BACKGROUND Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.)

SummaryIn contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6’s kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6’s central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a.

Key Points CDK6 acts as a transcriptional regulator to suppress Egr1 in HSCs and LSCs, allowing their activation. Cdk6 −/− HSCs fail to contribute to repopulation in competitive transplants, and BCR-ABLp210+Cdk6−/− LSCs fail to inflict disease.

In BCR-ABL1 ؉ leukemia, drug resistance is often associated with up-regulation of BCR-ABL1 or multidrug transporters as well as BCR-ABL1 mutations. Here we show that the expression level of the transcription factor STAT5 is another parameter that determines the sensitivity of BCR-ABL1 ؉ cells against tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, or dasatinib. Abelson-transformed cells, expressing high levels of STAT5, were found to be significantly less sensitive to TKI-induced apoptosis in vitro and in vivo but not to other cytotoxic drugs, such as hydroxyurea, interferon-␤, or Aca-dC. The STAT5-mediated protection requires tyrosine phosphorylation of STAT5 independent of JAK2 and transcriptional activity. In support of this concept, under imatinib treatment and with disease progression, STAT5 mRNA and protein levels increased in patients with Ph ؉ chronic myeloid leukemia. Based on our data, we propose a model in which disease progression in BCR-ABL1 ؉ leukemia leads to up-regulated STAT5 expression. This may be in part the result of clonal selection of cells with high STAT5 levels. STAT5 then accounts for the resistance against TKIs, thereby explaining the dose escalation frequently required in patients reaching accelerated phase. It also suggests that STAT5 may serve as an attractive target to overcome imatinib resistance in BCR-ABL1 ؉ leukemia. IntroductionMore than 99% of all patients with chronic myeloid leukemia (CML) and approximately 30% of acute lymphoid leukemia are characterized by the t(9;22)(q34;q11) translocation and the Philadelphia chromosome. Two chimeric oncogenic tyrosine kinase products, p185 BCR-ABL1 or p210 BCR-ABL1 , may be generated. 1,2 Whereas p210 BCR-ABL1 is associated with CML, p185 BCR-ABL1 is almost exclusively found in acute lymphoid leukemia. 3 The BCR-ABL1 oncoprotein promotes leukemia development by activating multiple signal transduction pathways that regulate cell proliferation, transformation, and survival. BCR-ABL1-induced acute lymphoid leukemia is characterized by an excess of lymphoblasts and progresses rapidly, whereas BCR-ABL1 ϩ CML is a stem cell-derived disease with distinct phases: chronic phase (CP), which may last for several years, accelerated phase (AP), and blast crisis (BC). 4 Therapy of BCR-ABL1-induced diseases was significantly improved by the development of small molecular weight inhibitors blocking the activity of the ABL1 kinase. Imatinib was the first substance, soon followed by other kinase inhibitors (TKIs), such as dasatinib and nilotinib. [5][6][7] All these TKIs target the enzymatic activity of the ABL1 tyrosine kinases. 6,8,9 Imatinib is now the standard first-line therapy for all CML patients. However, not all CML patients respond equally well. 10 Moreover, approximately 15% to 25% of the patients who initially responded well acquire resistance against imatinib during therapy. The percentage of nonresponders is even higher in AP. [10][11][12] These patients are treated with increased imatinib dosage (600-800 mg/day), secondge...

The G1 cell-cycle kinase CDK6 has long been thought of as a redundant homolog of CDK4. Although the two kinases have very similar roles in cell-cycle progression, it has recently become apparent that they differ in tissue-specific functions and contribute differently to tumor development. CDK6 is directly involved in transcription in tumor cells and in hematopoietic stem cells. These functions point to a role of CDK6 in tissue homeostasis and differentiation that is partially independent of CDK6's kinase activity and is not shared with CDK4. We review the literature on the contribution of CDK6 to transcription in an attempt to link the new findings on CDK6's transcriptional activity to cell-cycle progression. Finally, we note that anticancer therapies based on the inhibition of CDK6 kinase activity fail to take into account its kinase-independent role in tumor development.