Abstract The minor spliceosome (MiS) is a specialized RNA-processing machinery upregulated in cancer to promote expression of oncogenic genes. Here, we identify its catalytic component, U6atac snRNA, as a druggable vulnerability in prostate and breast cancers. U6atac knockdown triggers R-loop–mediated DNA damage while impairing DNA repair by downregulating key factors such as BRCA1, PARP1, TP53BP1, and CHK1/2, disabling both homologous recombination and non-homologous end joining. This dual effect sensitizes tumors to PARP inhibitors, cisplatin, and radiation, independent of BRCA status. Moreover, we uncover an adaptive resistance mechanism driven by extracellular vesicles enriched in U6atac, which amplify MiS activity and facilitate therapy escape; a process reversed by U6atac depletion. Across multiple in vitro and in vivo models, MiS targeting demonstrates tumor-selective activity with minimal toxicity. These findings position U6atac as a central regulator of genome stability and establish MiS targeting as a promising approach to potentiate genotoxic therapy and overcome resistance. Citation Format: Anke Katharina. Augspach, Mark Rubin, Paola Francica, Sven Rottenberg, Rahul Kanadia. Targeting Minor Splicing Disrupts DNA Repair and Overcomes Therapy Resistance in Prostate and Breast Cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (2Suppl): Abstract nr B003.
Augspach et al. (Tue,) studied this question.