Abstract 2006: Exploring a novel crosstalk between PTK7 and EPHA2 tyrosine kinase receptors in metastatic colorectal cancer

Abstract Colorectal cancer (CRC) remains a significant public health challenge, with an urgent need of novel prognostic biomarkers and therapeutic targets. PTK7, a membrane tyrosine pseudokinase and Wnt receptor, is overexpressed in CRC and linked to poor prognosis. In CRC, PTK7 expression correlates with metastatic progression and decreased survival in non-metastatic patients, positioning PTK7 as both a potential biomarker and a promising drug target. Notably, a targeted antibody-drug conjugate (ADC) against PTK7 has recently shown efficacy in inducing tumor regression in patient-derived xenograft (PDX) models of solid tumors and has progressed to a Phase I clinical trial with promising results. Other PTK7-targeting ADCs and anti-PTK7 CAR-T cells recently entered into preclinical development. At the plasma membrane, PTK7 can interact with a range of membrane receptors, including VEGFR, EGFR, ROR2, and Plexins, enhancing their signaling capabilities. Using biotin proximity labeling in HCT116 CRC cells, we identified a novel network of PTK7-associated membrane proteins. Among these, we focused on EPHA2, an active tyrosine kinase receptor also implicated in cancer. Our findings indicate that PTK7 and EPHA2 interact via their extracellular domains (ECDs), and that PTK7 depletion significantly upregulates EPHA2 expression and signaling in response to its ligand EFNA-1 in HCT116 cells. Additionally, PTK7 regulates EPHA2’s endosomal sorting through a Rab11-dependent mechanism, as well as its K63-linked ubiquitination and subsequent lysosomal degradation. Mechanistically, PTK7 does not appear to participate directly in EFNA-1 binding but instead promotes multimerization of active EPHA2, a crucial step in amplifying EPHA2 signaling. This study aims to elucidate the functional role of the interaction between PTK7 and EPHA2 in colorectal cancer progression. By mapping the PTK7:EPHA2 interface using AlphaFold3 predictions and introducing targeted deletions and mutations within the ECDs of both receptors, we seek to understand how disrupting this interaction influences tumorigenic and metastatic processes. Our findings indicate that PTK7 depletion triggers cell rounding and detachment, with a marked increase in total and activated EPHA2 levels, which may drive pro-metastatic activity in vivo. Outcomes may significantly impact the use of anti-PTK7-ADCs or CAR-T cells and orient new therapeutic strategies to design agents interfering with PTK7 and EPHA2 interaction and function. Citation Format: Charlotte Dessaux, Constantin Semenchenko, Luc Camoin, Stéphane Audebert, Emilie Baudelet, Avais Daulat, Flavio Maina, Jean-Paul Borg. Exploring a novel crosstalk between PTK7 and EPHA2 tyrosine kinase receptors in metastatic colorectal cancer [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 2006.