Dual inhibition of Jak2 and STAT5 enhances killing of myeloproliferative neoplasia cells

M Bar-Natan, EA Nelson, SR Walker, Y Kuang… - Leukemia, 2012 - nature.com
M Bar-Natan, EA Nelson, SR Walker, Y Kuang, RJ Distel, DA Frank
Leukemia, 2012nature.com
Myeloproliferative neoplasms (MPNs) are a group of clonal disorders that arise from the
transformation of hematopoietic stem cells. In 2005, several groups reported a single
acquired point mutation in the Janus kinase 2 (Jak2) gene in the majority of patients with
Philadelphia chromosome (Ph)-negative MPN. 1 The mutation is believed to have a critical
role in the pathogenesis of these disorders, 2, 3 and it has been suggested that most
patients harbor mutations (some of which remain to be identified) that potentially serve as a …
Myeloproliferative neoplasms (MPNs) are a group of clonal disorders that arise from the transformation of hematopoietic stem cells. In 2005, several groups reported a single acquired point mutation in the Janus kinase 2 (Jak2) gene in the majority of patients with Philadelphia chromosome (Ph)-negative MPN. 1 The mutation is believed to have a critical role in the pathogenesis of these disorders, 2, 3 and it has been suggested that most patients harbor mutations (some of which remain to be identified) that potentially serve as a molecular target for selective Jak2 inhibition. 4 Although small molecule Jak2 inhibitors are entering clinical trials, their ultimate efficacy is unclear. 5 In addition to the concern of insufficient inhibition of mutated Jak2 in vivo or the emergence of resistance through activation of complementary pathways, many MPNs contain other mutational events (for example, mutation in exon12 of Mpl, 6 or the KIT D618V mutation in patients with systemic mastocytosis7), and thus are not sensitive to Jak inhibitors. Therefore, the development of inhibitors to common mediators of diverse signaling pathways in this disease is very desirable. One convergence point of these pathways is the transcription factor STAT5. STAT5 regulates the expression of genes controlling key events such as cell cycle progression and survival. Thus, continuous STAT5 activation drives increased expression of these genes that directly contribute to leukemogenesis. Continued STAT5 activation appears to be necessary for tumor cell survival, 8 although normal cells are generally tolerant to the loss of STAT5 function. Consequently, STAT5 is an attractive target for cancer therapy. Increasing evidence indicates that STAT5 activation is required for Jak2 V617F-mediated transformation. 9 As STAT5 is a critical mediator of the effects of Jak2 V617F, the development of drugs that inhibit this transcription factor holds promise as a treatment for MPN. Furthermore, the dual inhibition of both STAT5 and Jak2 may yield better disease control. We previously identified the neuroleptic drug pimozide as an inhibitor of STAT5 transcriptional function in a cell-based screen. 10 In the present study, we evaluated the effect of pimozide alone and in combination with a Jak2 inhibitor on the biology of myeloproliferative cell lines. We examined a model system, Ba/F3 cells that had been reconstituted with erythropoietin receptor and the mutant form of Jak2 (BAFEJ), as well as the HEL and SET2 human leukemia cell lines that endogenously express Jak2 V617F. Treatment of each cell line with pimozide led to a decrease in tyrosine phosphorylation of STAT5 (Figure 1a and data not shown). Reflecting the fact that the phosphorylation of STAT5 is mediated by activated Jak2, the Jak2 inhibitor Jak inhibitor 1 also led to decreased phosphorylation of STAT5. STAT3 phosphorylation was only minimally affected by pimozide (Figure 1a). Thus, the principal effect of pimozide in these MPN cell lines is the inhibition of STAT5 phosphorylation. Three lines of evidence suggest that pimozide is not functioning as a classic kinase inhibitor. First, pimozide does not inhibit Jak family kinases in an isolated in vitro kinase assays (Table 1). Second, its pattern of inhibition of substrate phosphorylation catalyzed by extracts from Jak2 V617F-expressing cells using the PamChip tyrosine kinase microarray system (PamGene, Den Bosch, Netherlands) 11 is clearly distinct from that mediated by a Jak inhibitor (including no inhibition of the phopshorylation of the Jak2 peptide by pimozide; Figure 1b). Pimozide did inhibit phosphorylation of a peptide derived from Jak1, though this likely occurred through an indirect mechanism …
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