Many cancers require loss of p53 function for cancer progression, often mediated through loss of function mutations or homozygous deletions. Although it is the most frequently mutated gene in cancer, several cancer types lack TP53 mutations. Our preliminary data suggest that even in the absence of mutations, p53 may be inactivated in these cancers through repressive post translational modifications (PTMs). P53 is usually expressed at low levels in the cell, due to proteasomal targeting by MDM2. However, NTera2 cells express abnormally high levels of wildtype p53 protein, but p53 canonical activity is repressed. Recently, our lab discovered that this repression is mediated by the methyltransferases SMYD2 and KMT5A which methylate p53 at specific lysine residues. I found that knockdown of the methylase SMYD2 led to upregulation of hundreds of canonical p53 targets, further implicating a repressive role for p53 methylation. Intriguingly, it also led to robust downregulation of several well-known oncogenes, suggesting p53 methylation may be important for maintaining expression of certain oncogenic programs. Further, KD of the methyltransferase enzymes or p53 led to reduced cell growth in cancer cell lines with methylated (wildtype) p53. I hypothesize that methylation not only represses the tumor suppressor activity of p53, but through altered protein-protein interactions results in novel gain-of-function activity that drives tumor growth. To test this hypothesis, I propose the following aims: (1A) Determine the range of methylated p53 in cancer; (1B) Identify dependencies on p53 methylation for oncogenic growth; (1C) Identify transcriptional targets and genomic binding sites of methylated p53; (2A) Identify differential interacting protein partners of methylated p53; (2B) Identify how combinatorial post-translational modifications alter p53 protein-protein interactions. I will utilize genomic and proteomic approaches to study wildtype p53 in cancer cell lines, patient derived organoids and mouse models. Pharmacological inhibitors, shRNAs, overexpression systems and CRISPR induced base-editors will be used to create methylation deficient mutants in the p53 carboxy-terminal domain. RNA-seq, ChIP-seq and p53 IP followed by mass-spectrometry will be utilized to identify transcriptional targets, binding sites and interacting partners that are specific to methylated p53. Function will be assessed using proliferation, migration, invasion and xenograft assays. This study will make pioneering contributions to our understanding of basic p53 biology in cancer and will establish a new paradigm in the cancer field whereby post-translational modifications switch p53 from a tumor suppressor to an oncogene. Our long-term goal is to use this information to select patients for targeted therapy with drugs to inhibit the p53 methyltransferases, and thus restore p53?s tumor suppressor activity.
P53 is the most commonly mutated gene in cancer and loss of the protein drives cancer progression. There is evidence to suggest that even without a p53 mutation, p53 can be inactivated through additional modifications to the protein. Our work will vigorously study these modifications and will elucidate how they affect p53 function in cancer.
