Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1.

Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase-binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

Robust patient-derived xenografts of MDS/MPN overlap syndromes capture the unique characteristics of CMML and JMML.

Chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) are myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap disorders characterized by monocytosis, myelodysplasia, and a characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Currently, there are no available disease-modifying therapies for CMML, nor are there preclinical models that fully recapitulate the unique features of CMML. Through use of immunocompromised mice with transgenic expression of human GM-CSF, interleukin-3, and stem cell factor in a NOD/SCID-IL2Rγnull background (NSGS mice), we demonstrate remarkable engraftment of CMML and JMML providing the first examples of serially transplantable and genetically accurate models of CMML. Xenotransplantation of CD34+ cells (n = 8 patients) or unfractionated bone marrow (BM) or peripheral blood mononuclear cells (n = 10) resulted in robust engraftment of CMML in BM, spleen, liver, and lung of recipients (n = 82 total mice). Engrafted cells were myeloid-restricted and matched the immunophenotype, morphology, and genetic mutations of the corresponding patient. Similar levels of engraftment were seen upon serial transplantation of human CD34+ cells in secondary NSGS recipients (2/5 patients, 6/11 mice), demonstrating the durability of CMML grafts and functionally validating CD34+ cells as harboring the disease-initiating compartment in vivo. Successful engraftments of JMML primary samples were also achieved in all NSGS recipients (n = 4 patients, n = 12 mice). Engraftment of CMML and JMML resulted in overt phenotypic abnormalities and lethality in recipients, which facilitated evaluation of the JAK2/FLT3 inhibitor pacritinib in vivo. These data reveal that NSGS mice support the development of CMML and JMML disease-initiating and mature leukemic cells in vivo, allowing creation of genetically accurate preclinical models of these disorders.

Targeting BET Proteins Downregulates miR-33a To Promote Synergy with PIM Inhibitors in CMML.

PURPOSE: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi. EXPERIMENTAL DESIGN: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. RESULTS: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. CONCLUSIONS: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination.

Somatic gene mutations expose cytoplasmic DNA to co-opt the cGAS/STING/NLRP3 axis in myelodysplastic syndromes.

NLRP3 inflammasome and IFN-stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in myelodysplastic syndromes (MDSs). Gene mutations involving mRNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDSs through undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS BM specimens, we showed accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor cyclic GMP-AMP synthase. Cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling caused ISG induction, NLRP3 inflammasome activation, and maturation of the effector protease caspase-1. Deregulation of RNA polymerase III drove cytosolic R-loop generation, which upon inhibition, extinguished ISG and inflammasome response. Mechanistically, caspase-1 degraded the master erythroid transcription factor, GATA binding protein 1, provoking anemia and myeloid lineage bias that was reversed by cGAS inhibition in vitro and in Tet2-/- hematopoietic stem and progenitor cell-transplanted mice. Together, these data identified a mechanism by which functionally distinct mutations converged upon the cGAS/STING/NLRP3 axis in MDS, directing ISG induction, pyroptosis, and myeloid lineage skewing.

TFEB links MYC signaling to epigenetic control of myeloid differentiation and acute myeloid leukemia.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML) and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.

RAS mutations drive proliferative chronic myelomonocytic leukemia via a KMT2A-PLK1 axis.

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

Effects of Corynebacterium bovis on Engraftment of Patient-derived Chronic-Myelomonocytic Leukemia Cells in NSGS Mice.

Modeling chronic myelomonocytic leukemia (CMML) in immunodeficient NSGS mice relies on unique human CMML specimens and consistent murine engraftment. Only anecdotal comments have thus far supported the notion that research data may be altered by Corynebacterium bovis, an opportunistic cutaneous pathogen of immunodeficient mice. C. bovis disseminated by asymptomatic and clinically affected mice with hyperkeratotic dermatitis, resulting in resilient facility contamination and infectious recurrence. Herein we report that, compared with C. bovis PCR-negative counterparts, C. bovis PCR-positive NSGS mice developed periocular and facial hyperkeratosis and alopecia and had reduced metrics indicative of ineffective human CMML engraftment, including less thrombocytopenia, less splenomegaly, fewer CMML infiltrates in histopathologic sections of murine organs, and fewer human CD45+ cells in samples from murine spleen, bone marrow, and peripheral blood that were analyzed by flow cytometry. All CMML model metrics of engraftment were significantly reduced in the C. bovis PCR-positive cohort compared with the - negative cohort. In addition, a survey of comprehensive cancer center practices revealed that most murine facilities do not routinely test for C. bovis or broadly decontaminate the facility or its equipment after a C. bovis outbreak, thus increasing the likelihood of recurrence of invalidated studies. Our findings document that CMML engraftment of NSGS mice is diminished-and the integrity of murine research data jeopardized-by C. bovis infection of immunodeficient mice. In addition, our results indicate that C. bovis should be excluded from and not tolerated in murine facilities housing immunodeficient strains.