Abstract A041: Development of MTX-5FU-Gem-miR-15a as a novel miRNA-based therapeutic strategy for epithelial ovarian cancer (EOC)

Despite advances in therapeutic strategies, epithelial ovarian cancer (EOC) is still the most lethal gynecological malignancy due to late diagnosis, drug resistance, and high relapse rate (∼80%). Although PARP inhibitors (PARPi) like Olaparib have shown significant efficacy against BRCA-mutated tumors, many EOC cases are BRCA wild-type and are intrinsically resistant to these. Moreover, BRCA-mutant tumors also develop resistance to PARPi through BRCA reversion mutations or activation of ATR/CHK1/WEE1 axis. Therefore, novel therapeutic strategies are urgently needed to enhance therapeutic efficacy and overcome resistance mechanisms. Epigenetic alterations of microRNA (miRNA) also contribute to resistance mechanisms. miR-15a (hsa-miR-15a) is a tumor suppressor miRNA that downregulates several oncogenic proteins like WEE1, CHK1, YAP1, and BCL2. miR-15a is frequently downregulated in EOC patients, and restoring the level of miR-15a offers a promising approach for targeted therapy. To enhance therapeutic efficacy, stability, and tumor cell specificity, we engineered MTX-5FU-Gem-miR-15a, combining the therapeutic power of tumor suppressor miR-15a with methotrexate (MTX), 5-fluorouracil (5FU), and gemcitabine (Gem). We hypothesize that this modified miRNA will be able to overcome both intrinsic and acquired PARPi resistance in EOC. To determine its efficacy, Olaparib-resistant EOC cell lines (OVCAR-3 and UWB1.289) were established through stepwise dose escalation. Both parental and resistant cells were treated with MTX-5FU-Gem-miR-15a or respective controls and evaluated for cell viability (WST-1 assay), induction of apoptosis (Annexin V/PI staining), and cell cycle progression (PI staining). MTX-5FU-Gem-miR-15a significantly reduced cell viability in both parental (IC50: 0.5-5 nM) and resistant (IC50: 5-15 nM) OC cells without any delivery vehicle, induced S-phase arrest, and significantly increased apoptosis. The therapeutic efficacy of MTX-5FU-Gem-miR-15a was improved by over 1000-fold than that of Olaparib. Western blotting confirmed downregulation of miR-15a target genes. Notably, co-treatment of MTX-5-FU-Gem-miR-15a with olaparib exhibited additional synergistic anti-tumor effects. All experiments were conducted in biological triplicate. Statistical significance was determined using Student’s t-test (p < 0.05). To determine therapeutic efficacy in vivo, a metastatic model of EOC was established by injecting tumor cells via tail vein into female NOD/SCID mice. The mice were then treated with MTX-5FU-Gem-miR-15a (3.75 mg/kg) or vehicle control. Our results show that MTX-5FU-Gem-miR-15a treatment reduced EOC tumor volume by 5.5-fold (∼80%) compared to the control group and increased survival without any observable toxicity. Taken together, these results show that MTX-5FU-Gem-miR-15a inhibits tumor growth in both BRCA-mutant and BRCA-wild-type ovarian cancer, and it presents a promising novel miRNA-based therapeutic strategy with strong potential for overcoming drug resistance and enhancing treatment outcomes in both naïve and refractory EOC. Citation Format: Amartya Pal, Anushka Ojha, Jingfang Ju. Development of MTX-5FU-Gem-miR-15a as a novel miRNA-based therapeutic strategy for epithelial ovarian cancer (EOC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A041.