Rad52 DNA binding activity can be targeted to eliminate CML stem cells

BCR-ABL1 transforms hematopoietic stem cells into leukemia stem cells (LSCs) to induce chronic myeloid leukemia in chronic phase. Expression of BCR-ABL1 stimulates production of elevated levels of reactive oxygen species (ROS), which induce oxidative DNA damage. CML cells accumulate excessive amounts of ROS-induced DNA damage which can be converted to potentially lethal DNA double strand breaks (DSBs). BCR-ABL1 stimulates enhanced Rad51-mediated DSB repair by the homologous recombination repair (HRR) pathway. In these studies we show BCR-ABL1-transformed cells depend on Rad52-mediated HRR to promote repair of ROS-induced DSBs and that this activity is dependent on Rad52 binding to single-stranded DNA (ssDNA). Our results show in the absence of Rad52, BCR-ABL1-positive hematopoietic cells accumulated elevated numbers of DSBs as detected by enhanced gamma-H2AX foci formation compared to cells with wild-type Rad52 which resulted in a decrease in proliferation and expansion of the Rad52-null LSC population. Expression of wild-type Rad52 in Rad52-null cells decreased the accumulation of DSBs and restored expansion of the LSC population. Inhibition of ROS with the antioxidants Vitamin E or N-acetyl cysteine exerted similar effects on the LSC population of Rad52-null cells as restoration of wild-type Rad52. Our studies also show Rad52's ssDNA-binding activity is required for the proliferation of CML cells as evidenced by the accumulation of DSBs and impairment of clonogenic potential in cells in which the Rad52-F79A ssDNA-binding deficient mutant was expressed. Inhibition of Rad52 DNA binding activity by a peptide aptamer targeting Rad52-F79 resulted in a synthetic lethal phenotype in BCR-ABL1-positive cells due to impairment of the Rad52-dependent HRR pathway, as demonstrated by immunofluorescence and HRR repair assays. Altogether we identify Rad52 as a novel target in the treatment of CML, and other BRCA1- and/or BRCA2-deficient cancers, by showing induction of synthetic lethality in proliferating BCR-ABL1-positive cells in which Rad52 ssDNA-binding activity is inhibited.