Abstract 6227: Tumor cell heterogeneity may cause treatment failure of GPA33-targeted therapy in colorectal cancer

Abstract Purpose: Glycoprotein A33 (GPA33) is a membrane protein almost exclusively expressed in intestinal epithelia and in more than 95% of colorectal cancers (CRCs). This renders GPA33 a promising antigen for targeted therapy, but trials did not meet pre-specified response rates. We investigated intratumoral heterogeneity as a reason for low treatment efficacy, GPA33 regulation and the potential for pharmacological induction of GPA33 expression in vitro and in vivo to enhance treatment response. Methods: We assessed intratumoral heterogeneity of GPA33 in tissue specimens of 223 stage II CRCs using immunohistochemistry and immunofluorescence, and obtained scRNAseq transcriptomic analysis from 12 primary CRCs. We then manipulated WNT and PPARγ signaling pathways and transcription factor expression in colon cancer cells using specific inhibitors, siRNAs or inducible dominant negative TCF4 (dnTCF4), and analyzed the effects on GPA33 protein and RNA expression. Furthermore, we investigated the effects of WNT inhibition on GPA33 in vivo in NOD/SCID xenografts. Additionally, we developed anti-GPA33 CAR-T cells and measured their activation after co-cultivation with colon cancer cells using flow cytometry. Results: In the CRC cohort we found that GPA33 expressing cells had a differentiated phenotype and low WNT activity. GPA33-negative subpopulations were located at the infiltrative tumor edge and showed increased nuclear β-catenin. Downregulation of WNT pathway activity using inhibitors, siRNAs or inducible dominant negative TCF4 (dnTCF4), as well as activation of PPARγ signaling induced GPA33 expression in vitro. Forced expression of dnTCF4 also enhanced GPA33 expression in vivo in previously GPA33-negative tumor cell subpopulations in colon cancer xenografts. siRNA mediated knockdown of CDX1 and KLF4, two transcription factors that were previously implicated in GPA33 regulation, only moderately reduced GPA33 expression levels in vitro. Anti-GPA33 CAR-T cells activated in response to co-culture with GPA33-expressing CRC cells in vitro. Conclusion: Our analysis highlights a heterogenous expression of GPA33 in vivo as a limiting factor for anti-GPA33 therapy. WNT inhibition or PPARγ activation induced GPA33 expression in vitro and in vivo. Depletion of CDX1 and KLF4 had moderate effects on GPA33 only, indicating a complex regulation in colon cancer. We suggest that pharmacological induction of GPA33 expression may increase the efficacy of anti-GPA33 targeted therapy in patients with CRC. Citation Format: Teresa Börding, Tobias Janik, Markus Morkel, Christine Sers, David Horst. Tumor cell heterogeneity may cause treatment failure of GPA33-targeted therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6227.