Targeting Phosphotyrosine-315 In RAD51 Recombinase to Prevent BCR-ABL1 - Mediated Unfaithful Homeologous Recombination Repair.

Abstract 1190 Background: CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast phase (CML-BP) and cell lines transformed by non-mutated BCR-ABL1 kinase or tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) (Nowicki et al., Blood, 2006; Cramer et al., Cancer Res., 2008). DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS and radiation induced DSBs generated chromosomal aberrations in BCR-ABL1-positive leukemia cells (Koptyra et al., Leukemia, 2008), which may contribute to the malignant progression to CML-BP. We also showed that homologous recombination repair (HomoRR) driven by RAD51 recombinase is one of the major DSB repair mechanisms, which is stimulated by BCR-ABL1 (Slupianek et al., Molecular Cell, 2001). Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. Here we investigated if BCR-ABL1 can modulate RAD51 recombinase to corrupt the fidelity of recombination and if this process can be targeted to prevent genomic instability in leukemia cells. Result: To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease–inducible DSB site and a repair template displaying 1% sequence divergence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells, BCR-ABL1-32Dcl3 cells and BCR-ABL1(T315I)-32Dcl3 cells. BCR-ABL1 and BCR-ABL1(T315I) kinase caused about a 3-fold increase in HomeoRR activity implicating its role in the accumulation of chromosomal aberrations in CML cells. The magnitude of HomeoRR stimulation depended on BCR-ABL1 expression levels. RAD51 recombinase, a key regulator of recombination repair, forms a complex with BCR-ABL1 which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain of BCR-ABL1, but does not depend on its kinase activity. In fact, BCR-ABL1(K1172R) kinase-dead mutant formed complexes more abundantly with RAD51 than the kinase-active BCR-ABL1. BCR-ABL1-RAD51 complex formation resulted in direct phosphorylation of RAD51 on Y315 [RAD51(phosphoY315)], which is located in the vicinity of PP motifs in the C-terminal portion of RAD51. Phosphorylation-less C-terminal RAD51(Y315F) mutant formed a stronger complex with BCR-ABL1 than the wild-type form. Altogether, it appears that RAD51 PP bind to the SH3 domain of BCR-ABL1 kinase followed by quick phosphorylation of RAD51 on Y315 and disassembly of RAD51(phosphoY315) from the complex. Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. In fact RAD51(phosphoY315) was found in the nuclei of BCR-ABL1 leukemia cells, but not of parental cells and formed numerous foci on DSBs. Phosphorylation-less RAD51(Y315F) mutant abrogated foci formation and inhibited HomeoRR. Thus, BCR-ABL1 – RAD51(phosphoY315) pathway appears to promote unfaithful HomeoRR and chromosomal instability. To test if RAD51(phosphoY315) can be targeted to prevent unfaithful HomeoRR in leukemia cells, a peptide aptamer strategy was applied. Aptamer mimicking the RAD51(phosphoY315) fragment, but not that with the Y315F phosphorylation-less substitution inhibited RAD51 foci formation and HomeoRR activity in BCR-ABL1 leukemia cells. Conclusion: In summary, the BCR-ABL1-RAD51 axis may promote accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR-ABL1 kinase. We hypothesize that targeting BCR-ABL1-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BP progression. Furthermore, RAD51 and recombination is also affected by other oncogenic tyrosine kinases (OTKs) such as TEL-ABL1, TEL-PDGFR, ZNF198-FGFR1, TEL-JAK2, JAK2[V617F], NPM-ALK, IGF-1R, EGFR and FLT3-ITD suggesting that abrogation of OTK-mediated aberrant modulation of RAD51 may inhibit genetic instability and tumor progression. Disclosures: No relevant conflicts of interest to declare.