Combined Genetic Knockdown and High-Content Image Analysis-based Approach to Discovering Novel Inhibitors of Mutant p53-Stabilizing Factors in Ovarian Tumors

Authors

• DJ Schilling
• SD Mazur
• E Appella
• TS Karpova

Abstract

The tumor suppressor protein p53 is a transcription factor that helps maintain genomic integrity by regulating genes involved in cell-cycle arrest, the DNA-damage response, and apoptosis. TP53 mutations are the most common genetic aberration across all human cancer subtypes, with the highest incidence (>96%) in high-grade serous ovarian tumors. Gain-of-function (GOF) mutant p53 (mutp53) such as p53-R175H are characterized by excessive accumulation and the formation of stable aggregates within the cell, which can lead to oncogenic properties such as tumorigenesis stimulation and the development of chemoresistance. Tumor cells often become addicted to the malignant properties of GOF mutp53, making its degradation an appealing approach to targeted cell death. Recently published work identified the deubiquitinating enzyme (DUB) USP15 as a major contributor to the stabilization of p53-R175H. Quantitative image analysis using CellProfiler reveals that two hour in vitro administration of the broad spectrum DUB inhibitor PR-619 induces p53 degradation in ovarian cancer cells containing endogenously expressed p53-R175H, but has little to no effect on cells expressing wild-type. Due to off-target toxicities, broad spectrum DUB inhibitors are poor candidates for drug development, but were useful in identifying DUBs as a candidate target for increasing degradation of mutp53. We are developing a high-content image analysis-based assay to be used in combination with bioinformatics and genetic knockdown assays to discover inhibitors of mutp53-stabilizing factors.

Scientific Focus Area: Cancer Biology

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