We have shown previously that phosphorylation of Mdm2 by ATM and c-Abl regulates Mdm2-p53 signaling and alters the effects of DNA damage in mice, including bone marrow failure and tumorigenesis induced by ionizing radiation. Here, we examine the physiological effects of Mdm2 phosphorylation by Akt, another DNA damage effector kinase. Surprisingly, Akt phosphorylation of Mdm2 does not alter the p53-mediated effects of ionizing radiation in cells or mice but regulates the p53 response to oxidative stress. Akt phosphorylation of Mdm2 serine residue 183 increases nuclear Mdm2 stability, decreases p53 levels, and prevents senescence in primary cells exposed to reactive oxidative species (ROS). Using multiple mouse models of ROS-induced cancer, we show that Mdm2 phosphorylation by Akt reduces senescence to promote KrasG12D-driven lung cancers and carcinogen-induced papilloma and hepatocellular carcinomas. Collectively, we document a unique physiologic role for Akt-Mdm2-p53 signaling in regulating cell growth and tumorigenesis in response to oxidative stress.

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T-cell based immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that induction of cellular senescence and its accompanying senescence-associated secretory phenotype (SASP) can be a powerful way to enhance T cell infiltration into tumors and reactivate a different type of innate Natural Killer (NK) cell anti-tumor immunity that can mediate tumor control. Here, we found that the pancreas tumor microenvironment (TME) suppresses NK cell surveillance following therapy-induced senescence (TIS) with MEK and CDK4/6 inhibitor treatment through EZH2-mediated repression of pro-inflammatory SASP genes. Genetic or pharmacological inhibition of EZH2 or its methyltransferase activity enhanced the production of pro-inflammatory SASP factors and led to NK and T cell infiltration and immune-mediated tumor control in transplanted and genetically engineered PDAC mouse models. Mechanistically, EZH2 suppression induced expression of SASP factors CCL2 and CXCL9/10 necessary for lymophocyte chemotaxis into the pancreas TME. EZH2 levels were also associated with reduced NK cell numbers, SASP expression, and overall survival in a PDAC patient cohort. Taken together these results demonstrate that EZH2 mediates repression of the pro-inflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK and T cell surveillance in PDAC to achieve immune-mediated tumor responses. Citation Format: Loretah Chibaya, Katherine C. Murphy, Yvette Lopez Diaz, Kelly D. DeMarco, Haibo Liu, Sneha Gopalan, Melissa Faulkner, Junhui Li, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Elisa de Stanchina, Karl Simin, Lihua J. Zhu, Thomas G. Fazzio, Scott W. Lowe, Marcus Ruscetti. EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype and promotes immune surveillance in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C015.

The serine/threonine kinase Akt is activated by a myriad of growth signals and promotes cell proliferation, survival, and metabolism. Akt is frequently activated in a wide assortment of human hematologic malignancies and solid tumors. In vitro studies demonstrated that Akt phosphorylates the Mdm2 oncoprotein at ser166 and ser186 (163 and 183 in mice) to promote Mdm2 translocation into the nucleus. This enhances Mdm2-mediated p53 ubiquitination and the subsequent proteasomal-mediated degradation of p53. Wild-type p53 protein is a tumor suppressor that is activated in response to stress signals such as DNA damage, oxidative stress, and aberrant oncogene activation. Activated p53 induces the expression of genes that trigger cell cycle arrest, senescence, and apoptosis. This inhibits the growth of damaged and/or potentially oncogenic cells, underscoring the role of p53 in tumor suppression. However, the role of Akt-mediated phosphorylation of Mdm2 on p53 functions in vivo remains unknown. We hypothesize that Mdm2 phosphorylation by Akt at ser183 promotes cell growth and tumorigenesis by reducing p53 levels and function in vivo. Using CRISPR/Cas9-mediated gene editing, we generated mice with mutant Mdm2 protein that cannot be selectively phosphorylated at ser183, by substituting this serine with an alanine. Unlike Mdm2-/- mice that are embryonic lethal, Mdm2S183A mice are viable, fertile, and obtained at Mendelian ratios. There are no apparent growth/weight differences between unchallenged Mdm2WT and Mdm2S183A mice. Other stress-induced kinases like ATM and c-Abl phosphorylate Mdm2 to regulate the levels and functional activity of p53 in response to stress such as DNA damage. In contrast, Akt-mediated phosphorylation of Mdm2 does not alter p53 levels and function in response to DNA damage both in vitro and in vivo. However, under low-density stress plating, the ability of Mdm2S183A MEFs to form colonies is completely abrogated. In addition, Mdm2S183A MEFs fail to proliferate under normal cell culture conditions and exhibit salient features of senescence including positive staining for SA-β Gal. The expression of p53-target genes that mediate senescence is elevated in Mdm2S183A MEFs than in Mdm2WT MEFs. The severe proliferation defect and premature senescent phenotypes observed in Mdm2S183A MEFs are rescued by culturing cells at low oxygen tension with media supplemented with N-acetyl cysteine, a reactive oxygen species (ROS) scavenger. Our results from various assays examining the levels and effects of ROS suggest that under normal cell culture conditions, Mdm2S183A MEFs are more sensitive to oxidative stress compared to Mdm2WT MEFs. So far, our data suggest that Mdm2 phosphorylation by Akt at ser183 is required for proper regulation of the oxidative stress response and is necessary to promote cell growth under normal plating stress conditions. We are currently analyzing RNA-seq data to determine whether there are certain gene expression changes that could aid our quest to decipher the underlying mechanism responsible for the senescent phenotype of Mdm2S183A MEFs. In addition, we have crossed Mdm2S183A to lung and liver cancer models in which ROS plays a pivotal role to examine whether blocking Mdm2 phosphorylation by Akt alters the incidence and progression of tumors. Citation Format: Loretah Chibaya, Hong Zhang, Stephen N. Jones. Mdm2 phosphorylation by Akt is critical for modulating cellular responses to oxidative stress [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A23.