Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr–Abl signal transduction and oncogenesis

Abstract

Chronic myelogenous leukemia (CML) is defined by the presence of the Philadelphia (Ph) chromosome, which results in the expression of the 210 kDa Bcr–Abl tyrosine kinase. Bcr-Abl constitutively activates several signaling proteins important for the proliferation and survival of myeloid progenitors, including the Src family kinases Hck and Lyn, the Stat5 transcription factor and upstream components of the Ras/Erk pathway. Recently, we found that kinase-defective Hck blocks Bcr–Abl-induced transformation of DAGM myeloid leukemia cells to cytokine independence, suggesting that activation of the Src kinase family may be essential to oncogenic signaling by Bcr–Abl. To investigate the contribution of Src kinases to Bcr–Abl signaling in vivo, we used the pyrrolo-pyrimidine Src kinase inhibitors PP2 and A-419259. Treatment of the Ph⁺ CML cell lines K-562 and Meg-01 with either compound resulted in growth arrest and induction of apoptosis, while the Ph⁻ leukemia cell lines TF-1 and HEL were unaffected over the same concentration ranges. Suppression of Ph⁺ cell growth by PP2 and A-419259 correlated with a decrease in Src kinase autophosphorylation. Both inhibitors blocked Stat5 and Erk activation, consistent with the suppressive effects of the compounds on survival and proliferation. In contrast, the phosphotyrosine content of Bcr–Abl and its endogenous substrate CrkL was unchanged at inhibitor concentrations that induced apoptosis, blocked oncogenic signaling and inhibited Src kinases. These data implicate the Src kinase family in Stat5 and Erk activation downstream of Bcr–Abl, and identify myeloid-specific Src kinases as potential drug targets in CML.