Targeting BCR/ABL-RAD51 Interaction to Prevent Unfaithful Homeologous Recombination Repair.

Abstract Abstract 3272 Poster Board III-1 CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) and cell lines transformed by non-mutated BCR/ABL kinase or the tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress. In addition CD34+CD38- CML-CP and CD34+ CML-BC stem cell-enriched populations seem to display more DSBs than normal counterparts. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS- and radiation- induced DSBs induce chromosomal instability implicating enhanced, but unfaithful repair in BCR/ABL-positive leukemias [Leukemia, 2008]. We show here that BCR/ABL kinase (non-mutated and TKI-resistant mutants) facilitate recombination repair (RR) of DSBs. 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. To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease-;inducible DSB site and a repair template displaying 19% divergence sequence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells and BCR/ABL-positive counterparts. BCR/ABL kinase caused about 3-fold increase in HomeoRR activity implicating its role in accumulation of chromosomal aberrations in CML cells. RAD51, a key regulator of recombination repair, forms a complex with BCR/ABL which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL. SH3+SH2-CD domains of BCR/ABL form a pocket binding the PP regions of RAD51. Single amino acid substitutions in the BCR/ABL SH3+SH2-CD pocket, which disrupted binding to the RAD51 PP regions reduced complex formation with RAD51. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3+SH2-CD pocket mutant displayed slow proliferation rate and responded poorly to genotoxic stress despite intact kinase activity. On the other hand, disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) prevented its interaction with BCR/ABL SH3+SH2-CD pocket. Interestingly, expression of RAD51 PP-LL mutant abrogated the clonogenic capability of BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. BCR/ABL-RAD51 complex results in direct phosphorylation of RAD51 on Y315, which is located in the vicinity of PP motifs in the C-terminal portion (aa 271–339) of RAD51. C-terminal Y315F mutant formed more abundant complex with BCR/ABL that the wild-type form, but it did not restore the lost interaction of the PP/LL mutant. Thus, BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for disassembly of RAD51 from BCR/ABL. In concordance, RAD51[Y315F] mutant remained mostly in the cytoplasm, while the wild-type protein accumulated in the nucleus in BCR/ABL-positive cells in response to DSBs induced by genotoxic treatment. In addition to the regulation of BCR/ABL-RAD51 interaction, phospho-Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. To test this hypothesis we employed a peptide aptamer strategy targeting phospho-Y315 of RAD51. Peptides corresponding to the RAD51 fragment containing phospho-Y315, but not these with Y315F substitution reduced HomeoRR activity by approximately 2-fold in BCR/ABL-positive leukemia cells. Altogether, it appears that PP-regions of RAD51 interact with SH3+SH2-CD niche of BCR/ABL, which leads to phosphorylation of RAD51 on Y315 and disassembly of the complex. Phospho-Y315 stimulates abundant nuclear localization of RAD51 on DSBs, which disrupts the mechanisms responsible for preventing recombination using divergent templates resulting in unfaithful HomeoRR in BCR/ABL-positive leukemia cells. In summary, BCR/ABL-RAD51 interaction promotes survival and accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR/ABL kinase. We hypothesize that targeting BCR/ABL-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BC progression. Disclosures: No relevant conflicts of interest to declare.