Identification of regulators of mutant p53 accumulation in lymphoma using RNA interference

Abstract

In over 40% of all cancers, the key tumor suppressor p53 is inactivated via mutation. Mutant (mut) p53 can gain new properties (gain-of-function, GOF), which actively contribute to tumorigenesis. In many tumors, a massive accumulation of mutp53 protein is observed and a prerequisite for the GOF activity. Therefore, tumors often depend on sustained high levels of mutp53, which suggests that interfering with mutp53 accumulation may be exploitable in cancer therapy. However, the mechanisms that control excessive mutp53 stabilization are not fully understood. To this end, the main aim of this study was to identify regulators of mutp53 accumulation in Burkitt’s lymphoma (BL) as a model for a highly aggressive cancer. Despite the presence of functional MDM2, the main negative regulator of p53, mutp53 was found to be stabilized in BL. To identify proteins regulating mutp53 levels in an unbiased fashion, a flow cytometry-based RNA interference (RNAi) screen was conducted in a mutp53 BL cell line model. The primary screen hit was TRRAP (transformation/transcription domain-associated protein), a constituent of several histone acetyltransferase (HAT) complexes. TRRAP knock-down and knock-out resulted in depletion of mutp53 protein (but not mRNA) in lymphoma and colorectal cancer cell lines with a diverse spectrum of p53 mutations. Conversely, TRRAP overexpression increased mutp53 levels. Mass spectrometric analysis of the mutp53 interactome after TRRAP knock-down indicated that TRRAP silencing caused nuclear export of mutp53 and degradation via the MDM2-proteasome axis, suggesting targeting of mutp53 to the physiological p53 degradation machinery. Gene expression profiling after TRRAP knock-down showed a suppression of cell cycle-related genes and an induction of interferon signaling, which however did not contribute to mutp53 regulation. To map functional regions of TRRAP, a CRISPR/Cas9 mutagenesis approach (“CRISPR scanning”) was applied which identified a 109 amino acid region in the N-terminal HEAT repeat region crucial for mutp53 accumulation and cell survival. In wild-type p53 BL cells, TRRAP silencing attenuated p53 stabilization and activity upon genotoxic stress. Finally, to transfer the results from RNAi screening, a drug-based screening was performed and identified that inhibition of histone deacetylases (HDAC) and specifically HDAC1/2/3 decreased mutp53 levels to a surprisingly similar extent as TRRAP knock-down. In summary, this study identifies TRRAP as a key regulator of p53 levels and links histone-modifying complexes to p53 protein accumulation. Based on the GOF properties of mutp53, this may provide a basis for therapeutic targeting of mutp53 in lymphoma and other cancers.