Activating alleles of JAK3 in acute megakaryoblastic leukemia Denise K. Walters, 1,2,9 Thomas Mercher, 3,9 Ting-Lei Gu, 4,9 Thomas O’Hare, 1,2 Jeffrey W. Tyner, 2 Marc Loriaux, 5 Valerie L. Goss, 4 Kimberly A. Lee, 4 Christopher A. Eide, 2 Matthew J. Wong, 2 Eric P. Stoffregen, 2 Laura McGreevey, 6 Julie Nardone, 4 Sandra A. Moore, 3 John Crispino, 7 Titus J. Boggon, 8 Michael C. Heinrich, 2,6 Michael W. Deininger, 2 Roberto D. Polakiewicz, 4 D. Gary Gilliland, 3 and Brian J. Druker 1,2, * 1 Howard Hughes Medical Institute, Portland, Oregon 97239 2 Department of Hematology and Medical Oncology, Oregon Health & Science University, Portland, Oregon 97239 3 Brigham and Women’s Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115 4 Cell Signaling Technology Inc., 3 Trask Lane, Danvers, Massachusetts 01923 5 Department of Pathology, Oregon Health & Science University, Portland, Oregon 97239 6 Portland VA Medical Center, Portland, Oregon 97239 7 Ben May Institute for Cancer Research, University of Chicago, 924 East 57th Street, Chicago, Illinois 60637 8 Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, SHM B-302, New Haven, Connecticut 06520 9 These authors contributed equally to this work. *Correspondence: drukerb@ohsu.edu Summary 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 pro- teins 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. JAK3 A572V , JAK3 V722I , and JAK3 P132T each transform Ba/F3 cells to factor-inde- pendent growth, and JAK3 A572V 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. Introduction Tyrosine kinases comprise a family of 90 enzymes involved in the regulation of various cellular processes, including prolifera- tion, survival, differentiation, and motility (Krause and Van Etten, 2005). The activity of these kinases is normally tightly controlled. However, tyrosine kinases can become aberrantly activated by different mechanisms, including point mutation; fusion with un- related genes that lead to constitutive dimerization and activa- tion; and in the case of receptor tyrosine kinases, mutation in the juxtamembrane domain that results in constitutive kinase activation (Paul and Mukhopadhyay, 2004). Dysregulated tyro- sine 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, 2003; Tse et al., 2000; Yamamoto et al., 2001; Yokota et al., 1997). Despite the importance of dysregulated tyrosine kinases in cancer, the identification of specific oncogenes within complex activation pathways is difficult in malignant cells. Recently, pro- teomic approaches focusing on the phosphotyrosine content of the cell have demonstrated that large numbers of tyrosine-phos- phorylated proteins can be consistently identified in cancer cell lines. Indeed, Rush et al. (2005) and Walters et al. (2006) demon- strated that this methodology is capable of identifying activated tyrosine kinases even when the signaling pathways are un- known (Rush et al., 2005; Walters et al., 2006). A particularly interesting disease in which to search for acti- vated tyrosine kinases is AML. STAT5 has been found constitu- tively tyrosine-phosphorylated in the leukemic cells of approxi- mately 70% of AML patients (Birkenkamp et al., 2001; Hayakawa et al., 1998). The presence of FLT3 or KIT activating SIGNIFICANCE We used a mass spectrometry-based screen to identify activating alleles of JAK3 in acute megakaryoblastic leukemia (AMKL). Acti- vating alleles of JAK3 had not previously been detected in human cancer. The JAK3 A572V 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 muta- tions in cancer is warranted, and validates high-throughput mass spectrometry of phosphopeptides as a strategy for identification of therapeutic targets in cancer. A R T I C L E CANCER CELL 10, 65–75, JULY 2006 ª2006 ELSEVIER INC. DOI 10.1016/j.ccr.2006.06.002 65