Fig. 6: MEKi/PAPRi cytotoxicity converges in the upregulation of excess ROS.

A Intracellular reactive oxygen species (ROS) levels of MPM cells treated for 72 h with DMSO (vehicle), PARPi (5 µM), and MEKi (0.5 µM), alone and in combination. Data were normalized to vehicle control and shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Welch’s t-test (comparisons between treatment groups and vehicle control). MESO-1 cells treated with H₂O₂ (1 mM; 30 min) were used as the positive control. B N‑acetylcysteine (NAC) attenuates MEKi/PARPi cytotoxicity. Viability assay of MPM cells pre-incubated (12 h) with DMSO (vehicle) or NAC (5 mM) followed by further treatment (96 h) with trametinib (0.5 µM) and olaparib (5 µM). Data are shown as fold changes, with the viability of the cells treated with trametinib/olaparib/DMSO (vehicle) set as 1. *p < 0.05, **p < 0.01 by Welch’s t-test (n = 3). Representative clonogenic assay of BE261T cells treated with MEKi (0.1 µM) and PAPRi (5 µM) with or without NAC (5 mM) was shown to the right. C p-MEK1 (Ser217/221) protein level is significantly positively correlated with TIGAR and DJ1. The protein expression profile [reverse-phase protein array (RPPA)] was downloaded from The Cancer Proteome Atlas (TCPA) portal (https://tcpaportal.org/). D Volcano plots showing the genes downregulated [adjusted p-value (padj) <0.05 & log₂ FC < -2] (in green) and upregulated (padj < 0.05 & log₂ FC > 2) (in red) by H₂O₂, with the H₂O₂-upregulated genes determined as ROS responsive signature. The transcriptome dataset (GSE32335) of H₂O₂-treated cells was downloaded from the GEO. E PARP1 mRNA levels are significantly positively correlated with the ROS-responsive gene signature in MPM tumors. F, G High levels of PARP1 and ROS-responsive gene signature are significantly associated with poor survival in MPM patients. The P value is calculated by using the log-rank test. H Working model illustrating the results of this study. MEK hyperactivation is the molecular driver and a therapeutic target in MPM. MEKi evokes genomic and metabolic stress and triggers a protective mechanism involving the pleiotropic PARP1. A combination of MEK and PARP inhibitors leads to synergistic upregulation of ROS, which translates into incurable genomic and metabolic perturbations and subsequently apoptotic cell death.