscoring a need for more potent and durable treatment strategies. The early persistence of leukemic blasts during FLT3 inhibition is a key driver of resistance. We find that in combination, inhibitors of Lysine Specific Demethylase 1 (LSD1) potentiate the activity of FLT3 inhibitors, driving synergistic cell death. This novel therapeutic approach has the potential to drive deeper therapeutic responses in FLT3-Mutant AML, delaying or preventing the development of resistance. LSD1 is a dynamic DNA-associated protein that functions as a chromatin modifier and transcription factor. LSD1 removes methylation on both lysine 4 of histone H3 (H3K4), associated with transcriptional activation, and lysine 9 (H3K9), associated with transcriptional repression. Additionally, LSD1 has been reported to function as a transcription factor independent of its catalytic demethylase function. LSD1 inhibition reduces cell proliferation in several cancer types. In AML specifically, inhibition of LSD1 has been reported to activate enhancers associated with genes that promote differentiation. We hypothesized that combining LSD1 inhibition with FLT3 inhibition in FLT3-ITD AML would result in synergistic effects on cell viability through reactivating differentiation pathways and more strongly blocking proliferation. In this study, we aimed to examine the efficacy, transcriptional effects, and changes in chromatin dynamics when combining LSD1 inhibition with FLT3 inhibition in a FLT3-ITD mutant cell line and patient samples. We used matrix combination screening to determine that combining the FLT3 inhibitor Quizartinib with LSD1 inhibitors (GSK-2879552 or ORY-1001) synergistically represses cell viability in the FLT3-ITD mutant MOLM-13 cell line and in multiple primary AML samples. RNA-seq followed by Gene Set Enrichment Analysis revealed that combining LSD1 and FLT3 inhibition synergistically represses target genes of the oncogenic transcription factor MYC. This finding was corroborated through high-throughput genome-wide profiling of histone marks, using the recently developed technique Cleavage Under Targets and Tagmentation (CUT&Tag). Specifically, we discovered several promoter regions in which acetylation of lysine 27 of Histone H3 (H3K27Ac), associated with transcriptional activation, was repressed by combining LSD1 and FLT3 inhibition. The genes associated with these regions were strongly enriched for known MYC target genes. Through additional genomic profiling methods including ChIP-seq and ATAC-seq, we have established potential roles for several DNA-binding transcription factors including CEBPA, RUNX1, STAT5, and LSD1 itself, that may mediate repression of MYC function resulting from combining LSD1 and FLT3 inhibition. Together, our work establishes LSD1 and FLT3 inhibitor combinations as a promising treatment strategy in FLT3-ITD AML. Importantly, this study identifies combined FLT3 and LSD1 inhibition as an effective strategy to indirectly target MYC function, as MYC is often referred to as an "undruggable" target. Furthermore, it has the potential to drive deeper molecular responses in FLT3-mutant AML, decreasing the likelihood of treatment resistance. Disclosures Druker: Bristol-Myers Squibb: Research Funding; Blueprint Medicines: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Cepheid: Consultancy, 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; Millipore (formerly Upstate Biotechnology): Patents & Royalties; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talks: Consultancy; Oregon Health & Science University: Patents & Royalties; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; MolecularMD (acquired by ICON): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Iterion Therapeutics (formerly Beta Cat Pharmaceuticals): Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics Inc. (formerly Lorus): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; GRAIL: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Research Funding; ALLCRON: Consultancy, 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; Dana-Farber Cancer Institute: Patents & Royalties; EnLiven: Consultancy, Research Funding. Maxson:Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. This finding of mutation-synergy is broadly applicable other mutations that activate the JAK/STAT pathway or disrupt CEBPA function (i.e. activating mutations in JAK3 and Core Binding Factor translocations). The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation, confirming predictions from clinical sequencing data. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity.

Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of a subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity., Acute Myeloid Leukemia (AML) develops following multiple mutations of differing impact. Here, the authors show that activating mutations of CSF3R co-operate with loss-of-function mutations of CEBPA to promote AML development through an enhancer-dependent mechanism.

Responses to kinase inhibitors in AML are short-lived with the inevitable development of resistance. Combination therapy with agents from distinct functional classes is one strategy to overcome this resistance. Using AML cell lines and primary patient samples harboring oncogenic cKIT mutations we demonstrate that inhibition of the epigenetic regulator Lysine-Specific Demethylase 1 (LSD1) markedly augments the cytotoxic effect of the KIT inhibitor avapritinib. Transcriptomic and epigenetic profiling revealed synergistic suppression of Myc activity accompanied by a global loss of acetylation and decreased expression of key leukemia cell cycle genes. This epigenetic reorganization results from disruption of key transcription factor networks responsible for maintaining leukemia cell self-renewal and survival. These findings demonstrate that combined KIT and LSD1 inhibition is an exciting new therapeutic avenue for KIT mutant AML. Previously, we have shown targeting the leukemic epigenome as a possible mechanism to augment the efficacy of kinase inhibitors. Thus, we hypothesize that targeting both the cKIT and CBF mutations with combination therapy will result in a deeper molecular response. Kasumi-1 cells harbor mutant cKIT, making an ideal model to study this combination. We observed a synergistic response to a clinically available KIT inhibitor, avapritinib, and two different LSD1 inhibitors, ORY-1001 and GSK-LSD1. Colony formation of healthy CD34+ cells and cell growth of cKIT WT cells were not significantly altered by the combination, thus showing the specificity of this combination by targeting mutant KIT. Bulk RNA-seq and master regulator analysis revealed greater suppression of Myc target genes with dual inhibition of KIT and LSD1. Using Cleavage Under Targets and Tagmentation (CUT&Tag), we observed synergistic loss of H3K27Ac at the promoters of genes necessary for cell proliferation. Additionally, we used Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to interrogate Myc localization, and found a greater loss of Myc binding with dual inhibition, overlapping with the loss of acetylation at these loci. This study demonstrates the synergistic efficacy of combination therapy and identifies key biomarkers for drug activity, namely loss of histone acetylation and Myc binding. Collectively, combined KIT and LSD1 inhibition may be an effective therapeutic approach for cKIT mutant AML. Citation Format: Brittany M. Smith, Daniel J. Coleman, Lauren Maloney, Cody Coblentz, Zachary Schonrock, Joseph Estabrook, Theresa Lusardi, Brian J. Druker, Julia E. Maxson, Theodore P. Braun. Combined inhibition of cKIT and Lysine-Specific Demethylase 1 as a therapeutic strategy in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1133.

Single Cell RNA Sequencing Identifies a Crucial Role for ASXL1 in Neutrophil Development Additional sex combs-like 1 (ASXL1) is a polycomb-associated protein that is essential for normal hematopoiesis. ASXL1 is recurrently mutated across the spectrum of myeloid malignancies including myelodysplastic syndromes, myeloproliferative neoplasms and Acute Myeloid Leukemia. ASXL1 mutations are also found in the premalignant disorders clonal hematopoiesis of indeterminate potential and clonal cytopenias of indeterminate potential. In all cases, ASXL1 mutations are associated with more aggressive disease biology and resistance to treatment. Mutations in ASXL1 broadly dysregulate the hematopoietic system, opening chromatin at genes associated with differentiation and self-renewal, predisposing to malignant transformation. However, in spite of this, the specific role of ASXL1 at different phases of hematopoiesis remains unknown. Indeed, the development of therapeutic approaches for ASXL1-mutant malignancies will require a nuanced understanding of the role of ASXL1 in directing normal blood development to maximize on target effects and minimize toxicity. ASXL1 mutations are commonly identified in myeloid disorders with dysplasia. In the neutrophil lineage, morphologic dysplasia is associated with nuclear-cytoplasmic dyssynchrony, where neutrophils demonstrate differences in nuclear and cytoplasmic differentiation (i.e. hypolobated nuclei or hypogranular cytoplasm). Given its associated with dysplasia, we hypothesized that ASXL1 plays a fundamental role in neutrophil maturation. To investigate this, we performed single cell RNA sequencing (scRNA-seq) on lineage depleted bone marrow from MX-1 Cre/Asxl1FL/FL mice (Asxl1KO) or cre negative littermate controls (Asxl1WT). This analysis revealed a loss of multi-lineage differentiation potential in response to Asxl1 deletion with the most prominent effects noted in myeloid differentiation. Although the neutrophil-primed granulocyte-macrophage progenitors appeared relatively normal, a differentiation block was identified at the transition between promyelocytes and myelocytes. Specifically, Asxl1KO mice demonstrated a failure to normally upregulate specific granule constituents. Although key differentiation-associated transcription factors are present in the appropriate precursor populations, they appear to require normal Asxl1 function to effectively initiate transcription of specific granule genes. This is the first description of a crucial role for Asxl1 in terminal neutrophil differentiation. Furthermore, the failure to effectively upregulate specific granule genes in Asxl1 deficient mice may provide a mechanistic explanation for the dysplasia-associated hypogranular neutrophils present in dysplastic disorders with mutant ASXL1. Disclosures Druker: Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; CureOne: Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Monojul: Other: former consultant; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Gilead Sciences: Other: former member of Scientific Advisory Board; Celgene: Consultancy; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding.

Acute Myeloid Leukemia (AML) results from the stepwise accumulation of mutations from distinct functional classes, ultimately culminating in malignant transformation. Based on their oncogenic activity, mutations can be classified into three distinct groups. Class I mutations activate signaling pathways, produce uncontrolled proliferation, and in isolation produce a myeloproliferative phenotype. Class II mutations result from point mutations or chromosomal translocation events in lineage determining transcription factors, producing differentiation arrest and myelodysplasia in isolation. A classic example of oncogene synergy between distinct mutational classes can be found in the co-occurrence of mutations in the transcription factor CCAAT-enhancer binding protein alpha (CEBPA) with mutations in colony stimulating factor receptor 3 (CSF3R). Mutations in CEBPA occur in approximately 10% of AML where they block differentiation and convey favorable risk. In contrast, CSF3R mutations lead to constitutive receptor activation and uncontrolled neutrophil proliferation. In the absence of co-occurring Class II mutations, membrane proximal CSF3R mutations produce the myeloproliferative neoplasm chronic neutrophilic leukemia (CNL). Interestingly, patients with CEBPA mutant AML that also harbor an oncogenic CSF3R mutation have worse prognosis than those with wild type CSF3R. However, the mechanism underlying this oncogene synergy remains unknown. To model the co-occurrence of these mutations, we expressed CSF3RT618I (The most common membrane proximal CSF3R mutation) in fetal liver hematopoietic stem cells harboring compound heterozygous CEBPA mutations in the endogenous allele (CEBPAK/L). Mice transplanted with mutant CEBPA alone developed a long latency AML with a median survival of 60 weeks. In contrast, mice transplanted with mutant CSF3RT618I/CEBPAK/L cells developed a much more rapid AML with a median survival of 13 weeks. These results were corroborated in an orthogonal model in which mutant CSF3R and a C-terminal mutant CEBPA were retrovirally expressed prior to bone marrow transplant. To dissect the underlying mechanism, we performed a comprehensive transcriptomic and epigenetic analysis on cells expressing each mutation in isolation as well as the combination. This analysis revealed that mutant CSF3R activates a distinct set of enhancers that regulate genes associated with differentiation and drive neutrophil differentiation. Co-expression of mutant CEBPA blocks the activation differentiation-associated enhancers but is permissive to those associated with proliferation. Differentiation but not proliferation-associated enhancers are bound by wild type CEBPA. Thus, the dominant negative impact of mutant CEBPA at these enhancers explains its differential impact on differentiative and proliferative transcriptional programs. Enhancer activation precedes promoter activation and CEBPA mutations are thought to represent early events in AML initiation. The epigenetic mechanism underlying the observed oncogene synergy argues that CEBPA mutations must occur prior to CSF3R to impact differentiation. We therefore developed a retroviral vector system enabling temporal control of Cre-mediated oncogene expression. Using this system, we found that only when mutant CEBPA is expressed prior to mutant CEBPA is differentiation arrest observed. Furthermore, AML develops in vivo only when mutant CEBPA is expressed prior to mutant CSF3R. To develop novel therapeutic strategies for this subclass of AML with adverse prognosis, we performed medium throughput drug screening on CSF3R/CEBPA mutant AML cells and identified sensitivity to inhibitors of JAK/STAT signaling as well as Lysine Demethylase 1 (LSD1). In other subtypes of AML, LSD1 inhibitors activate enhancers associated with differentiation. We confirmed that LSD1 inhibition promotes neutrophilic differentiation in CSF3R/CEBPA and through epigenetic and transcription profiling establish that this occurs via the reactivation of differentiation-associated enhancers. We further found that the combination of ruxolitinib (JAK/STAT inhibitor) and GSK2879552 produce a complete hematologic response and double median survival in mice harboring CSF3R/CEBPA mutant AML. Thus, the combination of JAK/STAT and LSD1 inhibitors represents and exciting therapeutic strategy for CSF3R/CEBPA mutant AML. Disclosures Druker: Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Beat AML LLC: Other: Service on joint steering committee; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; CureOne: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding.

Juvenile myelomonocytic leukemia (JMML) is an aggressive, rare form of early childhood leukemia driven by Ras pathway mutations. Mutations in SET binding protein 1 (SETBP1) are a strong predictor of relapse in JMML, and are associated with reduced five-year event-free survival. Although some mechanisms of oncogenesis have been established for SETBP1 mutations, it remains unclear why they are associated with poor prognosis and relapse. The goal of this study was to understand how SETBP1 modulates the biology of Ras-driven leukemias and to determine whether there are therapeutic vulnerabilities of SETBP1-JMML that can be exploited. Here, we present novel findings on the synergy of SETBP1 and NRAS, and provide pre-clinical evidence for therapeutic intervention. To address our central question of how SETBP1 mutations modulate Ras pathway-driven leukemia, we first set out to determine whether mutant SETBP1 promotes the growth of hematopoietic progenitors in the context of a Ras pathway mutation. To this end, we performed mouse hematopoietic colony forming unit assays in the absence of exogenous cytokines. Both NRAS-G12D and PTPN11-E76K alone formed a modest number of colonies, and the addition of SETBP1-D868N significantly augmented colony number with either Ras pathway mutation. The combination of NRAS-G12D and SETBP1-D868Nconfer robust serial replating, indicating that the SETBP1-D868N promotes oncogenic transformation and progenitor self-renewal in the NRAS-G12D mutant cells. To understand how SETBP1 modulates therapeutic response, a novel NRAS/SETBP1-mutant cell line was generated and analyzed with a chemical screen of essential cell growth and survival pathways. This screen revealed dependencies on the mTOR/AKT/PI3K and Raf/MEK/ERK pathways. An immunoblot analysis revealed that mutant SETBP1 enhanced NRAS-driven ERK and mTOR pathway activation. Inhibitors of these pathways, such as rapamycin and trametinib were highly efficacious against our cell line. The combination of trametinib and rapamycin had sub-nanomolar efficacy in our NRAS/SETBP1-hematopoietic cell line and exhibited greater than bliss additivity at several time points. To evaluate the efficacy in vivo, our SETBP1/NRAS-mutant cell line was injected into mice. At the onset of disease, mice were given once-daily treatment of trametinib, rapamycin, combination treatment, or DMSO control. The median survival of mice receiving DMSO was 19.5-days post-transplant, compared to 21 days for rapamycin, 35 days for the combination treatment, and 42 days for trametinib. Treatment with trametinib significantly increased the median survival to beyond rapamycin or DMSO, doubling the survival time of the mice. Our data demonstrates that SETBP1 mutations accelerate NRAS-driven oncogenesis and enhance MAPK pathway activation by NRAS-G12D. Despite enhanced transforming potential, SETBP1-mutant cells are still sensitive to inhibitors of the RAS/ERK/MAPK pathway. Trametinib, an inhibitor of this pathway, doubles overall survival in our murine model of NRAS/SETBP1-mutant leukemia, thus providing encouraging pre-clinical data for the use of trametinib in SETBP1-mutant disease. Disclosures No relevant conflicts of interest to declare.