Letters to the Editor 1407 8 Yin W, Rossin A, Clifford JL, Gronemeyer H. Co-resistance to retinoic acid and 12 Green CL, Evans CM, Zhao L, Hills RK, Burnett AK, Linch DC et al. The prognostic TRAIL by insertion mutagenesis into RAM. Oncogene 2006; 25: 3735 -- 3744. significance of IDH2 mutations in AML depends on the location of the mutation. 9 Jenkins RB, Wrensch MR, Johnson D, Fridley BL, Decker PA, Xiao Y et al. Distinct Blood 2011; 118: 409 -- 412. germ line polymorphisms underlie glioma morphologic heterogeneity. Cancer 13 Boissel N, Nibourel O, Renneville A, Huchette P, Dombret H, Preudhomme C. Genet 2011; 204: 13 -- 18. Differential prognosis impact of IDH2 mutations in cytogenetically normal acute 10 Rice T, Zheng S, Xiao Y, Decker PA, McCoy LS, Smirnov I et al. Associations of myeloid leukemia. Blood 2011; 117: 3696 -- 3697. glioma risk loci by IDH mutation status. NeuroOncology 2011; 13(Suppl 3): iii27. 14 Patnaik MM, Hanson AA, Hodnefield JM, Lasho T, Finke C, Knudson RA et al. 11 Sanson M, Hosking FJ, Shete S, Zelenika D, Dobbins SE, Ma Y et al. Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic Chromosome 7p11.2 (EGFR) variation influences glioma risk. Hum Mol Genet syndromes: a Mayo Clinic study of 277 patients. Leukemia 2011; e-pub ahead of 2011; 20: 2897 -- 2904. print 28 October 2011; doi:10.1038/leu.2011.298. Dual inhibition of Jak2 and STAT5 enhances killing of myeloproliferative neoplasia cells Leukemia (2012) 26, 1407--1410; doi:10.1038/leu.2011.338; the principal effect of pimozide in these MPN cell lines is the published online 2 December 2011 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). Myeloproliferative neoplasms (MPNs) are a group of clonal Second, its pattern of inhibition of substrate phosphorylation disorders that arise from the transformation of hematopoietic catalyzed by extracts from Jak2 V617F-expressing cells using the stem cells. In 2005, several groups reported a single acquired point PamChip tyrosine kinase microarray system (PamGene, Den Bosch, mutation in the Janus kinase 2 (Jak2) gene in the majority of Netherlands)11 is clearly distinct from that mediated by a Jak patients with Philadelphia chromosome (Ph)-negative MPN.1 The inhibitor (including no inhibition of the phopshorylation of the mutation is believed to have a critical role in the pathogenesis of Jak2 peptide by pimozide; Figure 1b). Pimozide did inhibit these disorders,2,3 and it has been suggested that most patients phosphorylation of a peptide derived from Jak1, though this harbor mutations (some of which remain to be identified) that likely occurred through an indirect mechanism. Finally, other potentially serve as a molecular target for selective Jak2 inhibition.4 signaling pathways downstream of Jak2, such as the phosphor- Although small molecule Jak2 inhibitors are entering clinical trials, ylation of extracellular signal-regulated kinase mitogen-activated their ultimate efficacy is unclear.5 In addition to the concern of protein kinases (MAPK) are not inhibited by pimozide (as they are insufficient inhibition of mutated Jak2 in vivo or the emergence of with Jak inhibitor 1) in BAFEJ and HEL cells, and actually show a resistance through activation of complementary pathways, many small but reproducible increased activity (Figure 1c). This finding is MPNs contain other mutational events (for example, mutation in similar to that seen in cells transformed with BCR/ABL,10 and may exon12 of Mpl,6 or the KIT D618V mutation in patients with reflect a loss of a negative regulator of MAPK signaling that is systemic mastocytosis7), and thus are not sensitive to Jak inhibitors. STAT5 dependent.12 Although the exact mechanism by which Therefore, the development of inhibitors to common mediators of pimozide inhibits STAT5 phosphorylation is being elucidated, it diverse signaling pathways in this disease is very desirable. may involve direct effects on STAT5 or on a negative regulator of One convergence point of these pathways is the transcription STAT5 activation rather than by targeting a kinase. factor STAT5. STAT5 regulates the expression of genes controlling Consistent with the central role of STAT5 activation in driving key events such as cell cycle progression and survival. Thus, expression of genes underlying the malignant nature of these continuous STAT5 activation drives increased expression of these cells, inhibition of STAT5 phosphorylation by pimozide also led genes that directly contribute to leukemogenesis. Continued to a decrease in expression of STAT5 target genes (Figure 1d). STAT5 activation appears to be necessary for tumor cell survival,8 Significantly, these include two important pro-survival genes, although normal cells are generally tolerant to the loss of STAT5 Bcl-xl and Mcl1. Bcl-xl appears to be a key mediator in promoting function. Consequently, STAT5 is an attractive target for cancer Jak2-mediated survival in SET2 cells13 as inhibition of Jak2 signaling therapy. Increasing evidence indicates that STAT5 activation is blocked STAT5-mediated regulation of Bcl-xl mRNA level and required for Jak2 V617F-mediated transformation.9 As STAT5 is a reduced its protein expression. Mcl1 is also a critical survival factor critical mediator of the effects of Jak2 V617F, the development of in MPN cells. Specific STAT5-docking domains have a major role in drugs that inhibit this transcription factor holds promise as a inducing Mcl1 expression driven by a mutated FLT3 containing an treatment for MPN. Furthermore, the dual inhibition of both STAT5 internal tandem duplication (FLT3-ITD), a mutation found in about and Jak2 may yield better disease control. 20% of acute myelogenous leukemia patients, independent of We previously identified the neuroleptic drug pimozide as an JAK2 activation.14 inhibitor of STAT5 transcriptional function in a cell-based screen.10 Given that pimozide inhibits STAT5 tyrosine phosphorylation In the present study, we evaluated the effect of pimozide alone and decreases STAT5 target genes expression, we next deter- and in combination with a Jak2 inhibitor on the biology of mined whether pimozide also affects the viability of cells with myeloproliferative cell lines. We examined a model system, Ba/F3 activating mutations of Jak2. Pimozide caused a dose-dependent cells that had been reconstituted with erythropoietin receptor and decrease in viability of the three cell lines, with IC50 (half maximal the mutant form of Jak2 (BAFEJ), as well as the HEL and SET2 inhibitory concentration) values ranging from 4 mM for BAFEJ and human leukemia cell lines that endogenously express Jak2 V617F. SET2 to 10 mM for HEL cells (Figure 1e and data not shown). As Treatment of each cell line with pimozide led to a decrease in expected, pimozide also inhibits survival of parental Ba/F3 cells, tyrosine phosphorylation of STAT5 (Figure 1a and data not which are known to be dependent on STAT5. shown). Reflecting the fact that the phosphorylation of STAT5 is To determine the mechanism by which pimozide decreases mediated by activated Jak2, the Jak2 inhibitor Jak inhibitor 1 also viable cell numbers, BAFEJ and HEL cells were treated with led to decreased phosphorylation of STAT5. STAT3 phosphoryla- pimozide for 24 h, after which they were permeabilized and tion was only minimally affected by pimozide (Figure 1a). Thus, stained with propidium iodide followed by flow cytometric & 2012 Macmillan Publishers Limited Leukemia (2012) 1402 -- 1448 Letters to the Editor 1408 Figure 1. Pimozide inhibits STAT5 phosphorylation, decreases expression of STAT5 target genes and reduces viability of MPN cells. (a) HEL cells were treated with vehicle, pimozide (10 mM) or Jak inhibitor 1 (1 mM) for 5 h. Immunoblots were performed with the indicated antibodies. (b) Peptide phosphorylation profiles of HEL cells treated with pimozide or JAK inhibitor 1. Relative phosphorylation of each peptide is shown compared with control (orange represents 0.99--0.75 activity, red represents 0.74--0.50 activity). (c) HEL cells were treated with vehicle, pimozide (10 mM) or Jak inhibitor 1 (1 mM) for 5 h, after which immunoblots were performed to phosphorylated MAP kinase and total MAP kinase. Normalized band intensities are indicated. (d) HEL cells were treated with vehicle, pimozide (10 mM) or Jak inhibitor 1 (0.8 mM) for 6 h, after which RNA was harvested, and expression of the indicated genes was measured using quantitative reverse transcriptase PCR and normalized to the expression of HPRT. (e) HEL cells were treated with pimozide or Jak inhibitor 1 at the indicated concentrations for 48 h, after which viable cell number was quantitated by ATP-dependent bioluminescence. roles performed both by mutated Jak2 and STAT5 in MPNs, we Table 1. Pimozide does not directly inhibit Jak tyrosine kinase activity hypothesized that dual inhibition of both Jak2 and STAT5 may lead to enhanced effects on myeloproliferative cells. When HEL Kinase % Inhibition of kinase activity cells were treated with pimozide or Jak inhibitor 1, each of the drugs alone led to a 50% reduction in tyrosine phosphorylation JAK1 6 JAK2 À3 of STAT5. However, when cells were treated with the combina- JAK2 JH1 JH2 À2 tion, there was near-complete loss of STAT5 phosphorylation JAK2 JH1 JH2 V617F À3 (Figure 2a). Reflecting this combinatorial effect on STAT5 JAK3 À7 phosphorylation, treatment of HEL cells with the combination of pimozide and Jak inhibitor 1 also led to a greater reduction in the The effect of pimozide on the activity of selected tyrosine kinases was level of the pro-survival protein Mcl1 (Figure 2b). It also led to a analyzed using the SelectScreen Kinase Profiling service. greater reduction in viable cells over a range of combinations in HEL cells as well as with the BAFEJ and SET2 cell lines (Figure 2c analysis.