Abstract 1717: RAS and PP2A co-regulated phosphosite on KDM1A dictates its substrate specificity & transcriptional outcome

Abstract

Abstract RAS-mediated human cell transformation requires simultaneous inhibition of tumor suppressor phosphatase PP2A; however, the target phosphosites co-regulated by RAS and PP2A are poorly characterized. In this study, we describe the phosphoregulation of an epigenetic regulator, KDM1A, by RAS and PP2A. KDM1A is a lysine-specific demethylase that can act as a transcriptional activator or inhibitor, depending on its specific activity toward various histone marks. However, the post-translational modifications regulating the substrate specificity of KDM1A are poorly addressed. We found that KDM1A physically interacts with PP2A through a conserved binding motif, and that mutating the motif residues decreased its interaction with PP2A. Additionally, PP2A activation or RAS inhibition led to increased chromatin recruitment of KDM1A. This enhanced chromatin recruitment of KDM1A was recapitulated upon CRISPR/CAS9-mediated substitution of the PP2A/RAS-targeted serine to alanine. Using histone proteomics, we further identified that the RAS/PP2A-regulated phosphosite determines the substrate specificity of KDM1A. This is evidenced by an increased demethylation of histone H3K9me2/3, a marker of gene activation. The differential gene expression profile of the phosphorylation mutants of KDM1A predominantly showed increased gene expression, further validating our findings. The gene expression profiles of KDM1A phosphomutant RAS mutant cells demonstrate gene level specificity indicating that this newly discovered KDM1A phosphoswitch has an important role in specifying KDM1A function in cancer. Collectively, our results demonstrate that RAS and PP2A activities converge on phosphoregulation of epigenetic machinery in cancer cells. This RAS and PP2A co-regulated phosphosite on KDM1A dictates both its histone mark specificity and transcriptional outcome. Citation Format: Mukund Sharma, Anna Aakula, Jesse Kamila, Reetta Nätkin, Tiziana Bonaldi, Saverio Minucci, Matti Nykter, Jukka Westermarck. RAS and PP2A co-regulated phosphosite on KDM1A dictates its substrate specificity & transcriptional outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1717.