Abstract 125: Targeting colorectal cancer stem cells through GUCY2C expression

Abstract The intestinal cell surface receptor GUCY2C is universally over-expressed by colorectal tumors. Loss of expression of the intestinal hormones regulating GUCY2C silences this signaling pathway which dysregulates the cell cycle and DNA damage repair contributing to intestinal tumorigenesis. Thus, reconstituting GUCY2C activity with oral hormone replacement is being explored in clinical trials as a novel chemoprevention approach for colorectal cancer (CRC). Moreover, GUCY2C is being explored in clinical trials as an immunotherapeutic target reflecting its universal over-expression by metastatic CRC tumors. Here, we explore the expression and activity of GUCY2C in tumor stem cells (SCs) to define its potential as a therapeutic target to prevent therapeutic escape and recurrence in colorectal cancer. CRC-SCs were isolated from a library of patient-derived xenografts (PDXs) and in vitro cell lines by flow cytometry using aldehyde dehydrogenase (ALDH) activity and EpCAM expression. The ALDHHIEpCAMHI CRC-SC fraction comprised ∼1-10% of bulk tumor cells, and expressed the cardinal features of cancer SCs, including non-adherent tumor sphere and colony formation in vitro and tumor formation in immunodeficient mice compared to ALDHLOEpCAMHI differentiated tumor cells. ALDHHIEpCAM+ SCs universally express GUCY2C mRNA and protein, and expression levels are positively correlated to bulk PDX tumors of origin. GUCY2C is expressed on the surface of ALDHHIEpCAM+ SCs, which respond to the GUCY2C agonist linaclotide (Linzess™) by producing and secreting cyclic (c)GMP. Unexpectedly, ALDHHIEpCAM+ SC are hyper-responsive to linaclotide, and their hormone-induced cGMP accumulation is 5- to 10-fold greater than ALDHLOWEpCAMHI differentiated tumor cells. Universal expression, with amplified signaling, in CRC-SCs suggest that GUCY2C might be utilized as a therapeutic target. Indeed, linaclotide induces a durable block of proliferation in, and expansion of, ALDHHIEpCAM+ SCs, quantified by colony formation and expression of Ki67 in vitro. Similarly, ALDHHIEpCAM+ CRC-SCs are completely eliminated by GUCY2C-targeted chimeric antigen receptor (CAR) T cells in vitro. Taken together, these observations suggest that, beyond bulk tumor cells, GUCYC may have dual utilities as a therapeutic target in CRC-SCs. Thus, amplified GUCY2C signaling could be activated by agonists, like linaclotide, to suppress SC persistence and resistance to chemotherapy and potentially shift them to a differentiated phenotype susceptible to treatment. Moreover, metastatic CRC-SCs could be eliminated by GUCY2C-targeted immunotherapies, like CAR T cells, to prevent tumor persistence and recurrence. Citation Format: Jess Kopenhaver, Scott A. Waldman, Adam E. Snook, Trevor Baybutt, Lara Cheslow, Ross Staudt, Jasmine Alvarez. Targeting colorectal cancer stem cells through GUCY2C expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 125.