KEY POINTS
- Genome-wide CRISPR-Cas9 screening under 4 Gy × 3 and 2 Gy × 3 irradiation identified 182 high-confidence survival genes, with CHAF1A among the strongest hits. TCGA analysis subsequently showed CHAF1A upregulation across 18 cancer types, particularly lung squamous-cell and colorectal cancers.
- CHAF1A depletion in A549, H1299 and H1975 lung cancer models increased persistent γ-H2AX after 5 Gy, reduced clonogenic or cellular survival, and impaired both homologous recombination and non-homologous end joining reporter activity. RAD51 focus formation was particularly reduced after irradiation.
- Following DNA double-strand breaks, CHAF1A accumulated at damaged chromatin through an ATM-associated mechanism. ATM inhibition suppressed CHAF1A recruitment, while CHAF1A depletion itself reduced ATM phosphorylation, suggesting a bidirectional amplification loop rather than direct phosphorylation of CHAF1A by ATM.
- Mechanistically, CHAF1A directly interacted with RAD51, and irradiation strengthened this interaction. During S phase, PCNA facilitated CHAF1A-RAD51 association and RAD51 loading; outside S phase, CHAF1A preferentially interacted with KU70, providing a cell-cycle-dependent mechanism for supporting both major double-strand-break repair pathways.
- Re-expression of full-length CHAF1A restored RAD51 recruitment and γ-H2AX clearance, whereas a CHAF1A mutant lacking the RAD51-interacting region did not, linking the radiosensitivity phenotype directly to its DNA-repair function.
- In both A549 cell-derived and rectal-cancer patient-derived xenografts, tumors were treated with 8 Gy × 2 fractions. CHAF1A depletion combined with irradiation produced significantly greater tumor growth suppression and lower tumor weight than either intervention alone, with 5–6 mice per group.
- The patient-derived xenograft was proof-of-concept and came from a single rectal cancer specimen despite much of the mechanistic work being performed in lung cancer models. No clinically available CHAF1A-directed drug was evaluated, so therapeutic translation remains hypothetical.
CLINICAL TAKEAWAY
CHAF1A appears to function as a cell-cycle-dependent DNA repair switch that helps tumor cells survive irradiation by supporting RAD51-mediated homologous recombination and KU-mediated non-homologous end joining. It is an attractive radiosensitization target biologically, but the study remains several translational steps away from a treatment strategy.