Abstract A103: Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile
Authors: Brown Kate , Jenkins Lisa , Crooks Dan , Surman Deborah , Mazur Sharlyn , Xu Yuan , Arimilli Bhargav , Yang Ye , Lane Andrew , Durell Stewart , Fan Teresa , Schrump David , Marston Marston , Ripley Taylor , Appella Ettore , Lyons Gaelyn , Perciaccante Andrew , Dinan Jerry , Robello Marco , Nikolayevskiy Herman , O’Connor Robert , Appella Daniel 
Published: December 2023

Abstract
The tumor suppressor p53 is mutated (mt-p53) in over 50% of human cancers causing gain-of-function oncogenic effects, including metabolic changes that reduce tumor responsiveness to radio/chemotherapy. Common hot-spot mutations within the DNA-binding domain can be categorized as conformational (R175H) or DNA binding (R248W). NSC59984 has been characterized as a small molecule that targets mt-p53 for degradation and restores wt-p53 signaling. Using esophageal adenocarcinoma cells and CRISPR generated isogenic cell lines bearing matching hot-spot p53 mutations, we aim to understand how the molecular features of mt-p53 affect drug efficiency and enable the development of targeted therapies to limit cancer cell growth. We found that NSC59984 covalently modifies p53 by Michael addition at cysteine residues 124 and 229, which promote interactions that would stabilize the protein/DNA complex leading to increased p53 transcriptional activity. In cells, the effects of NSC59984 were substantially greater in cells harboring the R248W mutation compared with the R175H mutation. Treatment with NSC59984 reduced proliferation and increased apoptosis via the intrinsic mitochondrial pathway. It also induced changes in OXPHOS, ATP level, mitochondrial membrane potential, glycolysis, and lactate production. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. NSC59984 treatment increased G6PD activity, total NADPH levels, and expression of TIGAR. Knockout of TIGAR partially removed the antiproliferative effects of the drug and reduced G6PD levels in the p53-R248W cells. Incorporation of [13C6] into cellular metabolites suggests that p53-regulated transcription of TIGAR increased utilization of the pentose phosphate pathway and inhibited glycolysis at the fructose-6-P fructose-1,6-bisphosphate junction, supported by an increase in Hexokinase 2 and a decrease of phosphofructokinase-1. Thermal proteome profiling identified TIGAR as an additional reaction target of NSC59984, suggesting increased involvement in modulating these metabolic effects. Combining currently available therapeutic metabolic inhibitors with NSC59984 enhanced the antiproliferative effects in cells harboring p53-R248W creating a therapeutic window when compared to the wt-p53 expressing cells. This suggests these combinations could be used in a clinically relevant setting. Overall, this work has identified a distinctive mode of action for p53 reactivation resulting in not only transcriptional activity, but also a unique effect on cellular energetics. This study shows evidence of variation in responsiveness of different mt-p53 forms and allows the development of specific therapeutics directed to individuals for patient-centered precision medicine. Importantly, we have shown that targeting p53 signaling has significant effects on the metabolic profiles of cancer cells rendering them more vulnerable to neoadjuvant therapy.
Citation Format: Kate Brown, Lisa Jenkins, Dan Crooks, Deborah Surman, Sharlyn Mazur, Yuan Xu, Bhargav Arimilli, Ye Yang, Andrew Lane, Stewart Durell, Teresa Fan, David Schrump, Marston Marston, Taylor Ripley, Ettore Appella, Gaelyn Lyons, Andrew Perciaccante, Jerry Dinan, Marco Robello, Herman Nikolayevskiy, Robert O’Connor, Daniel Appella. Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A103.