Alternative splicing plays a key role for generating protein diversity and contribute to essential biological processes among them differentiation, cell cycle regulation and apoptosis. Alterations in alternative splicing was identified in several human diseases, including cancer. Bcl-x is a Bcl-2 family member which is alternatively spliced in two distinct transcripts generating two different proteins: Bcl-xL (Long) and Bcl-xS (Short), with antagonistic functions. The long isoform inhibits apoptosis, in contrast the short one activates it. Bcl-xL is highly expressed in different types of cancer. CML is a myeloproliferative disorder characterized by a reciprocal chromosomal translocation t(9;22), that generate Bcr-Abl chimeric protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cell proliferation, alteration in cell-cell and cell-matrix adhesion and resistance to apoptosis. It has been reported that in CML Bcl-x isoforms ratio is altered in favor of the anti-apoptotic one (Bcl-xL).We investigated the involvement of Bcr-Abl in regulating splicing events affecting Bcl-x pre-mRNA occurring in CML. By proteomic approach, based on strategy using GST-Pull Down assay, we attempted to gain insight into functional interactions occurring among the oncogenic kinase Bcr-Abl and RNA-binding proteins, identifying physical interactions among the adapter protein Nck-beta, the spliceosome ribonucleoprotein hnRNPA1 and Sam68 which are assembled to form a functional quaternary complex. These interactions were further confirmed by immunofluorescence staining showing co-localization of the proteins within peculiar nuclear splicing regions (nuclear speackles). Using RNA Pull Down assays we detected that the quaternary complex was able to specifically bind BCL-xL mRNA. Using several specific tyrosine kinase inhibitor such as BMS-354825, AMN-107 or STI-571, we identified a key role of BCR-ABL phosphorylation on integrity of the multimeric complex. Furthermore, STI-571 impairs the interaction between quaternary complex and Bcl-xL mRNA. As observed also for other spliced genes like MCL1, PP1γ; PASG, VRK and CENT1. The Bcr-Abl mediated Bcl-x splicing was also confirmed by quantitative PCR and immunofluorescence analysis. Bcr-Abl leads to increased Bcl-xL mRNA expression as proved either by Bcr-Abl ectopic overexpression or by a Bcl-x minigene assay. In conclusion, these data indicate that phosphorilated Bcr-Abl is part of a complex affecting Bcl-x alternative splicing, increasing the anti-apoptotic isoform in respect to the pro-apoptotic one. These data represent the first original report of a direct involvement of Bcr-Abl in splicing events, thus opening new perspectives in CML therapeutic treatment field.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4771. doi:10.1158/1538-7445.AM2011-4771

©2011 American Association for Cancer Research
2011