The hallmark of chronic myelogenous leukemia (CML) is a chromosomal translocation between the c-abl gene (chromosome 9) and the bcr gene (chromosome 22). This event gives rise to BcrAbl, a chimeric protein with constitutive tyrosine kinase activity that drives the pathogenesis of the disease. Imatinib, a Bcr-Abl kinase inhibitor is the frontline therapy in CML. Although imatinib is very effective in the chronic phase of CML, patients in advanced stages develop resistance. An increased understanding of the signaling pathways implicated in CML pathogenesis and imatinib resistance is critical to the development of improved therapies.
Previous work in our laboratory found that A-419259, a broad-spectrum Src family kinase (SFK) inhibitor induces growth arrest and apoptosis in CML cells, suggesting that SFKs are required for Bcr-Abl transformation of myeloid progenitors. Additionally, Hck couples BcrAbl to Stat5 activation in myeloid cells, which may contribute to survival. Furthermore, studies on samples from some imatinib-resistant patients found increased expression and activity of Hck and Lyn. In this dissertation, using two chemical genetic methods, I addressed the contribution of Hck to Bcr-Abl signaling and imatinib resistance. To explore the individual contribution of Hck to Bcr-Abl signaling, I developed an A419259-resistant mutant of Hck (Hck-T338M). Expression of Hck-T338M fully protected K562 CML cells from A-419259-induced apoptosis, an effect that correlated with sustained Stat5 activation. In addition, the Hck-T338M partially protected CML cells against the growth inhibition induced by A-419259. These studies suggest that Hck plays a non-redundant role as a key downstream survival partner for Bcr-Abl.
I also tested whether Hck overexpression was sufficient to induce imatinib resistance in CML cells. For this study, I developed a mutant of Hck (Hck-T338A) that is uniquely sensitive to NaPP1, an analog of the generic SFK inhibitor pyrrazolo-pyrimidine 1. Overexpression of Hck or Hck-T338A in K562 cells induced resistance to imatinib-dependent apoptosis and growth arrest. Furthermore, NaPP1 reversed imatinib resistance in K562-Hck-T338A cells, suggesting that Hck-induced imatinib resistance requires Hck kinase activity. Taken together, my work validates Hck as a target for the development of apoptosis-inducing drugs and that are likely to be effective in imatinib-resistant patients.
