Abstract PR004: CD73 is a novel repressor of mutant β-catenin oncogenic activity in endometrial cancer
Authors: Hirsch Rebecca M. , Zhang Xingyuan , Chiou Lilly F. , Premsankar Sunthoshini , Lee Hannah N. , Kurnit Katherine C. , Broaddus Russell R. , Vaziri Cyrus , Bowser Jessica L. 
Published: March 2024

Abstract
β-catenin is an important oncogene commonly (20-30%) activated by somatic missense exon 3 CTNNB1 mutations in low grade, early stage endometrioid-type endometrial cancer (EEC). Although exon 3 CTNNB1 mutation is associated with increased risk of recurrence in many (~50%) patients with low grade, early-stage EEC, an equal number of patients with β-catenin mutant tumors will not recur. It remains unclear why patient outcomes are so variable. The variability suggests there are unknown determinants of β-catenin mutant tumor aggressiveness. The purpose of this study was to evaluate CD73, a cell surface 5’-nucleotidase, as a critical factor controlling mutant β-catenin oncogenic activity in EEC. We previously identified CD73 downregulation in exon 3 CTNNB1 mutant EEC predicts recurrence and reported, using a highly homologous Xenopus exon 3 β-catenin mutant, that CD73 restrains mutant β-catenin to the membrane. For this study, we interrogated patient-relevant exon 3 CTNNB1 mutation frequencies in 5 publicly available databases and developed 7 (D32N, S33F, S33Y, G34R, S37C, S37F, and S45F) myc-tagged β-catenin mutant lentiviral vectors for expression in HEC-1-A and Ishikawa cells in which we applied CRISPR-Cas9 deletion or the re-expression of CD73. Reporter assays showed all patient-relevant β-catenin mutants at baseline induce transcriptional activity compared to endogenous levels. With CD73 loss, transcriptional activity for all mutants increased significantly in HEC-1-A cells (normally CD73+/+). With re-expression of CD73 in Ishikawa cells (normally CD73−/−), transcriptional activity of several but not all β-catenin mutants decreased, which provides evidence for the first time that β-catenin mutants are differentially controlled in endometrial cancer. Cell viability assays also demonstrated mutant-specific control by CD73. Using RNA-seq data from TCGA, we assessed whether β-catenin mutants in EECs lacking CD73 exhibited unique tumorigenic transcriptomes. Contrary to current paradigms for exon 3 mutant β-catenin, a dependency on expression of canonical Wnt/β-catenin gene targets was not observed in CD73-deficient β-catenin mutant tumors. In contrast, unique gene signatures (e.g., neuron biology and alcohol metabolic process) that have not previously been linked to β-catenin mutant EEC were identified. Specifically, loss of NAAA and SRD5A3 (encoding N-acylethanolamine-hydrolyzing acid amidase and steroid 5-alpha reductase 3, respectively) were found in β-catenin mutant CD73 low EECs. Loss of these genes are associated with poor outcomes in EEC. Ongoing computational studies of pooled EEC datasets are assessing the unique transcriptomes of individual patient-specific β-catenin mutants in the context of CD73 loss. Our results describe CD73 as a novel determinant controlling mutant β-catenin in EEC. Furthermore, we provide mechanistic rationale to explain the variability in outcomes in endometrial cancer patients with exon 3 β-catenin mutant tumors.
Citation Format: Rebecca M. Hirsch, Xingyuan Zhang, Lilly F. Chiou, Sunthoshini Premsankar, Hannah N. Lee, Katherine C. Kurnit, Russell R. Broaddus, Cyrus Vaziri, Jessica L. Bowser. CD73 is a novel repressor of mutant β-catenin oncogenic activity in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR004.