Integrative transcriptomic and CRISPRi screening identifies determinants of intrinsic radiosensitivity in breast cancer

Background: Breast cancer (BC) remains a leading cause of cancer-related mortality among women, and radiation therapy (RT) is a cornerstone of its management. Despite its efficacy, more than 15% of patients experience locoregional recurrence following RT, highlighting a critical need to define the molecular determinants of RT response and resistance. We hypothesized that transcriptomic changes that occur after ionizing radiation in intrinsically radiosensitive and radioresistant BC models would offer mechanistic insight into mediators of this differential response. To investigate this, we performed in vitro RNA-sequencing across a panel of BC cell lines spanning a spectrum of intrinsic radiosensitivity to characterize radiation-induced transcriptional programs. Recognizing that key mediators of radioresistance may not exhibit differential expression, we complemented this approach with a genome-wide CRISPR interference (CRISPRi) screen to identify additional functional targets that modulate radiosensitivity. Together, these studies aim to elucidate novel molecular pathways underlying RT resistance and inform future therapeutic strategies Methods: Eight BC cell lines were treated with 4 Gy RT and RNA was collected 24 hours after treatment for RNA-seq. Differential gene expression analysis with DESeq2 was performed, followed by pathway analysis with Advaita Bioinformatics' iPathwayGuide. For the CRISPRi experiments, dCas9-KRAB-expressing BT-549 cells were transfected with the Dolcetto library and treated with 3 fractions of 3 Gy RT. DNA was isolated for sequencing to determine sgRNA abundance 7 days after the last dose of radiation and analyzed with MAGeCK. Results: Radiation induced significant transcriptional changes across both radiosensitive and radioresistant models, with highly enriched pathways including cell cycle regulation, DNA replication, and the Fanconi anemia pathway. The CRISPRi screen identified canonical regulators of the DNA damage response as modulators of radiosensitivity, as well as less-characterized candidates such as the potassium channel modifier KCNG3 and the spliceosome component U2AF1 , with mechanistic studies now underway. Conclusions: Ionizing radiation activates transcriptomic programs predominantly involving expression changes in DNA damage response-replated pathways, consistent with top hits identified in the CRISPRi functional screen. Importantly, the screen also reveals previously underexplored mechanisms of radiosensitization, including perturbations in alternative splicing and potassium channel signaling. Together, these findings highlight biologic vulnerabilities that may underlie intrinsic radioresistance and nominate potential targets for therapeutic intervention in women at high risk of local BC recurrence.