Cancer-associated MDM2 W329G mutant attenuates ribosomal stress-mediated p53 responses to promote cell survival and glycolysis

Although amplification/overexpression is the predominant mechanism for the oncogenic properties of MDM2, an increasing number of MDM2 somatic missense mutations were identified in cancer patients with the recent advances in sequencing technology. Here, we characterized an MDM2 cancer-associated mutant variant W329G identified from patient samples that contain a wild-type p53 gene. We found that the MDM2 W329G mutant was resistant to the inhibitory effect of ribosomal protein L11 (RPL11) on MDM2-mediated p53 ubiquitination and degradation, in line with its defect on RPL11 binding. Using isogenic U2OS cells with or without endogenous mdm2 W329G mutation, we demonstrated that the expression of classic p53 targets induced by ribosomal stress signals was reduced in mutant cells. RNA-seq analysis revealed that upon 5-FU treatment, the p53 response was significantly impaired. Also, the 5-FU-mediated repression of genes in cell cycle progression and DNA replication was diminished in W329G mutant-containing cells. Physiologically, U2OS W329G cells were more resistant to cell growth inhibition induced by ribosomal stress and exhibited higher glycolytic rates upon 5-FU treatment. Together, our data indicated that cancer-associated MDM2 W329G mutant attenuates ribosomal stress-mediated p53 responses to promote cell survival and glycolysis. Overall design: To investigate the effect of MDM2 W329G mutation in p53 regulation, we established isogenic U2OS wild-type and MDM2 W329G cells using Crispr/Cas9-based genome editing. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells treated with DMSO or 5-FU (50 µM) for 24 hours. Two independent experiments were performed and RNA-seq data were generated by two different vendors. Comparative gene expression profilling analysis of RNA-seq data were carried out between 5-FU treated U2OS wild-type and MDM2 W329G cells as well as DMSO treated U2OS wild-type and MDM2 W329G cells.