Abstract 1746: Dual inhibition of TREX1 and polymerase theta uncovers a synergistic DNA repair vulnerability in BRCA-deficient tumors

Abstract Alternative End Joining (Alt-EJ) becomes a key DNA double-strand break (DSB) repair mechanism in tumors with homologous recombination (HR) deficiencies, including BRCA1/2-mutant cancers. Because these tumors depend heavily on POLQ-mediated repair, we sought to identify additional druggable regulators of Alt-EJ that could be leveraged for combination therapy. We used CRISPR-based perturbations, three Alt-EJ reporter systems, and DSB repair pathway analyses in HR-proficient and HR-deficient models. TREX1 subcellular localization after DNA damage was assessed using confocal microscopy and fractionation. To evaluate therapeutic potential, we tested a TREX1 inhibitor alone and in combination with the POLQ inhibitor ART558. Our results reveal TREX1 as a potent suppressor of Alt-EJ and a complementary regulator to POLQ. TREX1 loss markedly increased Alt-EJ frequency, enhanced POLQ-dependent repair, and shifted pathway choice toward more mutagenic mechanisms. Following DNA damage in mitosis, TREX1 translocated from the cytosol into the nucleus, indicating a direct role in DSB repair engagement. Pharmacologic TREX1 inhibition reduced Alt-EJ activity, and co-inhibition of TREX1 and POLQ produced strong synergy, leading to substantial suppression of Alt-EJ and significant loss of viability in BRCA2-deficient cells. Genetic co-depletion of TREX1 and POLQ similarly enhanced sensitivity to ionizing radiation, demonstrating that dual targeting amplifies therapeutic impact across modalities. These findings identify TREX1 as a druggable modulator of DSB repair and establish a mechanistically informed combination strategy in which TREX1 and POLQ inhibitors cooperatively exploit the repair dependence of HR-deficient tumors. Dual inhibition may represent a rational therapeutic approach for BRCA-deficient cancers that rely on Alt-EJ for survival. Citation Format: Roger Shen, Daniel S Higginson. Dual inhibition of TREX1 and polymerase theta uncovers a synergistic DNA repair vulnerability in BRCA-deficient tumors [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 1746.