Abstract 7557: Hypoxia-induced downregulation of MUC16: A key player in bispecific T-cell engager resistance in ovarian cancer
Authors: Xu Mengyao , Barakzai Syem K. , Kononenko Artem , Kumar Raj , Veillard Irva , Kim Eugene , Bond Timothy S. , Spriggs David R. , Rueda Bo R. , Yeku Oladapo 
Published: March 2024

Background: Ovarian cancer (OC) is a lethal disease due to limited treatment options and the development of multidrug resistance. Bispecific T-cell engager (BiTE) therapy represents a promising immunotherapeutic approach to OC. Our previous findings suggest that resistance to BiTE therapy in OC is a complex interplay of factors including loss of tumor-associated antigens and epithelial-mesenchymal transition (EMT). Since the peritoneum is a relatively hypoxic (HX) tumor microenvironment, we hypothesized that HX plays a role in the modulation of MUC16 and potential resistance to BiTE therapy.

Methods: After informed consent, samples were collected from patients with recurrent OC undergoing treatment with MUC16 BiTEs. For HX studies, we used OVCAR3 tumor cell lines which express full-length MUC16. Tumor cells were cultured under normoxic (NX) or HX conditions. The effect of HX on cell death was analyzed using an apoptosis assay. Cells were evaluated for changes in cell surface MUC16 expression by flow cytometry. MUC16 protein expression and EMT markers were analyzed by western blot. Proteasome inhibitors were used to stabilize MUC16 expression under HX conditions. Antibody internalization assays were conducted to assess MUC16 endocytosis and recycling.

Results: In patients who progressed on MUC16 BiTEs, we found decreased MUC16 expression on tumor samples isolated from ascites samples. Using OVCAR3 cells, we found significantly decreased MUC16 expression on ovarian cancer cells grown under HX conditions (Fold change: 0.4 vs NX, p&lt;0.001). Flow cytometry analysis revealed a time-dependent loss of MUC16 expression on the cell surface under HX (64.22% after 9 days HX vs 88.26% NX), highlighting the susceptibility of MUC16 to HX-induced downregulation. Treatment with proteasome inhibitors partially rescued MUC16 expression under HX conditions (Fold change: 1.4 vs NX, p&lt;0.001). Notably, HX did not induce necrotic cell death in these tumor cells. In addition to the loss of MUC16, tumor cells under HX conditions showed alterations in EMT markers. E-cadherin was downregulated with prolonged HX exposure (Fold change: 0.3 vs NX, p &lt; 0.001), while Vimentin (Fold change: 7.1 vs NX, p &lt; 0.001) and Snail (Fold change: 2.8 vs NX, p &lt; 0.001) were upregulated, indicating a potential shift towards a more aggressive phenotype. Epidermal growth factor receptor (EGFR) was also upregulated (Fold change: 4.9 vs NX, p &lt; 0.001), potentially contributing to enhanced survival and invasion of OC cells under HX stress. Finally, under HX conditions, MUC16 internalization was slower in response to antibody binding over time due to reduced cell antigen expression, corroborating the observed MUC16 loss.

Conclusion: Understanding hypoxia-induced MUC16 downregulation and its downstream effects on EMT markers and CA125 expression provides crucial insight into the mechanisms of BiTE therapy resistance in ovarian cancer.

Citation Format: Mengyao Xu, Syem K. Barakzai, Artem Kononenko, Raj Kumar, Irva Veillard, Eugene Kim, Timothy S. Bond, David R. Spriggs, Bo R. Rueda, Oladapo Yeku. Hypoxia-induced downregulation of MUC16: A key player in bispecific T-cell engager resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7557.