IntroductionMany cases of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are characterized by an activating point mutation of JAK2 (JAK2 V617F ). It has been generally accepted that JAK2 V617F -positive cells outpace normal hematopoietic cells as a result of constitutively active growth factor signaling 1 ; however, failure of JAK2 V617F to confer a significant competitive advantage over normal hematopoiesis in 2 independent knock-in MPN models 2,3 suggests that additional factors may be required to promote expansion of JAK2 V617F -positive cells in patients.As MPN patients overproduce certain proinflammatory cytokines known to suppress normal hematopoiesis, 4 it is conceivable that JAK2 V617F may protect mutant stem cells and progenitors from the apoptotic cues induced by these cytokines.In this context, we recently observed that TNF␣ levels are elevated in mice with retrovirally induced JAK2 V617F MPN. 5 The physiologic effects of TNF␣ are complex and cell type-dependent, ranging from stimulation of proliferation to induction of apoptosis. 6 TNF␣ negatively regulates the expansion and self-renewal of pluripotent hematopoietic stem cells (HSCs) 7,8 and has inhibitory effects on normal as well as some leukemic human hematopoietic progenitor cells. [9][10][11] TNF␣'s involvement in the evolution of leukemia is not without precedent. Studies in Fanconi anemia (FA) have implicated TNF␣ hypersensitivity as a central mechanism of clonal evolution and progression to acute myeloid leukemia. In the FA Complementation Group C murine model (Fancc Ϫ/Ϫ ) TNF␣ induces bone marrow failure 12 and can promote the evolution of somatically mutated TNF␣-resistant preleukemic stem cell clones. 13 Taking into account TNF␣'s role in clonal evolution and that elevated TNF␣ levels are present in human MPN we hypothesized that JAK2 V617F induces TNF␣ expression and simultaneously confers TNF␣ resistance to MPN progenitor cells. Methods Isolation and culture of primary cellsBlood mononuclear cells (MNCs) were obtained from peripheral blood samples of patients with polycythemia vera (PV) and essential thromobocythemia (ET), myelofibrosis (MF), or normal volunteers. CD34 ϩ cells were obtained from bone marrow of normal, PV and ET patients or peripheral blood of MF patients. All patients gave their informed consent in accordance with the Declaration of Helsinki to participate in the study, which was approved by the Institutional Review Boards of Oregon Health & Science University (OHSU), Portland Veterans Affairs Medical Center, Cornell University, and Freiburg University.Submitted April 13, 2011; accepted August 7, 2011. Prepublished online as Blood First Edition paper, August 22, 2011; DOI 10.1182 DOI 10. /blood-2011 An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''adverti...

The JAK2V617F mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2 V617F

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gainor loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.Bcr-Abl, a constitutively active tyrosine kinase, is the defining molecular feature of chronic myeloid leukemia (CML) (8). Biochemical studies and murine models have established that tyrosine kinase activity is essential to the transforming capacity of Bcr-Abl (7, 17). Consistent with this, inhibition of the BcrAbl kinase with imatinib, an Abl-specific kinase inhibitor, induces remissions in patients with CML (9, 10). Although responses in the early phases of CML are frequently durable, relapse is common in patients with advanced-phase CML. Mutations in the kinase domain (KD) of Bcr-Abl that impair imatinib binding have been identified as the major mechanism of acquired resistance (1,3,13,15,34,37). However, in some patients, drug-resistant mutants were detected prior to therapy (16,27,28,34,38). The proportion of mutant allele in these samples varied, from being detectable only with allele-specific PCR, which could detect as few as one cell in 100,000 (38), to representing 40% of the BCR-ABL message (34). These data suggest that some mutants may have a proliferative advantage over unmutated Bcr-Abl (herein referred to as native Bcr-Abl) even in the absence of imatinib, while others may be loss-offunction alleles that are selected only in the presence of imatinib. Mutations of the ATP binding loop (P-loop) of Abl are associated with significantly shorter survival than other mutations, regardless of their sensitivity to imatinib (3, 36), suggesting that certain Bcr-Abl mutations may directly contribute to disease progression by conferring a more aggressive phenotype. To test these hypotheses, we performed various biochemical and biological assays to compare five common KD mutants that comprise ca. 60% of Bcr-Abl m...

Chronic myelogenous leukemia (CML) is characterized by the presence of a BcrAbl fusion protein with deregulated tyrosine kinase activity that is required for maintaining the malignant phenotype. Imatinib, a selective inhibitor of Bcr-Abl, induces major cytogenetic remission (MCR) or complete cytogenetic remission (CCR) in the majority of patients with CML in first chronic phase. However, thorough re-evaluation of cytogenetics in a cohort of patients in MCR or CCR demonstrated clonal karyotypic abnormalities in more than 10% of cases, some of which were clinically associated with a myelodysplastic syndrome (MDS). Further analysis identified previous exposure to cytarabine and idarubicin as significant risk factors for the subsequent occurrence of abnormalities in Philadelphia chromosome-negative (Ph ؊ ) cells. To investigate if cytogenetically normal but clonal hematopoiesis might be present in other patients in cytogenetic remission, we studied X-chromosome inactivation as a marker of clonality by polymerase chain reaction analysis of the human androgen receptor (HUMARA). We find that imatinib restores a polyclonal pattern in most patients in CCR and MCR. Nonetheless, our results are consistent with the notion that targeted therapy of CML with imatinib favors the manifestation of Ph ؊ clonal disorders in some patients. They indicate that patients on imatinib should be followed with conventional cytogenetics, even after induction of

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The CD19 antigen is a promising target for immunotherapy of acute lymphoblastic leukemia (ALL), but CD19 relapses remain a major challenge in about 10% to 20% of patients. Here, we analyzed 4 CD19 ALL relapses after treatment with the CD19/CD3 bispecific T-cell engager (BiTE) blinatumomab. Three were on-drug relapses, with the CD19 escape variant first detected after only 2 treatment courses. In 1 patient, the CD19 clone appeared as a late relapse 19 months after completion of blinatumomab treatment. All 4 cases showed a cellular phenotype identical to the primary diagnosis except for CD19 negativity. This argued strongly in favor of an isolated molecular event and against a common lymphoid CD19 progenitor cell or myeloid lineage shift driving resistance. A thorough molecular workup of 1 of the cases with early relapse confirmed this hypothesis by revealing a disrupted CD19 membrane export in the post-endoplasmic reticulum compartment as molecular basis for blinatumomab resistance.

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BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent ki-nases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF SKP2 (SKP1/Cul1/F-box), the E3 ubiq-uitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G 1 arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2 V617F , and TEL-PDGFR, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic ty-rosine kinases. Mice that received transplants of BCR-ABL-infected SKP2 / marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2 / marrow. SKP2 / leukemic cells demonstrated higher levels of nuclear p27 than SKP2 / counterparts, suggesting that the attenu-ation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF SKP2 may be therapeutically useful. (Blood.