PF397 IDENTIFICATION OF PROTEIN‐PROTEIN INTERACTIONS NECESSARY FOR MAINTENANCE OF BCR‐ABL CORE COMPLEX

Abstract

Background:Approximately 50% of chronic myeloid leukemia (CML) patients in deep remission experience a return of clinical CML after withdrawal of tyrosine kinase inhibitors (TKIs), suggesting signaling of catalytically inactive BCR‐ABL. This allows for survival of cancer cells responsible for CML relapse after discontinuation of the TKI therapy. Elucidation of protein‐protein interactions within the BCR‐ABL signaling complex is crucial for understanding of the mechanism of residual signaling of TKI‐inhibited BCR‐ABL.Aims:This study aims on detailed characterization of BCR‐ABL interactions with members of its core complex, i.e. the signaling mediators Grb2, SOS1, SHC1, cCbl, CrkL, SHIP2, Sts1 and p85a.Methods:Peptide microarrays were used to map binding interfaces between BCR‐ABL and its interactors in high resolution. The microarray data were validated by in vivo co‐immunoprecipitation and proximity ligation assays carried out with BCR‐ABL deletion mutants and their respective interactors. Reporter assays were employed to analyze the downstream signaling of BCR‐ABL in cells.Results:We demonstrate that TKIs inhibit catalytic activity of BCR‐ABL, but do not completely abrogate its downstream signaling. Further, treatment with TKIs does not entirely dissolve the BCR‐ABL signaling complex consisting of Grb2, SOS1, SHC1, cCbl, CrkL and SHIP2. The interacting interfaces between BCR‐ABL and members of its core complex were mapped in high resolution. The data show that CrkL binds to proline‐rich regions located in C‐terminal, intrinsically disordered region of BCR‐ABL, that deletion of pleckstrin homology (PH) domain of BCR‐ABL diminishes interaction with SHC1, and that SHIP2 associates with the src‐homology (SH)3‐SH2‐tyrosine kinase (TK) domain of BCR‐ABL. We further demonstrate that BCR‐ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of at least three core complex members, the Grb2, SOS1 and cCbl. Introduction of Y177F substitution to BCR‐ABL blocks association with Grb2, SOS1 and cCbl, and reduces residual signaling of TKI‐inhibited BCR‐ABL.Summary/Conclusion:The protein‐protein interactions within the BCR‐ABL core complex were characterized in detail here. Our findings support the concept of targeting BCR‐ABL signaling in CML by inhibition of Y177 interaction interface.