Horacek, A. G.; Kueper, F. L.; Lu, R.; Lamont, I. P.; Tran, S.; Li, H.; Hockemeyer, D.

Reversion mutations that restore BRCA2 function represent a major mechanism of resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) in BRCA2-mutant cancers. Understanding and predicting these events could inform treatment strategies, identify patients at increased risk of acquiring PARPi resistance, and improve the clinical interpretation of secondary BRCA2 variants detected in resistant tumors. Here, we evaluated reversions in an isogenic cell system using CRISPR editing and long-read transcript analysis to define the principles governing BRCA2 reversion systematically. We find that local sequence context dictates the spectrum of reversions, whereas BRCA2 domain architecture determines which of these events confer PARPi resistance. Moreover, we characterize the two routes of BRCA2 reversion: DNA-level restoration of the reading frame and transcript-level rescue through altered splicing. Both mechanisms were frequently used to restore BRCA2 expression across exon boundaries. The detection of such events at multiple loci suggests that inter-exonic reversion may be more widespread than previously appreciated. Lastly, we identify a unique set of mechanisms that underlie exon 11 reversions. In this region, reversions arose through both large genomic deletions and recurrent splicing events, including isoforms predicted to remove all eight BRC repeats. These splicing-mediated reversions engaged cryptic donors and were conserved across distinct genotypes, including the founder mutation c.5946delT. These findings define a predictive reversion code that governs the paths to BRCA2 functional rescue, enabling anticipation of reversion-mediated PARPi resistance and interpretation of secondary BRCA2 variants in resistant tumors.