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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.

SUMMARY Studies on the role of TP53 mutation in breast cancer response to chemotherapy are conflicting. Here, we show that, contrary to dogma, MMTV-Wnt1 mammary tumors with mutant p53 exhibited a superior clinical response compared to tumors with wild-type p53. Doxorubicin-treated p53-mutant tumors failed to arrest proliferation leading to abnormal mitoses and cell death, while p53 wild-type tumors arrested, avoiding mitotic catastrophe. Senescent tumor cells persisted, secreting senescence-associated cytokines that exhibited autocrine/paracrine activity and mitogenic potential. Wild-type p53 still mediated arrest and inhibited drug response even in the context of a heterozygous p53 point mutation or absence of p21. Thus, we show wild-type p53 activity hinders chemotherapy response and demonstrate the need to reassess the paradigm for p53 in cancer therapy.

A B S T R A C T PurposeWe conducted a phase II study of pegylated interferon alfa-2a (PEG-IFN-␣-2a) in patients with essential thrombocythemia (ET) and polycythemia vera (PV). Patients and MethodsSeventy-nine patients (40 with PV and 39 with ET) have been treated. Median time from diagnosis to PEG-IFN-␣-2a was 54 months in patients with PV and 33 months in patients with ET. Eighty-one percent of patients had received prior therapy. The first three patients received PEG-IFN-␣-2a at 450 g weekly. As a result of poor tolerance, this dose was decreased in a stepwise manner to a current starting dose of 90 g weekly. Seventy-seven patients are evaluable and have been observed for a median of 21 months. ResultsThe overall hematologic response rate was 80% in PV and 81% in ET (complete in 70% and 76% of patients, respectively). The JAK2 V617F mutation was detected in 18 patients with ET and 38 patients with PV; sequential measurements by a pyrosequencing assay were available in 16 patients with ET and 35 patients with PV. The molecular response rate was 38% in ET and 54% in PV, being complete (undetectable JAK2 V617F ) in 6% and 14%, respectively. The JAK2 V617F mutant allele burden continued to decrease with no clear evidence for a plateau. The tolerability of PEG-IFN-␣-2a at 90 g weekly was excellent. ConclusionPEG-IFN-␣-2a resulted in remarkable clinical activity, high rates of molecular response, and acceptable toxicity in patients with advanced ET or PV. The ability of PEG-IFN-␣-2a to induce complete molecular responses suggests selective targeting of the malignant clone.

Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML. (1). Mutations in JAK2 have been identified in the majority of patients with PV, ET, and PMF (2-6), underscoring the importance of activated JAK-STAT signaling to the pathogenesis of chronicphase MPN. Despite the increasing use of empiric and targeted therapies, a subset of MPN patients transform to secondary acute myeloid leukemia (AML). Leukemic transformation occurs in 1%, 4%, and 20% of patients over a 10-y period in ET, PV, and PMF, respectively (7). MPN patients who develop leukemic transformation have a dismal outcome, with a median survival of less than 6 mo (8). Advanced age (>60 y) and exposure to chemotherapy increase the risk of leukemic transformation; however, the mechanisms and pathways that contribute to transformation from MPN to AML have not been well delineated. Importantly, the use of standard AML therapies, including induction chemotherapy, has not been shown to improve outcome for patients with post-MPN AML (8, 9). These data indicate a need for new models and improved therapeutic approaches to improve outcomes for patients who have transformed from MPN to AML and to identify genetic lesions associated with leukemic transformation.Genetic studies of paired samples before and after leukemic transformation have suggested there are at least two distinct routes for leukemic transformation. Some patients who present with a JAK2/MPL-positive MPN progress to JAK2/MPL-positive AML that is associated with the acquisition of additional genetic alterations (10-13). A second, more complex route to AML from MPN has been described in which a JAK2/MPL-positive MPN is followed by JAK2/MPL-negative AML (14, 15). Clonality studies using X-chromosome inactivation in informative females demonstrated that JAK2/MPL-positive MPN and JAK2/MPL-negative AML are clonally related, consistent with transformation of an antecedent, preJAK2...

Angiogenesis has been associated with the growth, dissemination, and metastasis of solid tumors. The aims of this study were to evaluate the vascularity and the levels of angiogenic factors in patients with acute and chronic leukemias and myelodysplastic syndromes (MDS). The numbers of blood vessels were measured in 145 bone marrow biopsies and the levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis growth factor-α (TNF-α), tumor growth factor-α (TGF-α), and hepatocyte growth factor (HGF) were determined in 417 plasma samples. Except for chronic lymphocytic leukemia (CLL), vascularity was significantly higher in all leukemias and MDS compared with control bone marrows. The highest number of blood vessels and largest vascular area were found in chronic myeloid leukemia (CML). VEGF, bFGF, and HGF plasma levels were significantly increased in acute myeloid leukemia (AML), CML, CLL, chronic myelomonocytic leukemia (CMML), and MDS. HGF, TNF-α, and bFGF but not VEGF were significantly increased in acute lymphoblastic leukemia (ALL). TNF-α levels were significantly increased in all diseases except for AML and MDS. No significant increase was found in TGF-α in any leukemia or MDS. The highest plasma levels of VEGF were in CML, and the highest plasma levels of bFGF were in CLL. The levels of HGF were highest in CMML. These data suggest that vascularity and angiogenic factors are increased in leukemias and MDS and may play a role in the leukemogenic process.

Key Points• Treatment with PEG- in PV and ET results in a high rate of complete hematologic and molecular responses.• Patients failing to achieve complete molecular remission tended to have higher frequencies of mutations in genes other than JAK2.Pegylated interferon a-2a (PEG-IFN-a-2a) has previously been shown to induce hematologic and molecular responses in patients with polycythemia vera (PV) or essential thrombocythemia (ET). Here we present a follow-up of a phase 2 trial with PEG-IFN-a-2a treatment in 43 PV and 40 ET patients with detailed molecular analysis. After a median follow-up of 42 months, complete hematologic response was achieved in 76% of patients with PV and 77% of those with ET. This was accompanied by complete molecular response (CMR) (ie, undetectable JAK2V617F) in 18% and 17%, of PV and ET patients, respectively. Serial sequencing of TET2, ASXL1, EZH2, DNMT3A, and IDH1/2 revealed that patients failing to achieve CMR had a higher frequency of mutations outside the Janus kinase-signal transducer and activator of transcription pathway and were more likely to acquire new mutations during therapy. Patients with both JAK2V617F and TET2 mutations at therapy onset had a higher JAK2V617F mutant allele burden and a less significant reduction in JAK2V617F allele burden compared with JAK2 mutant/TET2 wildtype patients. These data demonstrate that PEG-IFN-a-2a induces sustained CMR in a subset of PV or ET patients, and that genotypic context may influence clinical and molecular response to PEG-IFN-a-2a. (Blood. 2013;122(6):893-901)

The oncogenetic events that transform chronic myeloproliferative neoplasms (MPN) to acute myeloid leukemias (AML) are not well characterized. We investigated the role of several genes implicated in leukemic transformation by mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). Frequent mutations were identified in TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML, and all possible mutational combinations of these genes were also observed. Analysis of 14 patients for which paired samples from MPN and sAML were available showed that TET2 mutations were frequently acquired at leukemic transformation [6 of 14 (43%)]. In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. One case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a preexisting MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. Our findings argue that the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. Given the presence of sAML that have no preexisting JAK2/TET2/ASXL1/IDH1 mutations, our work indicates the existence of other mutations yet to be identified that are necessary for leukemic transformation. Cancer Res; 70(2); 447-52. ©2010 AACR.