The identification of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK kinase inhibitors, including ruxolitinib. Ruxolitinib reduces splenomegaly and systemic symptoms in myelofibrosis (MF) and improves overall survival; however the mechanism by which JAK inhibitors achieve efficacy has not been delineated. MPN patients present with increased levels of circulating pro-inflammatory cytokines, which are mitigated by JAK inhibitor therapy. We sought to elucidate mechanisms by which JAK inhibitors attenuate cytokine-mediated pathophysiology. Single cell profiling demonstrated that hematopoietic cells from MF models and patient samples aberrantly secrete inflammatory cytokines. Pan-hematopoietic Stat3 deletion reduced disease severity and attenuated cytokine secretion, with similar efficacy as observed with ruxolitinib therapy. By contrast, Stat3 deletion restricted to MPN cells did not reduce disease severity or cytokine production. Consistent with these observations, we found that malignant and non-malignant cells aberrantly secrete cytokines and JAK inhibition reduces cytokine production from both populations.
Although the majority of acute myeloid leukemia (AML) patients initially respond to chemotherapy, many patients subsequently relapse; the mechanistic basis for AML persistence following chemotherapy has not been delineated. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3Amut), are observed in AML1–3 and in individuals with clonal hematopoiesis in the absence of leukemic transformation4,5. DNMT3Amut AML patients have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy6,7, suggesting that DNMT3Amut cells persist and drive relapse8. Here we show that Dnmt3amut induces hematopoietic stem cell (HSC) expansion, cooperates with Flt3ITD and Npm1c to induce AML in vivo, and promotes resistance to anthracycline chemotherapy. In AML patients, DNMT3AR882 mutations predict for minimal residual disease (MRD), underscoring their role in AML chemoresistance. DNMT3Amut cells show impaired nucleosome eviction and chromatin remodeling in response to anthracyclines, resulting from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect leads to an inability to sense and repair DNA torsional stress, which results in increased mutagenesis. Our studies identify a critical role for DNMT3AR882 mutations in driving AML chemoresistance, and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.
DNMT3A mutations are observed in myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). Transplantation studies have elucidated an important role for Dnmt3a in stem cell self-renewal and in myeloid differentiation. Here we investigated the impact of conditional hematopoietic Dnmt3a loss on disease phenotype in primary mice. Mx1-Cre-mediated Dnmt3a ablation led to the development of a lethal, fully penetrant myeloproliferative neoplasm with myelodysplasia (MDS/MPN) characterized by peripheral cytopenias and by marked, progressive hepatomegaly. We detected expanded stem/progenitor populations in the liver of Dnmt3a-ablated mice. The MDS/MPN induced by Dnmt3a ablation was transplantable, including the marked hepatomegaly. Homing studies showed that Dnmt3a-deleted bone marrow cells preferentially migrated to the liver. Gene expression and DNA methylation analyses of progenitor cell populations identified differential regulation of hematopoietic regulatory pathways, including fetal liver hematopoiesis transcriptional programs. These data demonstrate that Dnmt3a ablation in the hematopoietic system leads to myeloid transformation in vivo, with cell autonomous aberrant tissue tropism and marked extramedullary hematopoiesis (EMH) with liver involvement. Hence, in addition to the established role of Dnmt3a in regulating self-renewal, Dnmt3a regulates tissue tropism and limits myeloid progenitor expansion in vivo.
• Jak2 deletion in PLTs andMKs leads to thrombocytosis due to dysregulated TPO turnover.• Jak2 loss in PLTs/MKs induces non-autonomous expansion of stem/progenitors, and specifically of MK-primed hematopoietic stem cells (HSCs).JAK inhibitor treatment is limited by the variable development of anemia and thrombocytopenia thought to be due to on-target JAK2 inhibition. We evaluated the impact of Jak2 deletion in platelets (PLTs) and megakaryocytes (MKs) on blood counts, stem/ progenitor cells, and Jak-Stat signaling. Pf4-Cre-mediated Jak2 deletion in PLTs and MKs did not compromise PLT formation but caused thrombocytosis, and resulted in expansion of MK progenitors and Lin 2 Sca1 1 Kit 1 cells. Serum thrombopoietin (TPO) was maintained at normal levels in Pf4-Cre-positive Jak2 f/f mice, consistent with reduced internalization/ turnover by Jak2-deficient PLTs. These data demonstrate that Jak2 in terminal megakaryopoiesis is not required for PLT production, and that Jak2 loss in PLTs and MKs results in non-autonomous expansion of stem/progenitors and of MKs and PLTs via dysregulated TPO turnover. This suggests that the thrombocytopenia frequently seen with JAK inhibitor treatment is not due to JAK2 inhibition in PLTs and MKs, but rather due to JAK2 inhibition in stem/progenitor cells. (Blood. 2014;124(14):2280-2284
Background:Malignant transformation requires the interaction of cancer cells with their microenvironment, including infiltrating leukocytes. However, somatic mutational studies have focused on alterations in cancer cells, assuming that the microenvironment is genetically normal. Because we hypothesized that this might not be a valid assumption, we performed exome sequencing and targeted sequencing to investigate for the presence of pathogenic mutations in tumor-associated leukocytes in breast cancers.Methods:We used targeted sequencing and exome sequencing to evaluate the presence of mutations in sorted tumor-infiltrating CD45-positive cells from primary untreated breast cancers. We used high-depth sequencing to determine the presence/absence of the mutations we identified in breast cancer-infiltrating leukocytes in purified tumor cells and in circulating blood cells.Results:Capture-based sequencing of 15 paired tumor-infiltrating leukocytes and matched germline DNA identified variants in known cancer genes in all 15 primary breast cancer patients in our cohort. We validated the presence of mutations identified by targeted sequencing in infiltrating leukocytes through orthogonal exome sequencing. Ten patients harbored alterations previously reported as somatically acquired variants, including in known leukemia genes (DNTM3A, TET2, and BCOR). One of the mutations observed in the tumor-infiltrating leukocytes was also detected in the circulating leukocytes of the same patients at a lower allele frequency than observed in the tumor-infiltrating cells.Conclusions:Here we show that somatic mutations, including mutations in known cancer genes, are present in the leukocytes infiltrating a subset of primary breast cancers. This observation allows for the possibility that the cancer cells interact with mutant infiltrating leukocytes, which has many potential clinical implications.
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