Abstract 4066: Epidemiology meets epitranscriptomics: Uncovering a novel <i>TERT</i> -antisense transcript regulated by RNA methylation and its role in cancer cell growth and proliferation

Abstract Human telomerase reverse transcriptase (TERT) maintains telomeres at chromosome ends and its dysregulation contributes to human health conditions. Multiple GWAS signals are detected within the TERT genomic region at chr 5p15.33. As a potential proxy for these signals, we investigated a highly polymorphic variable number tandem repeat (VNTR)2-1 within intron 2 of TERT, with 57-143 copies of a 42 bp repeat unit per allele. Functional annotation predicted an antisense intronic transcript with a variable-length open reading frame (2,505-5,211 bp; 834-2,038 aa). Within this TERT-antisense transcript, we predicted N6-methyladenosine (m6A) consensus motifs in each 42 bp repeat. The consensus repeats and m6A-deficient variants of VNTR2-1 were cloned into a m6A-deficient dual-luciferase reporter vector (m-psiCHECK-2) and expressed in the bladder cancer cell line UMUC3. Transient treatment with STM2457, an inhibitor of the m6A writer METTL3, reduced reporter expression, confirming that m6A motifs within VNTR2-1 are methylated by METTL3. VNTR2-1 expression was experimentally validated by 5’-RACE/Nanopore-seq in UMUC3. Additionally, RNA-FISH further visualized the transcript, showing co-localization with ribosomes and suggesting translation potential. We explored potential peptides produced from VNTR2-1 transcript by analysis of public mass spectrometry datasets using PepQuery and identified several unique VNTR2-1 peptide fragments in human tumors. Next, we cloned a synthetic FLAG-tagged gene containing three VNTR2-1 consensus repeats with native Kozak sequence and translation start/stop codons. Western blotting confirmed the translation of the VNTR2-1 peptide that was ablated by start codon mutation, suggesting that the native Kozak sequence is sufficient for VNTR2-1 translation. These results support VNTR2-1 as a peptide-producing transcript. RNA-seq following VNTR2-1 overexpression revealed upregulation of pathways related to DNA replication and cell cycle, while CRISPR-Cas9 knockout (V2.1KO) produced reciprocal downregulation of these pathways. To determine whether these transcriptional shifts translated to functional differences, we measured proliferation of UMUC3 as well as the lung cancer cell line A549 using xCELLigence Real-Time Cell Analysis. Overexpressing cells exhibited accelerated growth compared with wild type, whereas V2.1KO cells proliferated more slowly, mirroring the pathway-level changes. BrdU incorporation assays further corroborated impaired DNA replication in V2.1KO cells. In summary, we identified a novel antisense transcript produced by VNTR2-1 within a TERT intron that functions as a lncRNA and/or translated peptide to modulate cell proliferation, potentially contributing to cancer and other disease-related phenotypes. Citation Format: Brenen Papenberg, Oscar Florez-Vargas, Michelle Ho, Kaitlin Forsythe, Chia-Han Lee, Sam M. Mbulaiteye, Pedro J. Batista, Ludmila Prokunina-Olsson. Epidemiology meets epitranscriptomics: Uncovering a novel TERT-antisense transcript regulated by RNA methylation and its role in cancer cell growth and proliferation [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 4066.