Role of a childhood cancer-linked BRIP1/FANCJ germline variant in genomic instability and cancer cell vulnerability

Childhood cancer is frequently associated with inherited pathogenic variants of cancer predisposition genes. Using Whole-Exome Sequencing, we identified an inherited, monoallelic pediatric cancer-linked germline variant of DNA helicase FANCJ/BRIP1, BRIP1R162Q of unclear clinical significance. Intriguingly, in vitro helicase assays demonstrated that BRIP1R162Q encodes a hyperactive DNA helicase. In cells, stable expression of BRIP1R162Q in BRIP1-proficient cells selectively sensitized to ectopic DNA replication stress. Moreover, BRIP1R162Q expressing cells showed chronic, steady-state activation of DNA replication stress indicated by decreased fork speed, fork stalling and impaired fork symmetry. Moreover, BRIP1R162Q expression triggered genomic instability indicated by elevated {gamma}H2AX foci numbers and chromosomal aberrations, suggesting BRIP1R162Q as a variant driving genomic instability. Mechanistically, BRIP1R162Q mislocalizes upon replication stress, and leads to accumulation of DNA secondary G-quadruplex (G4) structures and R-loops. RNaseH1 expression, which resolves R-loops, also reduced G4 levels and relieved BRIP1R162Q-induced replication stress. Finally, BRIP1R162Q introduces exploitable vulnerabilities for targeted therapies using G4-ligand Pyridostatin and DNA damage kinase ATR and DNA-PK inhibition. Our findings propose a mechanistic framework of how a childhood cancer-linked monoallic, hypermorphic BRIP1 germline variant participates in cancer development, and suggest potential therapeutic treatment strategies.