ALBERT

Description: Acute myeloid leukemia (AML) is a deadly hematologic malignancy with poor prognosis, particularly in the elderly. Even among individuals with favorable-risk disease, approximately half will relapse with conventional therapy. In this clinical circumstance, the determinants of relapse are unclear, and there are no therapeutic interventions that can prevent recurrent disease. Mutations in the transcription factor CEBPA are associated with favorable risk in AML. However, mutations in the growth factor receptor CSF3R are commonly co-occurrent in CEBPA mutant AML and are associated with an increased risk of relapse. To develop therapeutic strategies for this disease subset, we performed medium-throughput drug screening on CEBPA/CSF3R mutant leukemia cells and identified sensitivity to inhibitors of lysine-specific demethylase 1 (LSD1). Treatment of CSF3R/CEBPA mutant leukemia cells with LSD1 inhibitors reactivates differentiation-associated enhancers driving immunophenotypic and morphologic differentiation. LSD1 inhibition is ineffective as monotherapy but demonstrates synergy with inhibitors of JAK/STAT signaling, doubling median survival in vivo. These results demonstrate that combined inhibition of JAK/STAT signaling and LSD1 is a promising therapeutic strategy for CEBPA/CSF3R mutant AML.

Description: 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.

Description: 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.

Description: SETBP1 (SET Binding Protein 1) mutations are associated with exceptionally poor prognosis in myeloid neoplasms. Despite this, SETBP1's role in oncogenesis remains incompletely understood. In this study, we find that SETBP1 leads to a marked upregulation of the Myc oncogene and associated pro-stem and progenitor programs through epigenetic dysregulation. We further identify an epigenetic modulatory drug that normalizes SETBP1-driven Myc overexpression and synergizes with disease-relevant therapy. SETBP1 has documented roles in both the regulation of tumor suppressor pathways and modulation of transcription. To understand how SETBP1 modulates leukemia biology and leverage this mechanistic insight to develop novel therapeutic strategies, we turned to a genetically well-defined model system, chronic neutrophilic leukemia (CNL). SETBP1 is mutated in approximately half of all cases of CNL, a myeloproliferative neoplasm characterized by the presence of signaling-activating mutations in Colony Stimulating Factor 3 Receptor (CSF3R). By expressing SETBP1 and CSF3R mutations in mouse hematopoietic progenitors, we have generated models of CNL that can be leveraged for mechanistic studies and drug development. In a hematopoietic colony forming unit (CFU) assay, murine hematopoietic progenitors co-expressing mutant SETBP1 and mutant CSF3R have a high proliferation phenotype compared to those with mutant CSF3R alone. When cells co-expressing mutant SETBP1 and CSF3R are transplanted into lethally irradiated mice, they develop a rapidly lethal disease relative to the control mice. This is associated with an expansion of the granulocyte lineage, quantified by complete blood count, flow cytometry and histology. We find that SETBP1 is essential for the induction of a pro-proliferative transcriptional program. One of the most prominent SETBP1-associated signatures is that of Myc target genes. Myc itself is one of the top differentially expressed genes driven by SETBP1. Congruent with its increased expression, we also find higher Myc transcriptional activity in cells overexpressing SETBP1. To better understand the epigenetic regulation of Myc and progenitor pathways by SETBP1, we employed a low input profiling methods called CUT&Tag to assess the activation of regulatory elements. We began by profiling two epigenetic marks associated with active enhancers-H3K4me1 and H3K27Ac. We also assessed activation of the Myc promoter by measuring H3K27Ac and H3K4me3. Together this data helps us to understand the epigenetic underpinnings for dysregulation of Myc-driven programs by SETBP1. Therapeutic strategies that normalize aberrant Myc activity may be effective against SETBP1-driven disease. We found that SETBP1 and CSF3R transformed hematopoietic progenitors are highly sensitive to inhibitors of the epigenetic regulator lysine specific demethylase 1 (LSD1). LSD1 inhibition restores Myc expression to physiological levels and represses Myc promotor activity in vitro. Furthermore, LSD1 inhibitors are highly synergistic with JAK inhibitors, which block signaling downstream of CSF3R. Together these data establish the importance of Myc activation in SETBP1-driven malignancies and identify a therapeutic approach to normalize aberrant Myc activity. In future, we will use the insight gained in this genetically well defined disease to inform studies on the mechanistic basis and therapeutic vulnerabilities of other SETBP1-mutant myeloid malignancies. Disclosures Maxson: Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.