Abstract Title BRCA1 Is Required for DNA2 Deficiency-Induced Replication Stress Response at the ALT Telomeres

Background Cancer cells bypass senescence and achieve replicative immortality by elongating their telomeres. They accomplish this through two primary mechanisms: telomerase activation and the Alternative Lengthening of Telomere (ALT) pathway. Approximately 85-90% of cancers utilize telomerase (TEL+), while 10-15% of cancers employ ALT (ALT+). Notably, about 60% of osteosarcomas rely on ALT for telomere maintenance. There are currently no targeted therapies for ALT+ cancers, highlighting the need for research into specific molecular targets for the ALT pathway. FDA-approved PARP inhibitors are effective for treating the BRCA1/2 mutated cancers, highlighting the importance of understanding BRCA1-deficient cells in ALT contexts. Previously, the Zhang lab showed that depletion of FANCM induces a robust replication stress response at the ALT telomeres, which requires a functional BRCA1, emphasizing BRCA1's critical function in this pathway. Most recently, the Zhang lab found that depletion of DNA2 also induces a robust replication stress response at the ALT telomeres. This study aims to elucidate the functional interplay between BRCA1 and DNA2 at the ALT telomeres.

Methods U2OS cancer cells underwent two rounds of siRNA transfection. The transfection was followed by immunofluorescent staining where the cells were seeded onto coverslips. The cells were then fixed with 3% paraformaldehyde containing 2% sucrose for 10 minutes, followed by treatment with Triton X-100 solution on ice for 5 minutes. Subsequently, the cells were stained with primary antibodies and the respective Alexa-488 and Alexa-546 conjugated secondary antibodies. Colocalization of DNA damage markers and TRF2 was quantified to determine telomere DNA damage in BRCA1 and DNA2 depleted U2OS cell lines.

Results Our study utilized siRNA transfection to investigate the roles of BRCA1 and DNA2 in ALT+ cancer cells. Depletion of BRCA1 and DNA2 followed by immunofluorescence staining revealed the recruitment of critical cell cycle regulator proteins - pChK1, pRPA, BLM, and yH2AX - to telomeres. Our results demonstrate an increase of pChK1, pRPA, BLM and yH2AX to the telomere in DNA2-depleted cells, however the recruitment does not occur in BRCA1-DNA2 co-depleted cells.

Conclusion These findings highlight BRCA1 and DNA2 as crucial regulators of telomeric replication stress in ALT+ cancers. BRCA1 is necessary for the DNA damage response in DNA2-depleted ALT+ cells. Understanding BRCA1 and DNA2-mediated pathways may provide a promising approach to disrupt ALT-specific telomere maintenance, potentially advancing therapeutic strategies tailored to ALT+ cancers and improving treatment outcomes in these malignancies.