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Malgorzata Nieborowska-Skorska^*, Mariusz A. Wasik, Artur Slupianek^*, Paolo Salomoni^*, Toshio Kitamura, Bruno Calabretta^*, and Tomasz Skorski^*

From the ^* Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and the Department of Hematopoietic Factors, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan

Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor–dependent myeloid precursor cells, STAT5 activation–deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation–deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor–independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor–independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation–defective BCR/ABL SH3+SH2 mutants to induce growth factor–independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor–independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more than one domain and document the important role of STAT5-regulated pathways in BCR/ABL leukemogenesis.

Key Words: oncoprotein • domains • cooperation • transformation • leukemia

Abbreviations used: aa, amino acid(s); BrdU, 5-bromo-2'-deoxyuridine; CML, chronic myelogenous leukemia; CNS, central nervous system; DAM, dominant-active mutant; DNM, dominant-negative mutant; EMSA, electrophoretic mobility shift assay; PI-3k, phosphatidylinositol-3 kinase; P.Tyr, phosphotyrosine; RT, reverse transcription; SH, src homology; STAT, signal transducer and activator of transcription; WT, wild-type.

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