Abstract A080: METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8
Authors: Yang Xiaojie , Zoabi Mohamad , Hausmann Simone , Flores Natasha M. , Lu Xiaoyin , Wu Jibo , Morales-Benitez Ana , Gozani Or , Mazur Pawel K. 
Published: November 2022

Abstract
Background: Our work focuses on the role of METTL21A, a member of the little studied seven β-strand family of candidate lysine methyltransferases (KMTs), and its role in the regulation of pancreatic ductal adenocarcinoma (PDAC) development. Using a meta-analysis of publicly available gene expression datasets, we found that METTL21A is significantly downregulated in PDAC versus normal tissue and the reduced expression predicts poor patient survival. Thus, there are intriguing correlations connecting METTL21A to PDAC pathogenesis; however, to date, there are no data on the role of METTL21A in cancer and an enzymatic function for METTL21A is poorly recognized. Methods: To test the hypothesis that METTL21A plays a role in PDAC initiation and progression, we generated conditional Mettl21a knockout mice and crossed to PDAC models: p48Cre/+ KrasG12D/+ (Kras vs. Kras;Mettl21a) and p48Cre/+ KrasG12D/+ p53fl/fl (Kras;p53 vs. Kras;p53;Mettl21a). Furthermore, we studied the impact of METTL21A depletion on human PDAC cell lines and PDX growth in vivo and in vitro. To identify the substrate of METTL21A enzymatic activity we performed methylation assays followed by mass spectrometry analysis. Results: Histopathological analysis of pancreatic tissues of Kras and Kras;Mettl21a mice at 6 months of age revealed that loss of METTL21A significantly accelerates pancreatic cancer initiation. Additional studies using Kras;p53 and Kras;p53;Mettl21a mice demonstrated that METTL21A depletion accelerates PDAC progression and results in a significantly shorter overall survival. In congruence with those observations, knockdown of METTL21A in human PDAC cell lines leads to the robust increase in cell proliferation, enhances colony formation ability in vitro and xenograft growth in vivo. In contrast, cells rescued with ectopic expression of wildtype METTL21A but not with enzyme-dead mutant METTL21A showed significant growth impairment in vitro and in vivo. Next, our in vivo methylation assays identified HSPA1 and HSPA8 (members of the HSP70 protein family) as substrates of METTL21A in PDAC cells. Our subsequent analysis showed that METTL21a specifically tri-methylates HSPA1 and HSPA8 at lysine 561 (K561me3). Next, to directly evaluate the role of METTL21A mediated methylation of HSPA1/A8 at K561 in regulating cancer phenotype, we knocked out HSPA1/A8 and complemented with expression of wildtype or K561A mutant HSPA1/A8 in PDAC cell lines. We found that only methylation-resistant HSPA1/A8 increased cell proliferation and tumor growth in vivo. Together, these results argue that in PDAC, the principal physiologic activity of METTL21A is generation of HSPA1/A8 K561me3, which suppresses cancer growth. Conclusion: Our research revealed that METTL21A is a novel tumor suppressor that inhibits the initiation and progression of PDAC through methylation of the heat-shock proteins HSPA1/8 substrate-binding domain.
Citation Format: Xiaojie Yang, Mohamad Zoabi, Simone Hausmann, Natasha M. Flores, Xiaoyin Lu, Jibo Wu, Ana Morales-Benitez, Or Gozani, Pawel K. Mazur. METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8 [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 A080.