Abstract Epithelial ovarian cancer (EOC) is the deadliest gynecological cancer, mainly because of late diagnosis, high relapse (∼70%), and drug resistance, including resistance to PARP inhibitors (PARPi). Olaparib, a common PARP inhibitor, is most effective in homologous recombination-deficient (HRD) tumors. However, resistance often develops by activating other DNA repair pathways, such as the ATR/CHK1/WEE1 axis. Several studies show that blocking this pathway can restore olaparib sensitivity in resistant EOC models. miR-15a is often decreased in EOC and targets important regulators of DNA repair, cell-cycle progression, and survival, like WEE1, CHK1, BCL2, and YAP1, indicating that restoring miR-15a could help re-sensitize resistant tumors to PARPi. To enhance therapeutic efficacy, stability and tumor cell specificity, we engineered a multimodal MTX-5-FU-Gem-miR-15a combining the therapeutic power of tumor suppressor miR-15a with methotrexate (MTX), 5-fluorouracil (5-FU), and gemcitabine (Gem). MTX improves tumor specificity by targeting overexpressed folate receptor in EOC cells. Furthermore, the incorporation of 5-FU and Gem allows the construct to both disrupt DNA repair pathways and increase DNA damage burden. Hence, we hypothesized that miR-15a mimic treatment could suppress these pathways and re-sensitize resistant EOC to PARPi.MTX-5-FU-Gem-miR-15a robustly inhibited viability of olaparib-resistant EOC cells (IC50: 5–15 nM) without a delivery vehicle, induced S-phase arrest, and markedly increased apoptosis. The therapeutic efficacy of MTX-5-FU-Gem-miR-15a was improved by over 1000-fold compared to that of Olaparib. Combining MTX-5-FU-Gem-miR-15a with olaparib was significantly more effective than either agent alone. (BLISS Synergy score: 13.2). Transcriptomic profiling revealed downregulation of pathways linked to PARPi resistance, and suppression of pro-survival signaling including PI3K–AKT, mTOR, WNT pathways, consistent with broad disruption of resistance-associated networks. The mimic also retained potency in cancer stem–like (CSCs) spheroids (IC50: ∼3 nM) without any delivery vehicle. To evaluate therapeutic efficacy in vivo, a metastatic olaparib-resistant EOC model was established by tail-vein injection of olaparib resistant EOC cells into NOD/SCID mice. MTX-5-FU-Gem-miR-15a treatment (3.75 mg/kg) reduced tumor burden by 5.5-fold (∼80%) and significantly improved survival without observable toxicity or weight loss. Statistical significance was determined using Student’s t-test or two-way ANOVA (p 0.05). Overall, these findings demonstrate that MTX-5FU-Gem-miR-15a is a powerful multimodal miRNA-based therapeutic as it has the potential to exert strong antitumor effects in EOC and overcome drug resistance, supporting its potential as a next-generation strategy to improve patient outcomes. Citation Format: Amartya Pal, Anushka Ojha, Hersh Bendale, Iwao Ojima, Jingfang Ju, . Developing MTX-5-FU-Gem-miR-15a as a multimodal therapeutic strategy to overcome olaparib resistance in epithelial ovarian cancer 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 1370.
Pal et al. (Fri,) studied this question.