DMG-14. RAD52 inhibition induces DNA damage by accumulating R-loops and increases radiosensitivity in diffuse midline glioma

Diffuse midline glioma (DMG) is one of the most devastating childhood cancers. There is a critical need for new therapeutics for improving treatment outcomes for DMG patients. We performed genome-wide CRISPR/Cas9 screening and identified RAD52 as a potential therapeutic target in DMG cells. Genetic and pharmacologic inhibition of RAD52 activity suppressed DMG cell proliferation. Western blotting revealed that RAD52 inhibition increased DNA double-strand breaks (DSBs) marker γH2AX while it decreased repair markers BRCA1 and RAD51. Also, RAD52 inhibition causes a sustained level of phosphorylated RAD50 and γH2AX in irradiated DMG cells over 24 hours. Immunocytochemistry (ICC) of γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. Similarly, comet assays showed that radiation increased comet tail formation, indicating increased unrepaired DNA damage. DNA repair assay showed that RAD52 inhibition suppressed DNA DSB repair through a homologous recombination pathway. RNA sequencing revealed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Importantly, RAD52 inhibition increased the radiosensitivity of DMG cells. To understand the mechanism of RAD52 inhibition on DNA damage, we performed dot plot assay and ICC of S9.6 which recognizes DNA-RNA hybrids (also known as R-loops) and found accumulation of R-loop formation in DMG cells treated with RAD52 inhibitor. Finally, the combination therapy of RAD52 inhibitor and radiation inhibited tumor growth and increased survival of mice bearing DMG patient-derived xenografts, outperforming either monotherapy. Together, our results highlight that RAD52 inhibition induces DNA damage by accumulating R-loops, results in reducing DMG cell proliferation, while also increase DMG radiosensitivity, and providing a rationale for developing combination therapy with radiation for the treatment of DMG.