MAGE proteins are cancer testis antigens (CTAs) that are characterized by highly conserved MAGE homology domains (MHDs) and are increasingly being found to play pivotal roles in promoting aggressive ...cancer types. MAGE-A4, in particular, increases DNA damage tolerance and chemoresistance in a variety of cancers by stabilizing the E3-ligase RAD18 and promoting trans-lesion synthesis (TLS). Inhibition of the MAGE-A4:RAD18 axis could sensitize cancer cells to chemotherapeutics like platinating agents. We use an mRNA display of thioether cyclized peptides to identify a series of potent and highly selective macrocyclic inhibitors of the MAGE-A4:RAD18 interaction. Co-crystal structure indicates that these inhibitors bind in a pocket that is conserved across MHDs but take advantage of A4-specific residues to achieve high isoform selectivity. Cumulatively, our data represent the first reported inhibitor of the MAGE-A4:RAD18 interaction and establish biochemical tools and structural insights for the future development of MAGE-A4-targeted cellular probes.
Peptides have historically been underutilized for covalent inhibitor discovery, despite their unique abilities to interact with protein surfaces and interfaces. This is in part due to a lack of ...methods for screening and identifying covalent peptide ligands. Here, we report a method to identify covalent cyclic peptide inhibitors in mRNA display. We combine co- and post-translational library diversification strategies to create cyclic libraries with reactive dehydroalanines (Dhas), which we employ in selections against two model targets. The most potent hits exhibit low nanomolar inhibitory activities and disrupt known protein–protein interactions with their selected targets. Overall, we establish Dhas as electrophiles for covalent inhibition and showcase how separate library diversification methods can work synergistically to dispose mRNA display to novel applications like covalent inhibitor discovery.
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IJS, KILJ, NUK, PNG, UL, UM
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.
Abstract Uncovering mechanisms that contribute to the unique pathobiology of endometrial cancer has the potential to reveal opportunities for improving outcomes. CD73, a cell surface 5’nucleotidase ...that generates adenosine, has emerged as an attractive therapeutic target; inhibiting CD73 therapeutically restores antitumor immune responses. While anti-CD73 agents appear promising for several cancer types, the biology of CD73 in endometrial cancer is proving to be complex and distinct from other solid tumors. Here, we present two unpublished studies of novel roles for CD73 that diverge from current paradigms. Critically, we show how the unique biology of CD73 in endometrial cancer can be leveraged to improve patient outcomes. We previously reported that although CD73 is upregulated in many tumors, supporting tumorigenesis, CD73 is downregulated in aggressive (grade 3 endometrioid and serous) endometrial carcinomas and its loss is associated with poor outcomes. First, we show through comprehensive immunohistochemistry (n=119) and TCGA computational analyses of microsatellite stable grade 2 endometrioid endometrial carcinomas with intact mismatch repair that loss of CD73, as opposed to its upregulation (as seen in other tumors), reprograms endometrial cancer cells to develop immunosuppressive features. CD73 cancer cell downregulation results in proinflammatory cytokine/chemokine (e.g., CXCL10, CXCL9, CCL8, IL11, IL32) upregulation. Consequently, to counterbalance the inflammatory environment, cancer cells upregulate immunosuppressive genes (e.g., PD-L1). Second, we reveal that CD73 critically restrains the oncogenic transcriptional activity of mutant β-catenin. Mutations in exon 3 of CTNNB1 (the gene that encodes β-catenin) occur in ~26% of low grade endometrioid endometrial carcinomas. CD73 sequesters exon 3 mutant β-catenin (β-cateninEX3) to the membrane, dampening its oncogenic transcriptional activity. A clinical challenge with β-cateninEX3 mutant endometrioid tumors is that half of these patients recur and will have poor outcomes. We show low CD73 expression in endometrioid tumors independently predicts disease recurrence and therefore CD73 may serve as a predictive biomarker where none currently exists. Moreover, we have identified that CD73 differentially controls different patient-specific β-cateninEX3 mutants. Understanding the unique mechanisms of CD73 signaling in endometrial cancer will reveal opportunities for precision medicine strategies. Our studies highlight the continued need and opportunities that exist from focused efforts elucidating the molecular mechanisms driving endometrial cancer. Citation Format: Jessica L. Bowser, Hannah N. Lee, Rebecca H. Hirsch, Xingyuan Zhang, Russell R. Broaddus, Katherine C. Kurnit, Suzanne M. Crumley, Sunthoshini Premsankar, Emily M. Rabjohns, Lilly F. Chiou, Cyrus Vaziri. Harnessing the unique biology of CD73 for improving endometrial cancer outcomes 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 IA022.
