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, BRIP1 R162Q of unclear clinical significance. Intriguingly, in vitro helicase assays demonstrated that BRIP1 R162Q encodes a hyperactive DNA helicase. In cells, stable expression of BRIP1 R162Q in BRIP1- proficient cells selectively sensitized to ectopic DNA replication stress. Moreover, BRIP1 R162Q expressing cells showed chronic, steady-state activation of DNA replication stress indicated by decreased fork speed, fork stalling and impaired fork symmetry. Moreover, BRIP1 R162Q expression triggered genomic instability indicated by elevated gH2AX foci numbers and chromosomal aberrations, suggesting BRIP1 R162Q as a variant driving genomic instability. Mechanistically, BRIP1 R162Q 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, BRIP1 R162Q 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.