Abstract Diffuse pleural mesothelioma (DPM) remains a lethal malignancy with limited benefit from existing therapies, underscoring the urgent need for molecularly precise treatments that act directly at the disease interface. Previously, we identified WEE1 and PKMYT1 as consistently overexpressed in DPM and demonstrated that their combined inhibition induces synthetic lethality. Here, we evaluate a dual-siRNA therapeutic strategy targeting this kinase pair and position it within a platform engineered for localized RNA delivery. Across three DPM subtypes (epithelioid, sarcomatoid, and biphasic), siRNA constructs achieved robust knockdown of WEE1 and PKMYT1, with 70% reduction in mRNA levels at 48-96 h post-transfection. Single-gene silencing induced only modest reductions in cell viability, with 70-80% survival across most lines relative to controls. In contrast, combined WEE1/PKMYT1 knockdown consistently elicited a marked decrease in viability, ranging from 60% to 85% depending on the cell line (p 0.001). Cell-cycle analysis revealed that dual knockdown, unlike individual siRNAs, induced significant G2/M arrest accompanied by a depletion of S-phase cells, indicating disruption of DNA damage checkpoints. Flow cytometry demonstrated a 3- to 5-fold increase in apoptotic cell fractions compared with single knockdowns and controls (p 0.0001), confirming synergistic induction of programmed cell death. These data show that although individual siRNAs exert limited effects, their combination produces robust anti-proliferative, pro-apoptotic, and cell-cycle disruptive phenotypes consistent with synthetic lethality. Collectively, these data validate dual-siRNA synthetic lethality as a promising therapeutic approach for DPM and underscore the potential of a nanoparticle-hydrogel delivery system that we have developed for precise, localized RNA therapy. This work paves the way for RNA nanomedicine in mesothelioma and other anatomically restricted, treatment-refractory cancers. Citation Format: Yelixza I. Avila, Anand Singh, Vivek Singh, So-Hyun Yoon, Smrity Sahu, Nathanael Pruett, Chuong D. Hoang. Dual siRNAs for precision targeting of synthetic lethal vulnerabilities in Diffuse Pleural Mesothelioma 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 2505.
Avila et al. (Fri,) studied this question.