Abstract

Purpose Clinical control of oral squamous cell carcinoma (OSCC) is constrained by heterogeneous radiosensitivity driven by divergent DNA damage response (DDR) programs. The architecture and functional contribution of alternative end-joining (Alt-EJ), an error-prone DNA double-strand break (DSB) repair pathway frequently upregulated in cancer, to radiation resistance remains poorly defined. Methods and Materials We profiled microRNAs in radioresistant OSCC clones and performed multi-omic integration across an institutional OSCC cohort, an external OSCC cohort from GEO, the TCGA pan-cancer tumors, and cell lines characterized by Sanger-GDSC to infer DDR characteristics, genomic scar features, drug sensitivity, and radiotherapy outcomes. DSB repair capacity and pathway usage were validated using various functional assays, including Alt-EJ reporters and droplet digital PCR quantification of microhomology-mediated repair events. Core Alt-EJ effectors such as PARP1 and POLQ were perturbed genetically and pharmacologically. Therapeutic efficacy of PARP or POLQ inhibition with or without irradiation was tested in a syngeneic OSCC model, followed by bulk tumor transcriptomics to assess pathway engagement. Results Upregulation of miR-21-5p was not only selectively detected in radioresistant OSCC, but also modulates radiosensitivity in vitro and in vivo, and associated with inferior post-radiotherapy survival. A calibrated miR-21-5p target-gene signature tracked Alt-EJ activity across patient and mouse tumors and cancer cell lines, correlated with microhomology-mediated indels and broader genomic scarring, and predicted sensitivity to clinically available PARP inhibitors. Functionally, enforced miR-21-5p expression increased Alt-EJ usage and accelerated DSB repair, whereas inhibition or depletion of key Alt-EJ effectors reduced repair efficiency and restored radiosensitivity. In vivo, Alt-EJ targeting with PARP or POLQ inhibitor abrogated miR-21-5p-driven radiation resistance