Activation Mechanisms of STAT5 by Oncogenic Flt3-ITD.

Abstract

Mutations in the receptor tyrosine kinase Flt3 represent one of the most common known genetic lesions in AML. Internal tandem duplication (ITD) mutations clustered in the juxtamembrane domain are the most frequent and best characterized mutations found in Flt3. We and others have previously reported that oncogenic activation of Flt3 by ITD mutations activate aberrant signaling including activation of STAT5, up-regulation of STAT targets like-Pim and SOCS proteins and repression of the myeloid transcription factors Pu.1 and c/EBP-alpha (Mizuki et al., Blood 96: 3907–14, 2000; Mizuki et al., Blood 101: 3164–73, 2003). Earlier studies have observed activation of Src and Jak kinases by Flt3-ITD but the mechanisms of STAT5 activation by Flt3-ITD and the role of Src or Jak kinases in this process remains unclear. Using the specific small molecule inhibitors PP-1 and PP-2, we analyzed the role of Src kinases in Flt3-ITD mediated STAT5 activation. Treatment of Flt3-ITD expressing 32D cells with PP-1 and 2 had only marginal effect on Flt3-ITD induced STAT5 activation, which was due to a proportional inhibition of Flt3 kinase by these inhibitors. Similar results were obtained with AG490, reportedly a specific inhibitor of Jak2. However, use of cell a line deficient for all Src family kinases revealed a Src-independent activation of STAT5 by Flt3-ITD. Also, using cell lines deficient for Jak2 or Tyk2 we show that Flt3-ITD induced STAT5 activation independent of Jak2 or Tyk2 kinases. To rule out a possible redundant role of different Jak kinases in Flt3-ITD mediated STAT5 activation, we used SOCS1, a target of STAT5 and a potent molecular inhibitor of all Jak kinases. Surprisingly, retroviral transduction of 32D cells with SOCS1 inhibited IL-3 but not Flt3-ITD mediated proliferation of these cells. Similar results were obtained with SOCS3, another inhibitor of Jak kinases. Detailed signaling analyses of stable 32D cells co-expressing Flt3-ITD and SOCS1 revealed that activation of the STAT5 and up-regulation of STAT5 target gene Pim-2 by Flt3-ITD is completely resistant to SOCS1. In contrast, expression of SOCS1 severely inhibited IL-3 mediated STAT5 activation. Furthermore, using purified recombinant STAT5 and Flt3 kinase in in-vitro kinase assays, we show that STAT5 is indeed a direct target of Flt3. Taken together, our data show that Flt3-ITD directly activates STAT5 independent of Src or Jak kinases, providing the mechanistic basis of Flt3-ITD induced STAT5 activation. Furthermore, our finding that Flt3-ITD up-regulates SOCS proteins but activate STAT5 in a SOCS1 resistant manner may have important implications for Flt3-ITD-mediated modification of cytokine signaling.