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• W2119674879 abstract "Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3A572V, JAK3V722I, and JAK3P132T each transform Ba/F3 cells to factor-independent growth, and JAK3A572V confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations. Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3A572V, JAK3V722I, and JAK3P132T each transform Ba/F3 cells to factor-independent growth, and JAK3A572V confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations. We used a mass spectrometry-based screen to identify activating alleles of JAK3 in acute megakaryoblastic leukemia (AMKL). Activating alleles of JAK3 had not previously been detected in human cancer. The JAK3A572V mutation occurs at a conserved residue in the pseudokinase domain, providing mechanistic insights into the role of this domain in regulation of the JAK3 catalytic domain. Recent development of JAK3-selective small molecule inhibitors raises the possibility of therapeutic intervention in this subset of patients. This study thus provides insights into the molecular pathogenesis of AMKL, suggests that a more broadly based screen for JAK3 mutations in cancer is warranted, and validates high-throughput mass spectrometry of phosphopeptides as a strategy for identification of therapeutic targets in cancer. Tyrosine kinases comprise a family of 90 enzymes involved in the regulation of various cellular processes, including proliferation, survival, differentiation, and motility (Krause and Van Etten, 2005Krause D.S. Van Etten R.A. Tyrosine kinases as targets for cancer therapy.N. Engl. J. Med. 2005; 353: 172-187Crossref PubMed Scopus (1122) Google Scholar). The activity of these kinases is normally tightly controlled. However, tyrosine kinases can become aberrantly activated by different mechanisms, including point mutation; fusion with unrelated genes that lead to constitutive dimerization and activation; and in the case of receptor tyrosine kinases, mutation in the juxtamembrane domain that results in constitutive kinase activation (Paul and Mukhopadhyay, 2004Paul M.K. Mukhopadhyay A.K. Tyrosine kinase—Role and significance in Cancer.Int. J. Med. Sci. 2004; 1: 101-115Crossref PubMed Google Scholar). Dysregulated tyrosine kinases have been shown to have a significant role in a variety of cancers, including leukemia. In the case of acute myeloid leukemia (AML), mutations in FLT3 or c-KIT have been implicated as aberrations that confer a proliferative advantage to hematopoietic progenitors (Stirewalt and Radich, 2003Stirewalt D.L. Radich J.P. The role of FLT3 in haematopoietic malignancies.Nat. Rev. Cancer. 2003; 3: 650-665Crossref PubMed Scopus (670) Google Scholar, Tse et al., 2000Tse K.F. Mukherjee G. Small D. Constitutive activation of FLT3 stimulates multiple intracellular signal transducers and results in transformation.Leukemia. 2000; 14: 1766-1776Crossref PubMed Scopus (181) Google Scholar, Yamamoto et al., 2001Yamamoto Y. Kiyoi H. Nakano Y. Suzuki R. Kodera Y. Miyawaki S. Asou N. Kuriyama K. Yagasaki F. Shimazaki C. et al.Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies.Blood. 2001; 97: 2434-2439Crossref PubMed Scopus (936) Google Scholar, Yokota et al., 1997Yokota S. Kiyoi H. Nakao M. Iwai T. Misawa S. Okuda T. Sonoda Y. Abe T. Kahsima K. Matsuo Y. Naoe T. Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines.Leukemia. 1997; 11: 1605-1609Crossref PubMed Scopus (382) Google Scholar). Despite the importance of dysregulated tyrosine kinases in cancer, the identification of specific oncogenes within complex activation pathways is difficult in malignant cells. Recently, proteomic approaches focusing on the phosphotyrosine content of the cell have demonstrated that large numbers of tyrosine-phosphorylated proteins can be consistently identified in cancer cell lines. Indeed, Rush et al., 2005Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (927) Google Scholar and Walters et al., 2006Walters D.K. Goss V.L. Stoffregen E.P. Gu T.L. Lee K. Nardone J. McGreevey L. Heinrich M.C. Deininger M.W. Polakiewicz R. Druker B.J. Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes.Leuk Res. 2006; (Published online February 4, 2006)https://doi.org/10.1016/j.leukres.2006.01.001Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar demonstrated that this methodology is capable of identifying activated tyrosine kinases even when the signaling pathways are unknown (Rush et al., 2005Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (927) Google Scholar, Walters et al., 2006Walters D.K. Goss V.L. Stoffregen E.P. Gu T.L. Lee K. Nardone J. McGreevey L. Heinrich M.C. Deininger M.W. Polakiewicz R. Druker B.J. Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes.Leuk Res. 2006; (Published online February 4, 2006)https://doi.org/10.1016/j.leukres.2006.01.001Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A particularly interesting disease in which to search for activated tyrosine kinases is AML. STAT5 has been found constitutively tyrosine-phosphorylated in the leukemic cells of approximately 70% of AML patients (Birkenkamp et al., 2001Birkenkamp K.U. Geugien M. Lemmink H.H. Kruijer W. Vellenga E. Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts.Leukemia. 2001; 15: 1923-1931Crossref PubMed Scopus (117) Google Scholar, Hayakawa et al., 1998Hayakawa F. Towatari M. Iida H. Wakao H. Kiyoi H. Naoe T. Saito H. Differential constitutive activation between STAT-related proteins and MAP kinase in primary acute myelogenous leukaemia.Br. J. Haematol. 1998; 101: 521-528Crossref PubMed Scopus (62) Google Scholar). The presence of FLT3 or KIT activating mutations can account for STAT5 phosphorylation in up to 35% of patients. However, the mechanism of constitutive STAT5 phosphorylation remains unclear in a significant percentage of patients lacking these mutations. As STAT5 tyrosine phosphorylation is a marker for tyrosine kinase activation, we focused our investigations on AML cell lines with constitutive STAT5 phosphorylation for analysis by phosphopeptide mass spectrometry. Using this approach, we identified several activated tyrosine kinases in an acute megakaryoblastic leukemia (AMKL) cell line that lacked FLT3 or KIT mutations yet possessed constitutive STAT5 phosphorylation. Subsequent siRNA-induced downregulation experiments validated the role of candidate kinases identified by LC-MS/MS in the activation of STAT5. These studies guided DNA sequence analysis of candidate genes and enabled rapid identification of an A572V mutation in the pseudokinase domain of JAK3. Correlation of the structural location of A572 with biochemical data implicates the structural integrity of the pseudokinase catalytic cleft region as potentially important for proper JAK signaling. These findings provided a rationale for DNA analysis of the entire coding sequence of JAK3 in AMKL patients and resulted in identification of two additional JAK3 mutations. Each of these mutant JAK3 alleles resulted in constitutive activation of JAK3, phosphorylation of STAT5, and transformation of the hematopoietic cell line Ba/F3 to factor-independent growth. Furthermore, JAK3A572V recapitulated certain key features of megakaryoblastic leukemia in a murine model of disease. The baseline phosphorylation status of a spectrum of human AML cell lines was assessed, and STAT5 was constitutively phosphorylated in the AMKL cell line CMK (Figure 1A). CMK cells are a megakaryoblastic cell line derived from a patient with Down's syndrome and AMKL (Sato et al., 1989Sato T. Fuse A. Eguchi M. Hayashi Y. Ryo R. Adachi M. Kishimoto Y. Teramura M. Mizoguchi H. Shima Y. et al.Establishment of a human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute megakaryoblastic leukaemia.Br. J. Haematol. 1989; 72: 184-190Crossref PubMed Scopus (159) Google Scholar). Activating FLT3 or KIT mutations are commonly responsible for constitutive activation of STAT5 in AML; however, denaturing-HPLC analysis revealed that the CMK cell line does not possess activating mutations in FLT3 or KIT. To identify the upstream activator of STAT5, we used a phosphoproteomic approach in which CMK cell lysates were enzymatically digested with two different proteases to maximize the number of phosphopeptides identified after immunoprecipitation and subsequent analysis by LC-MS/MS mass spectrometry (Rush et al., 2005Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (927) Google Scholar). Using this approach, we identified a total of 348 nonredundant phosphopeptides corresponding to 274 nonredundant phosphorylation sites in 210 proteins (Table S1 in the Supplemental Data available with this article online). Among these proteins, we detected numerous phosphorylated peptides corresponding to tyrosine kinases (Table 1), including JAK3, which was found to be phosphorylated in two sites, including Y980, within the activation loop—an indication of JAK3 activation in these cells. Other kinases, such as TYK2, JAK2, c-Kit, and c-Abl were also found to be tyrosine phosphorylated in the CMK cell line by mass spectrometry.Table 1Tyrosine residues phosphorylated on kinases in CMK cellsProtein nameNCBI accession no.NCBI siteTrypsinChymotrypsinProtein kinaseA6rNP_009215§309•NM23NP_00026052•TIF1-βNP_005753458•Protein kinase, dual specificityDYRK1ANP_001387§145•DYRK1ANP_001387319•DYRK1ANP_001387§321•Protein kinase, ser/thr (nonreceptor)BcrNP_004318§644•Cdc2NP_001777§15••Cdc2NP_001777§19•CDK2NP_001789§15•CDK2NP_001789§19•CDK3NP_001249§15•CDK3NP_001249§19•CDK5NP_004926§15•DNA-PKNP_008835779•ERK1NP_002737§204•ERK2NP_002736§187•GSK3-βNP_002084§216••HIPK2NP_689909§352••HIPK3NP_005725§359••JNK1NP_002741§185•p38-αNP_001306§182••p38-γNP_002960185•PKCDNP_006245§313•PKCDNP_006245374•PRP4NP_003904§849••Protein kinase, tyrosine (nonreceptor)AblNP_005148§393••AckNP_005772§518••AckNP_005772§859•BtkNP_038510§223••BtkNP_038510225••BtkNP_038510344••BtkNP_038510361•FerNP_005237§714••FgrNP_005239§523•FynNP_002028§420•FynNP_002028§531•HckNP_002101§411•Jak2NP_004963§570•Jak3NP_000206§785•Jak3NP_000206§980•LckNP_005347§394•LynNP_002341194•LynNP_002341316••LynNP_002341473•LynNP_002341§508•Pyk2NP_004094§579•SrcNP_005408§530•SykNP_003168§323•Tyk2NP_003322§292•ZAP70NP_001070§292•ZAP70NP_001070§492•ZAP70NP_001070§493•Protein kinase, tyrosine (receptor)AxlNP_001690694•KitNP_000213§936•Tyrosine-phosphorylated kinases identified by mass spectrometry in CMK cells. §, published sites. Open table in a new tab Tyrosine-phosphorylated kinases identified by mass spectrometry in CMK cells. §, published sites. Activated JAK proteins are known to directly phosphorylate STAT proteins (Flores-Morales et al., 1998Flores-Morales A. Pircher T.J. Silvennoinen O. Gustafsson J.A. Sanchez-Gomez M. Norstedt G. Haldosen L.A. Wood T.J. In vitro interaction between STAT 5 and JAK 2; dependence upon phosphorylation status of STAT 5 and JAK 2.Mol. Cell. Endocrinol. 1998; 138: 1-10Crossref PubMed Scopus (20) Google Scholar, Fujitani et al., 1997Fujitani Y. Hibi M. Fukada T. Takahashi-Tezuka M. Yoshida H. Yamaguchi T. Sugiyama K. Yamanaka Y. Nakajima K. Hirano T. An alternative pathway for STAT activation that is mediated by the direct interaction between JAK and STAT.Oncogene. 1997; 14: 751-761Crossref PubMed Scopus (137) Google Scholar). Therefore, we next determined whether the pan-JAK inhibitor JAK Inhibitor I would affect the growth and viability of the CMK cell line. Treatment of CMK cells with the pan-JAK inhibitor resulted in a significant decrease in cell proliferation and viability (Figure 1B). However, treatment of CMK cells with imatinib, which inhibits both c-ABL and c-KIT, had little effect on CMK cell proliferation (Figure 1C). This indicated that JAK proteins, but not c-KIT or c-ABL, were essential for CMK growth and viability. Treatment of CMK cells with JAK Inhibitor I also impacted tyrosine phosphorylation of STAT5, p42/44 mitogen-activated protein (MAP) kinase, and STAT3 and was associated with an increase in the apoptotic death rate (Figures 1D and 1E). This effect could not be attributed to nonspecific toxicity of the inhibitor, as growth, viability, or STAT5 phosphorylation was not inhibited by the JAK Inhibitor I in K562 cells that express the BCR-ABL fusion protein. We next assessed whether JAK2, JAK3, or TYK2 contributed an essential role in growth and viability of CMK cells using a siRNA approach. Expression of JAK2, JAK3, or TYK2 was individually downregulated with specific siRNAs. Immunoblot analysis revealed that the expression of these proteins was specifically and significantly reduced by approximately 95% at 48 hr following transfection of the respective JAK family member siRNA into CMK cells (Figure 2A). We observed that downregulation of JAK3, but not JAK2 or TYK2, resulted in inhibition of CMK cell growth (Figure 2B). Moreover, treatment with JAK3 siRNA resulted in inhibition of STAT5 tyrosine phosphorylation and increased apoptosis of CMK cells, while downregulation of JAK2 or TYK2 had no effect (Figures 2A and 2C). In addition, we observed constitutively phosphorylated JAK3 in CMK cells following immunoprecipitation and phosphotyrosine immunoblot (Figure 2D). These findings indicated that activated JAK3 was essential for growth and survival of CMK cells. We next analyzed JAK3 for activating mutations. Constitutively phosphorylated JAK3 migrated at the expected molecular weight in CMK cells (Figure 2), indicating that point mutation rather than gene rearrangement was a likely mechanism for activation. Subsequent DNA sequence analysis identified a heterozygous C to T mutation at nucleotide position 1774 that is predicted to result in substitution of valine for alanine at amino acid position 572 in the JH2 domain of JAK3 (Figure 3A). To assess the transforming ability of the JAK3A572V mutation, we transduced Ba/F3 cells with either MSCV-IRES-EGFP (MIG)-JAK3WT or MIG-JAK3A572V. Twenty-four hours after transduction, GFP-expressing cells were selected by flow cytometry, plated in liquid culture in the absence of IL-3, and counted daily to assess IL-3-independent growth. As shown in Figure 3C, expression of JAK3A572V conferred IL-3-independent growth to Ba/F3 cells, whereas JAK3WT-transduced cells retained dependence on IL-3 for proliferation. In addition, JAK3A572V also conferred IL-3-independent growth to the 32D myeloid cell line (data not shown). Biochemical analysis in Ba/F3 cells revealed that JAK3 as well as downstream targets STAT5, phosphoinositol-3-kinase-Akt, and MAP kinase (p42/44) were each constitutively phosphorylated in cells transduced with JAK3A572V, but not in cells transduced with JAK3WT (Figure 3B). As a functional correlate to the observed increase in JAK3 phosphorylation, JAK3A572V exhibited increased kinase activity compared with JAK3WT in an in vitro immunoprecipitation kinase assay (Figure S1). Together, these results indicate that the JAK3A572V mutation results in constitutive activation of the JAK3 tyrosine kinase and can transform cytokine-dependent hematopoietic cell lines in vitro. These findings indicated that activating mutations in JAK3 contribute to the pathogenesis of AMKL. To further address this possibility, we screened 19 AMKL patient samples, of which 3 were derived from Down's syndrome and 16 from non-Down's syndrome patients. While none of these patients had the JAK3A572V mutation, two had other JAK3 mutations, one having a heterozygous V722I substitution in the JH2 pseudokinase domain and the second having a heterozygous P132T change in the JH6 domain of the receptor binding region (Figure 4A and Table S2). None of these 19 AMKL patient samples had the common myeloproliferative disorder mutation JAK2V617F. Of note, as for the AMKL patient from whom the CMK cell line was derived, the patient with the JAK3V722I allele had Down's syndrome and harbored a loss-of-function mutation in the GATA-1 gene (Wechsler et al., 2002Wechsler J. Greene M. McDevitt M.A. Anastasi J. Karp J.E. Le Beau M.M. Crispino J.D. Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome.Nat. Genet. 2002; 32: 148-152Crossref PubMed Scopus (579) Google Scholar). Transduction of Ba/F3 cells with MIG-JAK3WT, MIG-JAK3V722I, or MIG-JAK3P132T confirmed that, similar to JAK3A572V, the JAK3V722I and JAK3P132T alleles also conferred IL-3-independent growth to Ba/F3 cells as well as constitutive JAK3 and STAT5 phosphorylation (Figures 4B and 4C). We next developed a structural model to assess the role of the JAK3 mutations in constitutive kinase activation. The structure of the JAK3 JH1 catalytic domain, but not the JAK3 JH2 domain, has been solved (Boggon et al., 2005Boggon T.J. Li Y. Manley P.W. Eck M.J. Crystal structure of the Jak3 kinase domain in complex with a staurosporine analog.Blood. 2005; 106: 996-1002Crossref PubMed Scopus (143) Google Scholar). Although the JAK3 kinase JH2 (pseudokinase) domain lacks several residues critical to phosphotransferase activity, this domain is expected to adopt the overall protein architecture characteristic of tyrosine and serine/threonine kinases. We used the Swiss-model automated comparative protein modeling server to construct a homology model of the JAK3 JH2 domain using the crystal structures of the epidermal growth factor receptor (Stamos et al., 2002Stamos J. Sliwkowski M.X. Eigenbrot C. Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor.J. Biol. Chem. 2002; 277: 46265-46272Crossref PubMed Scopus (925) Google Scholar), insulin receptor (Parang et al., 2001Parang K. Till J.H. Ablooglu A.J. Kohanski R.A. Hubbard S.R. Cole P.A. Mechanism-based design of a protein kinase inhibitor.Nat. Struct. Biol. 2001; 8: 37-41Crossref PubMed Scopus (195) Google Scholar), Zap-70 (Jin et al., 2004Jin L. Pluskey S. Petrella E.C. Cantin S.M. Gorga J.C. Rynkiewicz M.J. Pandey P. Strickler J.E. Babine R.E. Weaver D.T. Seidl K.J. The three-dimensional structure of the ZAP-70 kinase domain in complex with staurosporine: implications for the design of selective inhibitors.J. Biol. Chem. 2004; 279: 42818-42825Crossref PubMed Scopus (51) Google Scholar), and Syk (Atwell et al., 2004Atwell S. Adams J.M. Badger J. Buchanan M.D. Feil I.K. Froning K.J. Gao X. Hendle J. Keegan K. Leon B.C. et al.A novel mode of Gleevec binding is revealed by the structure of spleen tyrosine kinase.J. Biol. Chem. 2004; 279: 55827-55832Crossref PubMed Scopus (175) Google Scholar) tyrosine kinases (Protein Data Bank accession codes 1M17, 1GAG, 1U59, and 1XBC, respectively) as a model. These tyrosine kinases have sequence identity to the JAK3 JH2 domain search sequence (Q501 to Q812) of 28%, 28%, 33%, and 33%, respectively. All four template crystal structures for the prediction of the JAK3 JH2 domain are active conformation tyrosine kinase domains. The model therefore assumes a conformation that is consistent with an active conformation kinase. The predicted structure of the JAK3 JH2 domain highlights the expected locations of two residues discussed in this study. A572 is predicted to lie in the pseudokinase domain C helix (Figure 5). The model predicts that A572 is on the cleft side of the C helix located at the same position as the catalytic glutamic acid in active kinase domains. While the preferred conformation of the JAK3 pseudokinase domain activation loop is not known at this point, the model presented here is in an active kinase conformation. Correlation of the structural location of A572 with the data presented in this paper would suggest a role for the “catalytic cleft” region of the pseudokinase domain in autoregulation. Alternatively, the activation loop may fold in an autoinhibited-like conformation, potentially packing against A572 in a manner that could be disrupted by an A572V mutation. The second residue that is located in the pseudokinase domain, V722, is predicted to be located C-terminal to the short kinase fold F helix (Figure 5). In the model, this residue is expected to be abutting the short D helix and partially surface exposed; however, a putative mechanism of activation for the V722I substitution is not readily apparent from the structural model. Hence, molecular modeling of the A572V mutation is consistent with functional data indicating an activating phenotype; however, numerous JAK3 pseudokinase domain mutations have been identified that induce loss-of-function phenotypes despite being constitutively phosphorylated (Chen et al., 2000Chen M. Cheng A. Candotti F. Zhou Y.J. Hymel A. Fasth A. Notarangelo L.D. O'Shea J.J. Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: evidence for interactions between the kinase and pseudokinase domains.Mol. Cell. Biol. 2000; 20: 947-956Crossref PubMed Scopus (106) Google Scholar, Notarangelo et al., 2001Notarangelo L.D. Mella P. Jones A. de Saint Basile G. Savoldi G. Cranston T. Vihinen M. Schumacher R.F. Mutations in severe combined immune deficiency (SCID) due to JAK3 deficiency.Hum. Mutat. 2001; 18: 255-263Crossref PubMed Scopus (77) Google Scholar). As such, it will be important to assess the position of these residues in the context of a solved structure of the composite JAK3 JH1/JH2 complex. We used a murine model to further characterize the transforming potential of JAK3A572V. C57BL/6 mice were transplanted with bone marrow (BM) cells that were retrovirally transduced with either MIG-JAK3WT or MIG-JAK3A572V. JAK3A572V mice exhibited significantly decreased survival compared with JAK3WT mice (Figure 6A) and showed several phenotypic characteristics of megakaryoblastic leukemia. There was splenomegaly in JAK3A572V versus JAK3WT animals (476 ± 94 mg versus 98 ± 6 mg, respectively). Multiparameter flow cytometry showed an approximate 8-fold expansion in the megakaryocyte-erythroid progenitors (MEP), defined as lineage−, c-KIT+, Sca1−, CD34low, and CD16/CD32low in spleens derived from JAK3A572V compared with JAK3WT animals. As a functional correlate of this finding, we observed an expansion in megakaryocyte colony formation in JAK3A572V compared with JAK3WT animals (Figure 6B). In addition, we detected abnormally high numbers of megakaryocytes infiltrated in the spleen and liver of JAK3A572V transplanted mice as determined by staining for the megakaryocyte-specific marker von Willebrand factor (vWF) (Figure 6D and Figure S2B). To further characterize the effect of JAK3A572V on megakaryocyte maturation, we assessed terminal differentiation by ex vivo culture of bone marrow cells from wild-type or mutant mice for 4 days in the presence of thrombopoietin (TPO) and stem cell factor (SCF). In both JAK3A572V and JAK3WT, mature proplatelet-forming megakaryocytes were observed in the cultures. Nevertheless, ploidy analysis on CD41+ cells showed a significant decrease in the median ploidy of megakaryocytes from JAK3A572V compared to JAK3WT mice, and an increase in megakaryocytes in S phase (Figure 6C). Despite the increased incidence of megakaryocytes in spleen and liver, JAK3A572V mice exhibited normal platelet counts in peripheral blood (Figure S2C). Also, secondary transplantation of either bone marrow or spleen cells into sublethally irradiated mice did not result in visible disease in the recipient animals (data not shown). Taken together, these findings indicate that JAK3A572V recapitulates several, but not all, of the phenotypic characteristics of AMKL, including splenomegaly, expansion of the megakaryocyte progenitors, and impaired megakaryocyte differentiation as assessed by analysis of polyploidization. In addition to the megakaryocyte phenotype in JAK3A572V-transduced animals, we observed marked leukocytosis that was comprised primarily of lymphocytes (Figure 6D). There was also lymphocytic infiltration of bone marrow, liver, spleen, lungs, and lymph nodes (Figure S2A and data not shown). Immunophenotypic analysis did reveal a marked expansion of CD8+ cells in the thymus, peripheral blood, bone marrow, and spleen in JAK3A572V compared with JAK3WT animals (Figure 6F and data not shown). These findings are consistent with a coexistent T cell lymphoproliferative disorder, in addition to the aberrancies in the megakaryocytic lineage induced by JAK3A572V. Using a screening strategy involving phosphopeptide immunoprecipitation followed by LC-MS/MS mass spectrometry, we have identified a JAK3 mutation, JAK3A572V, associated with human cancer in the megakaryoblastic cell line CMK. These findings prompted DNA sequence analysis of primary cells derived from patients with AMKL and identified two additional activating alleles of JAK3: JAK3V722I and JAK3P132T. Expression of these mutations transforms Ba/F3 cells to factor-independent growth that is associated with constitutive activation of the respective JAK3 allele in the absence of cytokine, and activation of known downstream effectors of JAK signaling, including STAT5, ERK, and AKT. While this report demonstrates an activating mutation of JAK3 associated with leukemia, several studies have previously suggested an association of JAK3 with cancer phenotypes in the absence of known mutations. Initially, JAK3 expression was found preferentially in hematopoietic cells, including samples of B cell lymphomas, supporting a possible involvement of JAK3 in transformation of hematopoietic cells (Tortolani et al., 1995Tortolani P.J. Lal B.K. Riva A. Johnston J.A. Chen Y.Q. Reaman G.H. Beckwith M. Longo D. Ortaldo J.R. Bhatia K. et al.Regulation of JAK3 expression and activation in human B cells and B cell malignancies.J. Immunol. 1995; 155: 5220-5226PubMed Google Scholar). Also, JAK3 splice variants were discovered in cancer cells from hematopoietic and epithelial origin, and JAK3 was found to be important for activation of the proto-oncogenes c-fos, c-myc, and bcl-2 (Kawahara et al., 1995Kawahara A. Minami Y. Miyazaki T. Ihle J.N. Taniguchi T. Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.Proc. Natl. Acad. Sci. USA. 1995; 92: 8724-8728Crossref PubMed Scopus (98) Google Scholar, Lai et al., 1995Lai K.S. Jin Y. Graham D.K. Witthuhn B.A. Ihle J.N. Liu E.T. A kinase-deficient splice variant of the human JAK3 is expressed in hematopoietic and epithelial cancer cells.J. Biol. Chem. 1995; 270: 25028-25036Crossref PubMed Scopus (48) Google Scholar). In addition, peripheral T lymphocytes infected and transformed with HTLV-1, a major cause of adult T cell leukemia, were subsequently shown to exhibit constitutively phosphorylated JAK3, and this event was shown to correlate with IL-2-independent growth (Migone et al., 1995Migone T.S. Lin J.X. Cereseto A. Mulloy J.C. O'Shea J.J. Franchini G. Leonard W.J. Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I.Science. 1995; 269: 79-81Crossref PubMed Scopus (500) Google Scholar, Xu et al., 1995Xu X. Kang S.H. Heidenreich O. Okerholm M. O'Shea J.J. Nerenberg M.I. Constitutive activation of different Jak tyrosine kinases in human T cell leukemia virus type 1 (HTLV-1) tax protein or virus-transformed cells.J. Clin. Invest. 1995; 96: 1548-1555Crossref PubMed Scopus (102) Google Scholar). Finally, oligomerization of JAK3 induced by fusion with TEL, although an artificial construct not found in patients so far, has been shown to constitutively activate JAK3, resulting in factor-independent growth of BaF3 cells as well as activation of downstream signaling partners, including STAT1, -3, and -5 (Lacronique et al., 2000Lacronique V. Boureux A. Monni R. Dumon S. Mauchauffe M. Mayeux P. Gouilleux F. Berger R. Gisselbrecht S. Ghysdael J. Bernard O.A. Transforming properties of chimeric TEL-JAK proteins in B" @default.
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