Abstract 422: Novel approach to destabilization of oncogenic mutant p53 and therapeutic implications

Introduction: Mutant p53 drives several hallmarks of cancer through a gain-of-function oncogenic program. The oncogenicity of mutant p53 includes sustained proliferation, increased survival and resistance to therapy, increased invasion and metastasis, and genomic instability. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. We and others observe that proteasome inhibitors cause paradoxical depletion of mutant p53 in lung cancer cell lines. Objectives: In this project, we sought to determine the mechanism(s) of mutant p53 depletion by proteasome inhibition and its therapeutic implications in lung cancer. Methods: Human lung cancer cell lines bearing mutant p53 and wildtype p53 (WTp53) were used in this study. An isogenic panel of cell lines with mutant p53, WTp53 and a vector control was also used to determine mutant p53 dependency of the observed effects. Western blotting, immunoprecipitation, siRNA technology, DNA transfection were used for protein detection, protein-protein interactions, gene knock-down and overexpression respectively. Cycloheximide chase assay was used to determine protein degradation. The cancer genome atlas (TCGA) analysis was performed using the cBioPortal platform. Results: Proteasome inhibitors resulted in depletion of mutant p53 in the lung cancer cell line H1975. This effect was also confirmed using the isogenic model described above. Depletion of mutant p53 occurred through a degradation mechanism and was time dependent. While WTp53 accumulated at initial timepoints of proteasome inhibition, mutant p53 did not accumulate but instead depleted with time. This suggested that the proteasome is not the primary mechanism for mutant p53, in contrast to WTp53. Since autophagy is an alternative mechanism of protein degradation in eukaryotes, we evaluated for, and found evidence of autophagy upon proteasome inhibition. We further showed that autophagic degradation of mutant p53 could involve the heat shock protein 70. Our TCGA data analysis showed proteasome genes amplification in mutant p53 lung cancers, suggesting a possible functional dependency. Furthermore, RNA sequencing analysis showed upregulation of most proteasome genes upon stable expression of the R273H mutant in p53-null lung cancer cell line. Finally, in vitro cell viability assa