Phosphoproteomic Analysis of Primary Acute Myeloid Leukemia Cells Reveals Redundant Roles for Src Family Kinases in AML Survival

Abstract

Recent results have demonstrated that multiple signal transduction pathways are activated in acute myeloid leukemia (AML) cells, however, the tyrosine kinase(s) that phosphorylates these signaling proteins is not identified. We have analyzed AML cells using a phosphoproteomics screen and demonstrate that the Src family kinases, Lyn, Lck and Fgr, are phosphorylated on their activation sites in AML samples. Expression and activation of Lyn has been previously confirmed. Evaluation of Lck demonstrated that Lck is expressed to a variable degree but consistently in AML samples (n=20). Lck kinase assays show activation of Lck in 17/20 samples tested at levels above the level of activation detectable in normal CD34+ progenitor cells. Lyn and Lck both contribute to AML cell growth as siRNA depletion of either kinase leads to decreased leukemia colony forming activity. Interestingly, both Lyn and Lck contribute to phosphorylation of STAT5 as STAT5 phosphorylation is decreased but not abrogated by siRNA modification of either kinase alone. Consistent with the necessity for this signaling pathway for optimal AML cell growth, siRNA knockdown of STAT5 leads to decreased expression of both STAT5A and STAT5B, decreased expression of the STAT5 target protein, Bclxl and decreased AML colony forming ability. Based on this data, we have studied the FDA approved compound, Dasatinib, and demonstrate that Dasatinib decreases AML colony formation in 4 out of 5 samples tested. Overall, these results demonstrate that SFK's act redundantly to regulate STAT5 phosphorylation and AML cell growth in primary cells and that phosphoprotein analysis is a robust approach to identify new targets for therapy of malignancy. Src family kinase inhibitors may be valuable in the therapy of AML.