Abstract 632: Cross-resistance of myeloid cells transformed with Bcr-Abl imatinib-resistant mutant T315I to Src-family kinase inhibitors

Abstract

Abstract Bcr-Abl, the constitutively active protein-tyrosine kinase arising from the Philadelphia chromosome translocation, plays a key role in the etiology of chronic myelogenous leukemia (CML). Treatment of CML with the Abl kinase inhibitor imatinib has been a clinical success in the chronic phase of the disease. However, treatment failure in the blast crisis phase is attributable to imatinib resistance caused by point mutations in the kinase domain of Bcr-Abl. One of the most clinically challenging resistance mutations involves substitution of the gatekeeper threonine with isoleucine (T315I). This residue is located within the ATP-binding site and is essential for imatinib binding. Src family kinases (SFKs) are important mediators of Bcr-Abl signal transduction and oncogenesis in CML. Bcr-Abl binds to and activates multiple SFKs in CML cells, including Hck and Lyn. In turn, active SFKs directly phosphorylate regulatory tyrosine residues in the SH3 domain, the SH2-kinase linker, and the Bcr-derived portion of Bcr-Abl. Previous work from our group showed that global inhibition of SFK activity with the ATP-competitive pyrrolo-pyrimidine A-419259 leads to growth arrest and apoptosis in CML cell lines and in primary CD34+ CML cells. In addition, expression of an Hck mutant with engineered resistance to A-419259 protects CML cells against the antiproliferative and apoptotic effects of this compound. Other studies also correlate CML progression and drug resistance with overexpression and activation of Hck and Lyn. Given the important role of SFKs in Bcr-Abl-mediated signaling and drug resistance, we studied the effect of A-419259 on human TF-1 myeloid cells transformed with clinically relevant imatinib-resistant forms of Bcr-Abl (E255V, Y253H and T315I). Proliferation of TF-1 cells transformed with Bcr-Abl E255V and Y253H was markedly inhibited by A-419249. The anti-proliferative effects of the inhibitor correlated with suppression of overall SFK activity. However, TF-1 cells transformed with the gatekeeper mutant T315I were completely cross-resistant to the SFK inhibitor A-419259. Surprisingly, SFK activity in TF-1 cells expressing Bcr-Abl T315I was not inhibited by this compound. Immunoprecipitation experiments show that endogenous Lyn remains active in A-419259-treated TF-1 cells transformed with Bcr-Abl T315I. In addition, over-expressed Hck remains active in TF-1/T315I cells upon A-419259 treatment, but is completely inhibited in TF-1 cells transformed with wild-type Bcr-Abl. These results suggest that in general, SFKs are important targets to overcome imatinib resistance in CML. However, our data also suggest a new mechanism of Bcr-Abl T315I-induced resistance to drugs that target SFKs in CML. We speculate that this effect may be due to an acquired ability of Bcr-Abl T315I to phosphorylate SFKs at novel sites that interfere with drug action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 632.