Effect of MUC16 Blockade using the Humanized AR9.6 Antibody in Patient Derived Organoid Models of PDAC

Pancreatic Ductal Adenocarcinoma (PDAC) represents nearly 90% of all pancreatic cancer cases. 49,830 of the 62,210 patients diagnosed in 2022 are estimated to succumb to the malignancy. Early diagnosis of the disease is uncommon as most patients present with symptoms when the cancer is late-stage and metastatic. This decreases the likelihood of successful surgical resection and increases the dependency on standard of care chemotherapy leads to which is met with therapeutic resistance, demonstrated by the 5-year post-diagnosis survival rate of a mere 11.5%. Mucin-16, a heavy glycosylated transmembrane protein is overexpressed in more than 65% PDAC cases. AR9.6 is an anti-MUC16 antibody that has been recently humanized after evidence of its therapeutic potential was found in an orthotopic study utilizing the murine version of the antibody. The HuAR9.6 antibody efficiently binds MUC16 expressed on tumor cells, and can both inhibit downstream oncogenic signaling and elicit tumor killing by signaling the immune system to the tumor. In this project, we used RNA sequencing to evaluate the MUC16 mediated transcriptomic changes by using the humanized AR9.6 antibody in patient-derived organoid models of PDAC. To begin this study, organoids were developed using tumor cells from a primary PDAC tumor with a high MUC16 profile obtained from rapid autopsy patient #142 from the Rapid Autopsy Program at UNMC. The organoids were then treated in triplicates using a monoclonal antibody HuIgG as an isotype control due to its lack of specificity, and the test arm of study, HuAR9.6. These samples were treated with 40 ug/mL of the antibodies for a 24-hour period, post which RNA isolation was performed. RNA sequencing and subsequent Gene Ontology and KEGG enrichment analysis revealed a downregulation of genes involved in the Hippo signaling pathway, fat digestion and absorption and TGF-β signaling. Based on this gene expression profiling, we hypothesize that HuAR9.6 can slow tumor progression by downregulating the Hippo and TGF-β signaling pathways. In the future, we aim to robustly validate these results at the level of the proteome and assess if these results can be reproduced in multiple patient samples with the hope to translate this antibody to the clinic to be used in PDAC patients who have a high MUC16 expression.https://digitalcommons.unmc.edu/surp2022/1038/thumbnail.jp

Truncated O-Glycan-Bearing MUC16 Enhances Pancreatic Cancer Cells Aggressiveness via α4β1 Integrin Complexes and FAK Signaling

Elevated levels of Mucin-16 (MUC16) in conjunction with a high expression of truncated O-glycans is implicated in playing crucial roles in the malignancy of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms by which such aberrant glycoforms present on MUC16 itself promote an increased disease burden in PDAC are yet to be elucidated. This study demonstrates that the CRISPR/Cas9-mediated genetic deletion of MUC16 in PDAC cells decreases tumor cell migration. We found that MUC16 enhances tumor malignancy by activating the integrin-linked kinase and focal adhesion kinase (ILK/FAK)-signaling axis. These findings are especially noteworthy in truncated O-glycan (Tn and STn antigen)-expressing PDAC cells. Activation of these oncogenic-signaling pathways resulted in part from interactions between MUC16 and integrin complexes (α4β1), which showed a stronger association with aberrant glycoforms of MUC16. Using a monoclonal antibody to functionally hinder MUC16 significantly reduced the migratory cascades in our model. Together, these findings suggest that truncated O-glycan containing MUC16 exacerbates malignancy in PDAC by activating FAK signaling through specific interactions with α4 and β1 integrin complexes on cancer cell membranes. Targeting these aberrant glycoforms of MUC16 can aid in the development of a novel platform to study and treat metastatic pancreatic cancer