A cell-penetrating antibody inhibits human RAD51 via direct binding

Abstract

RAD51, a key factor in homology-directed repair (HDR), has long been considered an attractive target for cancer therapy, but few specific inhibitors have been found. A cell-penetrating, anti-DNA, lupus autoantibody, 3E10, was previously shown to inhibit HDR, sensitize tumors to radiation, and mediate synthetic lethal killing of BRCA2-deficient cancer cells, effects that were initially attributed to its affinity for DNA. However, as the molecular basis for its ability to inhibit DNA repair, we report that 3E10 directly binds to the N-terminus of RAD51, sequesters RAD51 in the cytoplasm, and impedes RAD51 binding to DNA. Further, we generate separation-of-function mutations in the complementarity-determining regions of 3E10 revealing that inhibition of HDR tracks with binding to RAD51 but not to DNA, whereas cell penetration is linked to DNA binding. The consequences of these mutations on putative 3E10 interactions with RAD51 and DNA are correlated with in silico molecular modeling. Taken together, the results identify 3E10 as a novel inhibitor of RAD51 by direct binding, accounting for its ability to suppress HDR and providing the molecular basis to guide pre-clinical development of 3E10 as an anti-cancer agent.

Regulation and pharmacological targeting of RAD51 in cancer

“…The Glazer group discovered an autoantibody to RAD51 called 3E10 ( 183 ), which was originally identified as an autoantibody associated with lupus and is considered benign to noncancerous cells ( 184 ). 3E10 directly interacts with the N-terminus of RAD51, and this interaction prevents RAD51 from assembling as a filament onto ssDNA ( 183 ).…”

“…The Glazer group discovered an autoantibody to RAD51 called 3E10 ( 183 ), which was originally identified as an autoantibody associated with lupus and is considered benign to noncancerous cells ( 184 ). 3E10 directly interacts with the N-terminus of RAD51, and this interaction prevents RAD51 from assembling as a filament onto ssDNA ( 183 ). An expanded role for 3E10 was demonstrated as 3E10 exposure results in increased toxicity in PTEN-deficient glioma and melanoma cancer cells, which already have a significant propensity for DNA damage ( 185 ).…”

Synthetic lethality of a cell-penetrating anti-RAD51 antibody in PTEN-deficient melanoma and glioma cells

“…We recently reported that 3E10 inhibits HDR and does so through a physical interaction with RAD51, resulting in a functional RAD51 inhibition [23]. Based on prior work suggesting that PTEN loss causes a reduction in NHEJ, the other major cellular pathway of DNA DSB repair [18], we sought to test the effect of the 3E10 on PTEN deficient cells.…”

“…Numerous other pharmacological strategies are being advanced to inhibit DNA repair, and most utilize small molecules. As an alternative, our group has recently discovered that treatment of human cells with the cell-penetrating autoantibody, 3E10, inhibits DNA DSB repair by HDR through a physical interaction between 3E10 and RAD51 [23]. We demonstrated that 3E10 inhibits RAD51 accumulation on ssDNA and RAD51-dependent DNA strand exchange.…”

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

“…Recently described cell penetrating antibodies also operate through a similar mechanism (Pastushok et al, 2019;Turchick et al, 2017Turchick et al, , 2019. Inhibitors that suppress the D-loop activity of RAD51 have also been reported (Budke et al, 2019;Lv et al, 2016), although several optimized versions also exhibit DNAintercalating activity (Budke et al, 2019).…”