Abstract 2845: Midasin overexpression drives intrinsic endocrine resistance by stabilizing DNA damage

Abstract Midasin (MDN1), a AAA-ATPase protein critical to ribosome biogenesis, has emerged as a key player in the progression of triple-negative breast cancer (TNBC), a subtype characterized by its heterogeneity, absence of therapeutic targets, and poor prognosis. Previous work from our lab and others, demonstrated that estrogen receptor negative (ER-) breast cancer patients with high MDN1 expression are positively correlated with a worse relapse-free survival compared to their ER+ counterparts and breast tumors with MDN1 mutations are associated with high tumor mutation burden and unfavorable prognosis. Additionally, MDN1 alterations are associated with increased expression of a signature of proteins involved in cell cycle dysregulation and genomic instability. Thus, we hypothesize that MDN1 overexpression drives intrinsic endocrine resistance by stabilizing DNA damage which promotes tumor cell survival and progression. To gain mechanistic insight into the role of MDN1 in ER- breast cancer, we employed, Ribozinoindole-2 (i.e., Rbin-2), a potent MDN1 pharmacologic inhibitor to study two TNBC cell lines, which express high (MDA-MB-468) and low (MDA-MB-231) levels of MDN1. Both cell lines were treated with 1-12 μM Rbin-2, and after 72 hour, 12 µM Rbin-2 treatment induced a 50% reduction in cell viability. Western blot analyses were conducted, and MDA-MB-468 cells expressed a 1.5-fold increase in MDN1 expression compared to the MDA-MB-231 cells. Rbin-2 treatment reduced MDN1 expression by 30-50% in both cell lines. To gain an in depth understanding of the impact on MDN1 alterations on the fate of the cell, we conducted a series of in silico studies and observed that breast cancer patients with alterations in MDN1 are associated with various forms of DNA damage including higher mutation counts, microsatellite instability, and fractions of the genome that are altered. To confirm this finding, a focused DNA damage antibody array study was performed which revealed that 12 μM Rbin-2 treatment decreased mismatch repair proteins and DNA damage checkpoint markers. We validated this finding by immunoblots and Rbin-2 treatment reduced MSH2, MSH6, and phospho-p53, by 13.8-15.6% in MDA-MB-231 cells and 20-25% in MDA-MB-468 cells further supporting the in silico and protein array data. Immunofluorescence and immunoblotting of γH2AX, a marker for DNA double-strand breaks, confirmed that MDN1 inhibition caused a 55-75% increase in DNA damage in both cell lines, suggesting that MDN1 plays a key role in maintaining genomic stability and regulating DNA damage responses in TNBC. Given these findings elevated MDN1 expression may drive DNA repair dysregulation, thereby providing a survival advantage for de novo endocrine resistant breast cancer. These preliminary studies suggest that MDN1 may represents a potential therapeutic strategy to exploit TNBC's inherent genomic instability and improve treatment outcomes. Citation Format: Manasa Kotina, Afia Ohemeng, Mounika Pamukuntla, Bipika Banjara, Alicia Hudson, A. Micheal Davidson, Jillian L. Pope, Selina Darling-Reed, Syreeta L. Tilghman. Midasin overexpression drives intrinsic endocrine resistance by stabilizing DNA damage [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 2845.