Abstract 4199: Defining roles of telomerase reverse transcriptase in DNA damage response

Abstract Telomerase Reverse Transcriptase (TERT), the catalytic subunit of telomerase, is essential for telomere maintenance, elongating the G-strand of telomeric DNA using its associated RNA template (TR). Recruitment of TERT to telomeres is mediated by the shelterin component TPP1. While TERT is highly expressed in cancer cells, efforts to target its telomeric function have shown limited clinical efficacy. Recent studies have identified noncanonical roles for TERT, particularly in the DNA damage response (DDR), suggesting alternative therapeutic opportunities. Notably, TERT inhibitors sensitize cancer cells to radiotherapy, though the mechanisms underlying this effect remain unclear. To address this gap, we established a mutant TERT phenotyping system to study how TERT variants affect the DDR. Using a tetracycline-inducible shRNA-miR knockdown system in MCF7 and HeLa cells, we targeted endogenous TERT and complemented with transfected wildtype or mutant TERT, validating expression by Western blot. Cells expressing only transfected TERT were irradiated with 6 Gy to induce double-strand breaks (DSBs), and DDR effects were assessed by 53BP1 or γH2AX foci resolution, cell survival, and senescence onset. Knockdown of TERT led to persistent DNA damage foci, confirming a role for TERT in DSB repair. Foci resolution was restored by WT TERT but not by the catalytic mutant TERT D712A, V713I. Similarly, the TERT R132E, K78E mutant, defective in TPP1 binding, exhibited persistent foci, implicating TERT-TPP1 interactions in DDR. Supporting this, TPP1 mutants unable to bind TERT also caused persistent DNA damage foci. Combined with evidence that shelterin complexes, including TPP1, localize rapidly to nontelomeric DSBs after DNA damage, these results suggest that TPP1 recruits TERT to DSBs, analogous to its role at telomeres. Ongoing studies aim to elucidate how TERT's catalytic activity promotes DSB repair and to explore TERT as a target for sensitizing cancer cells to therapy. Citation Format: Leon L. Zhou, Stephen J. Kron. Defining roles of telomerase reverse transcriptase in DNA damage response [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 4199.