Cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2 hydroxyglutarate (2HG) production. Mutational and epigenetic profiling of a large AML patient cohort revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. Furthermore, expression of 2HG-producing IDH alleles in cells induced global DNA hypermethylation. In the AML cohort, IDH1/2 mutations were mutually exclusive with mutations in the α-ketoglutarate-dependent enzyme TET2, and TET2 loss-of-function mutations associated with similar epigenetic defects as IDH1/2 mutants. Consistent with these genetic and epigenetic data, expression of IDH mutants impaired TET2 catalytic function in cells. Finally, either expression of mutant IDH1/2 or Tet2 depletion impaired hematopoietic differentiation and increased stem/progenitor cell marker expression, suggesting a shared proleukemogenic effect.

The identification of somatic activating mutations in JAK21–4 and in the thrombopoietin receptor (MPL)5 in the majority of myeloproliferative neoplasm (MPN) patients led to the clinical development of JAK2 kinase inhibitors6,7. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, but does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic JAK2 inhibition. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible, such that JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, murine models, and patients treated with JAK2 inhibitors. RNA interference and pharmacologic studies demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors.

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.

Circulating exosomes contain a wealth of proteomic and genetic information, presenting an enormous opportunity in cancer diagnostics. While microfluidic approaches have been used to successfully isolate cells from complex samples, scaling these approaches for exosome isolation has been limited by the low throughput and susceptibility to clogging of nanofluidics. Moreover, the analysis of exosomal biomarkers is confounded by substantial heterogeneity between patients and within a tumor itself. To address these challenges, we developed a multichannel nanofluidic system to analyze crude clinical samples. Using this platform, we isolated exosomes from healthy and diseased murine and clinical cohorts, profiled the RNA cargo inside of these exosomes, and applied a machine learning algorithm to generate predictive panels that could identify samples derived from heterogeneous cancer-bearing individuals. Using this approach, we classified cancer and precancer mice from healthy controls, as well as pancreatic cancer patients from healthy controls, in blinded studies.

Platelets play a key role in atherogenesis and its complications. Both hypercholesterolemia and increased platelet production promote athero-thrombosis; however, a potential link between altered cholesterol homeostasis and platelet production has not been explored. Transplantation of bone marrow (BM) deficient in ABCG4, a transporter of unknown function, into Ldlr−/− mice resulted in thrombocytosis, accelerated thrombosis and atherosclerosis. While not detected in lesions, Abcg4 was highly expressed in BM megakaryocyte progenitors (MkP). Abcg4−/− MkPs displayed defective cholesterol efflux to HDL, increased cell surface levels of thrombopoietin (TPO) receptor (c-MPL) and enhanced proliferation. This appeared to reflect disruption of the negative feedback regulation of c-MPL levels and signaling by E3 ligase c-CBL and cholesterol-sensing LYN kinase. HDL infusions reduced platelet counts in Ldlr−/− mice and in a mouse model of myeloproliferative neoplasm, in a completely ABCG4-dependent fashion. HDL infusions may offer a novel approach to reducing athero-thrombotic events associated with increased platelet production.