Abstract 6977: ETV6 is an unexplored partner of the Chr.8 q24.3 amplicon-embedded immortalizing oncogene TONSL in tumorigenesis

Abstract

Abstract Approximately 20% of breast cancers exhibit recurring genomic amplification involving chromosome 8q24.3. In our previous study, we identified the Tonsoku-like DNA repair protein (TONSL) within this amplicon as an immortalizing oncogene of breast epithelial cells, that can promote Estrogen Receptor-positive (ER+) breast adenocarcinomas when combined with other breast cancer-associated oncogenes. Our findings indicated that TONSL-amplified breast cancer cells are dependent on TONSL for tumor growth and exhibited deregulated homologous recombination and cell cycle pathways. High levels of TONSL in primary breast cancers, particularly ER+ breast cancers, were associated with poor outcomes. Although TONSL itself is not druggable, owing to its lack of enzymatic activity, molecules that disrupt the TONSL interactome can be developed. Towards this goal, we performed immunoprecipitation (IP) with TONSL antibody using protein lysates from TONSL-immortalized primary breast epithelial cells and subjected the immunoprecipitates to mass spectrometry. We identified several proteins selectively enriched with the TONSL antibody, the most significant of which was ETS variant transcription factor 6 (ETV6). ETV6 is a modular protein consisting of a C-terminal DNA-binding ETS domain and N-terminal point (PNT) domain and is known to play a role as a transcriptional repressor during embryonic development and hematopoiesis. We confirmed these data by IP followed by western blot analysis of primary breast epithelial cells and TONSL amplified MDA-MB-436. Furthermore, we utilized ClusPro to model the TONSL-ETV6 interaction and predicted that the TONSL’s ankyrin repeats docks onto ETV6’s PNT in a favorable manner. To investigate the functional significance of this interaction, we evaluated ETV6-regulated genes using RNA-seq. Our analysis revealed that ETV6-regulated genes were downregulated following TONSL knockdown and vice versa. Most ETV6 target genes affected upon TONSL manipulation are involved in Epithelial-Mesenchymal Transition (EMT), particularly TWIST1, SNAI2 and ZEB1. Previous studies have demonstrated a role of ETV6 in limiting EMT by repressing the expression of EMT-associated transcription factors. Our findings revealed that TONSL overexpression in primary breast epithelial cells led to a significant increase in these EMT gene expression, whereas TONSL knockdown in MDA-MB-436 cells resulted in a moderate decrease. These findings suggest that TONSL binds to ETV6 and restricts its ability to bind to the regulatory regions of EMT-associated genes, leading to a transcriptional surge of EMT-associated genes leading to tumor progression. Further studies will contribute to our understanding of the role of the chr.8q24.3 amplicon, specifically the TONSL gene, in initiating/promoting breast tumorigenesis and the development of novel drugs that target the TONSL interactome. Citation Format: Aditi S. Khatpe, Emma Doud, Amber Mosely, Kathy D. Miller, Harikrishna Nakshatri. ETV6 is an unexplored partner of the Chr.8 q24.3 amplicon-embedded immortalizing oncogene TONSL in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6977.