Abstract 1469: Deciphering mechanisms of intrinsic radioresistance in breast cancer: Insights from transcriptomics and CRISPRi screens

Abstract Background: Breast cancer (BC), the most common cancer globally and a leading cause of death in women, often utilizes radiation therapy (RT) as part of treatment, with over 85% of patients receiving RT after breast-conserving surgery. Still, over 15% of women experience local recurrence despite RT, underscoring the need to better understand the molecular mechanisms driving RT response and resistance. We hypothesized that transcriptomic changes that occur after ionizing radiation in intrinsically radiosensitive and resistant BC models would offer mechanistic insight into mediators of this differential response. To test this, we conducted in vitro and in vivo RNA-seq experiments to characterize transcriptomic changes following RT across a panel of BC cell lines of varying levels of intrinsic radiation sensitivity. Furthermore, we conducted a genome-wide CRISPRi screen to identify genes which are critical to radiation survival and drive radioresistance. Methods: Eight BC cell lines representing all intrinsic subtypes were treated with 4 Gy RT and RNA was collected 24 hours after treatment for RNA-seq. For 2 cell lines, we also conducted in vivo mouse xenograft experiments in which mice received fractionated daily RT (6 fractions of 2 Gy each). Differential gene expression analysis with DeSeq2 was performed, followed by pathway analysis with Advaita Bioinformatics’ iPathwayGuide. For the CRISPRi experiments, we used the Dolcetto library in two TNBC cell lines: BT-549 and HCC-1937. Cells were transfected with the library and then treated with 3 fractions of 3 Gy RT. DNA was isolated for sequencing after 7 days and analyzed with MaGeCK to determine sgRNA abundance. Results: An average of 935 differentially expressed genes were noted in the in vitro models (S.D. 906); 142 and 324 were found in the in vivo models. Among the most highly enriched pathways from the transcriptomic data in both resistant and sensitive cell lines after RT, we found processes including cell cycle, cellular senescence, and estrogen signaling. Pathways uniquely differentially expressed in the in vivo samples for both cell lines include IL-17 signaling and transcriptional misregulation in cancer. From the CRISPRi screen, we identified genes involved in similar pathways, as well as canonical regulators of DNA damage response. Conclusions: Ionizing radiation induces transcriptomic expression changes that differ between intrinsically radiosensitive and resistant BC models. These data, coupled with results from the CRISPRi radiosensitivity screen, identify pathways that offer potential insight into the mechanisms underlying intrinsic radioresistance and suggest biologic vulnerabilities that may be targeted to more effectively treat women at a high risk of local BC recurrence. Ongoing studies are exploring the potential of therapeutic targeting of these pathways when combined with RT in breast cancer. Citation Format: Breanna McBean Priyanka S. Rana Benjamin Hauk Anna R. Michmerhuizen Samuel Lichtman-Mikol Reine Abou Zeidane Vesna Markovic Kari Wilder-Romans Mingfang Tao Alan P. Boyle Corey Speers. Deciphering mechanisms of intrinsic radioresistance in breast cancer: Insights from transcriptomics and CRISPRi screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1469.