Regulation of Signal Transducers and Activators of Transcription by Src Family Kinase in AML.

Abstract

Specific Signal Transducers and Activators of Transcription (STAT) family members are constitutively activated in leukemia where they contribute to leukemia self-renewal and apoptosis resistance. STAT5 activation in chronic myeloid leukemia (CML) is driven by the Bcr-Abl and Lyn kinases, whereas in acute myelogenous leukemia (AML), STAT5 and STAT3 activation is demonstrable under the control of as yet undefined upstream regulators. To investigate the regulation of STAT activation in AML, we evaluated the role of Src family kinases in AML cell lines and primary patient specimens. Inhibition of leukemia cell prolifaration by Src inhibitors was evaluated in 7 cell lines (U937, HEL, KG-1a, THP-1, OCI-AML3, HL-60 and NB-4). Treatment with either PD180970 or SKI-606 significantly suppressed proliferation and decreased viability in a concentration-dependent manner in two cell lines. U937 cells displayed sensitivity comparable to K562 CML cells, whereas KG-1a cells demonstrated moderate inhibition. Corresponding DNA-STAT5 binding activity assessed by electrophoretic mobility shift assay (EMSA) showed constitutively STAT5 activation in 3 cell lines (U937, HEL and KG-1a), with inhibition of STAT5 DNA binding By Src inhibitors in U937 and KG-1a cells, while STAT5 activity was not impaired in HEL cells. STAT5 protein phosphorylation assessed by immunoblot was suppressed by the Src inhibitors in the sensitive cell lines. Moreover, Src inhibition decreased Bcl-XL expression, a transcriptional target of STAT5, and induced apoptosis in U937 cells. Immunoblot assessment of phosphorylation of the activation loop tyrosine in the two major src kinases, c-Src and Lyn, confirmed inhibition of autophosphorylation in U937 cells and K562 cells by PD180970 and SKI-606, while phosphorylation was unchanged in HEL cells. Inhibition of src kinases was rapid (< 30 min), and preceeded inhibition of STAT5 activation. Analysis of primary AML patient cells revealed that 29 % (5 of 17) of patient cells have constitutively-activated STAT5, and 47 % (8 of 17) have constitutively-activated STAT3. More importantly, some primary AML patient cells were sensitive to Src inhibitor. These data suggest that Src activation of STAT5 has a pathogenetic role in AML cells. Investigation of Src inhibitors merits clinical investigation in patients with AML.