Abstract PR02: Loss of TET2 activity results in epigenetic instability and drives PDAC molecular subtypes
Authors: Eyres Michael , Lanfredini Simone , Willenbrock Frances , Thapa Asmita , Blake Andrew , Burns Adam , Sabbagh Ahmad , Abraham Aswin , Maughan Timothy , Soonawalla Zahir , Schuh Anna , Mukhergee Somnath , O'Neill Eric 
Published: March 2020

Abstract
Background: mRNA datasets have defined two molecular subtypes of pancreatic ductal adenocarcinoma (PDAC), classical and squamous, with distinct clinical characteristics and raising the possibility of subtype specific therapies (Collisson et al., 2019). The aggressive squamous subtype is characterized by poorer patient survival and loss of endodermal differentiation markers GATA6 and PDX1, but neither PDAC subtypes nor metastasis can be explained by genetic mutations alone (Reiter et al., 2018). Conversely, widespread epigenetic reprogramming is associated with progression to more aggressive phenotypes (McDonald et al., 2017), suggesting that aggressive molecular subtype is likely to be epigenetically driven. The role of DNA 5’methylcytosine (5’mc) in PDAC has been previously unclear due to an inability to distinguish this mark from the reciprocal activation mark DNA 5’hydroxymethylcytosine (5’hmC), mediated by Ten-eleven-translocation (TET) enzymes. Moreover, 5’hmc is dynamically regulated during pancreatic differentiation, but a role during PDAC initiation or progression has not been previously addressed.
Methods: PDAC patients who underwent surgical resection at the Churchill Hospital, Oxford (n=146) had FFPE tumor tissue mutation comprehensively profiled by illumina hotspot array (300X), mRNA microarray, and the first epigenetic separation of 5’mc and 5’hmc on FFPE via oxidative bisulphite sequencing (oxBS) and illumina EPIC arrays. iCluster was used to highlight epigenetic PDAC subtypes and compare to existing TCGA datasets. Orthotopic models were employed to test whether the Squamous subtype is defined by 5’hmc loss and if this can be converted to the classical subtype in vivo.
Results: Compared to healthy tissue, PDAC tumors demonstrate loss of 5’hmC at genes critical for pancreatic development and associated with PDAC progression, including MAPK signaling and TP53 targets. 5’hmC was preferentially lost in aggressive molecular subtypes (squamous) compared to the classical subtypes and was associated with SMAD4 mutations and reduced TET2. Overexpression of TET2 in squamous tumor cells restored 5’hmc and the expression of classical associated genes (e.g., GATA6 and PDX1), suggesting that 5’hmC is a master epigenetic regulator of PDAC molecular subtypes. Notably, we find TET2 stability is regulated by glucose levels, implying the widespread hypoglycemia seen in PDAC patients may contribute to progression of aggressive subtypes through loss of 5’hmC. Concomitantly, as metformin and vitamin C independently enhance endogenous TET2 activity, we find that this combination acts synergistically on squamous tumors to increase 5’hmC and expression of a classical-subtype phenotype, suggesting subtype switching is achievable in vitro and in vivo.
Conclusion: These results identify 5’hmC as a regulator of molecular subtype that can be targeted in vivo using well-tolerated drugs.
This abstract is also being presented as Poster A39.
Citation Format: Michael Eyres, Simone Lanfredini, Frances Willenbrock, Asmita Thapa, Andrew Blake, Adam Burns, Ahmad Sabbagh, Aswin Abraham, Timothy Maughan, Zahir Soonawalla, Anna Schuh, Somnath Mukhergee, Eric O'Neill. Loss of TET2 activity results in epigenetic instability and drives PDAC molecular subtypes [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR02.