Targeting Alternative End-Joinging to Overcome miR-21-5p Mediated Radioresistance in OSCC

Radiation resistance remains a major barrier to effective radiotherapy in oral squamous cell carcinoma (OSCC), yet its molecular basis is incompletely defined. Here, we uncover activation of the alternative end-joining (alt-EJ) DNA repair pathway as a key driver of miR-21-5p-mediated radioresistance in OSCC. Comprehensive miRNA profiling of radioresistant OSCC clones revealed distinct expression signatures marked by robust upregulation of miR-21-5p and miR-486-5p, coupled with reduced miR-320b and miR-1248. Functional assays, including clonogenic survival and syngeneic mouse tumor models, demonstrated that enforced expression of miR-21-5p markedly enhanced radioresistance, accompanied by sustained DNA damage response signaling and improved double-strand break (DSB) repair capacity. Transcriptomic and genomic analyses of the TCGA-HNSC cohort revealed that high miR-21-5p expression correlates with suppression of canonical DNA repair targets, poor radiotherapy outcomes, elevated tumor mutational burden (TMB), and an increased frequency of microhomology-mediated insertions/deletions, consistent with heightened alt-EJ activity. Cross-validation in independent OSCC datasets and local tumor panels confirmed that miR-21-5p-overexpressing tumors exhibit strong activation of alt-EJ gene programs, including upregulation of PARP1, POLQ, and associated accessory factors. Analysis of diverse cancer cell line databases further established that miR-21-5p activity positively correlates with alt-EJ gene expression independent of microenvironmental influence. Mechanistically, miR-21-5p overexpression shifted DSB repair dependence from homologous recombination and classical non-homologous end-joining toward alt-EJ. Pharmacologic or genetic suppression of alt-EJ components (PARP1 or POLQ) disrupted DSB resolution and effectively restored radiosensitivity in miR-21-5p-high OSCC cells. Notably, radiosensitization by PARP inhibition was abrogated in POLQ-deficient cells, confirming a POLQ-dependent mechanism. In vivo, miR-21-5p-driven OSCC tumors exhibited enhanced expression of alt-EJ signatures, increased radioresistance, and high TMB. Combination treatment with irradiation and PARP blockade suppressed tumor growth, diminished alt-EJ gene expression, and prolonged survival in a syngeneic mouse model. Collectively, these findings establish a novel miR-21-5p/alt-EJ signaling axis as a central determinant of radioresistance in OSCC. Targeting alt-EJ represents a promising therapeutic strategy to overcome miR-21-5p-mediated treatment failure and improve radiotherapy efficacy in head and neck cancers. Citation Format: Lin Ma, Nilupaier Tayier, Weitao Hu, Junyang Chen, Yanhe Li, Lu Wen, Yonghui Luo, Xinghan Li, Qi Liu. Targeting alternative end-joining overcomes miR-21-5p-mediated radioresistance in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4648.