Developing a Multimodal miR-15a Mimic to Overcome Gemcitabine and RAS(ON) Inhibitor Daraxonrasib Resistance in Pancreatic Ductal Adenocarcinoma

Gemcitabine remains a mainstay of pancreatic ductal adenocarcinoma (PDAC) treatment, while the RAS(ON) inhibitor daraxonrasib has recently demonstrated improved survival in previously treated metastatic disease. However, acquired resistance to both therapies remains a major clinical challenge. Here, we developed MTX-5-FU-Gem-miR-15a, a multimodal microRNA (miRNA)-based therapeutic, by chemically modifying tumor-suppressive miR-15a with methotrexate (MTX), 5-fluorouracil (5-FU), and gemcitabine (Gem) to combine sequence-specific gene regulation with disruption of nucleotide metabolism. MTX-5-FU-Gem-miR-15a exhibited low-nanomolar potency across five PDAC cell lines and retained activity in gemcitabine- and daraxonrasib-resistant models under vehicle-free conditions, substantially exceeding that of stoichiometrically matched free antimetabolites. Treatment induced S-phase accumulation, increased DNA damage and apoptosis, and suppressed checkpoint and survival-associated proteins, including WEE1, CHK1, YAP1, and BCL-2. Transcriptomic and proteomic analyses revealed increased DNA replication, replication-stress, and repair-associated programs, together with reduced oncogenic signaling. In gemcitabine-resistant cells, treatment restored the gemcitabine-activating enzyme dCK, reduced ABCC1 and ABCC3 expression, and suppressed resistance-associated signaling programs, indicating partial remodeling of resistance-associated pathways. MTX-5-FU-Gem-miR-15a also synergized with daraxonrasib (mean Bliss synergy score, 22.52) and activated tumor-cell-intrinsic STING-TBK1 signaling, accompanied by increased interferon stimulated gene expression and IFN-β and CXCL10 secretion. Finally, MTX-5-FU-Gem-miR-15a retained low-nanomolar potency in three patient-derived PDAC organoids and significantly suppressed metastatic tumor growth in vivo at 4 mg/kg. Together, these findings support a multimodal miRNA therapeutic strategy that combines replication-associated DNA damage, checkpoint protein suppression, and innate immune activation-associated responses to target therapeutic resistance in PDAC.