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We have previously identified that in the absence of MET amplification or mutation, RTK hyperphosphorylation, indicative of its active state, may contribute to resistance to the EGFR-directed therapeutic, cetuximab in colorectal cancer (CRC). In our in vitro 3D cultures and in vivo xenografts, we showed that inhibition of MET (and RON) by crizotinib can overcome cetuximab resistance in CRC lines and xenografts. We now show that MET may get hyperphosphorylated by the autocrine expression and maturation of its ligand, HGF. This non-genetic mode of cetuximab resistance contrasts with the usual genetic modes of CRC cetuximab resistance, with majority of them being RAS/RAF mutations. Exogenous addition as well as autocrine overexpression of HGF led to cetuximab resistance, which could be overcome by MET inhibition by crizotinib. Interestingly, HGF/HGFL are synthesized as inactive precursors and require cleavage by proteases (HGFA, Matriptase, and Hepsin) to be biologically active. A survey of TCGA datasets indicated that HGF/HGFL were overexpressed in several CRC CMS subtypes, while the endogenous inhibitor of HGF proteases, HAI-I, was downregulated across all CRC CMS subtypes. We next expressed human full-length HGF in cetuximab-sensitive CC cells and observed that HGF overexpression imparts cetuximab resistance. Moreover, cetuximab resistance induced by upstream HGF overexpression could be overcome by the downstream MET inhibition. Next, in a subset of KRAS WT CRC patient-derived xenografts (PDXs) we tested and found that at majority of PDXs exhibit elevated pMET/pRON levels. RNA-seq-based principal component analysis showed that the maximal differences in expression among PDXs could be explained by their pMET/pRON phosphorylation status. Next, we generated nude mice xenografts for one PDX with high pMET/pRON levels and showed that cetuximab inhibition alone was not sufficient to inhibit regrowth of tumors after cessation of drug treatment, but a combined cetuximab/crizotinib treatment was, indicating endogenous MET/RON activation contributes to cetuximab resistance. In a parallel tumor stratification strategy, we have optimized conditions to generate [18F]crizotinib and are testing its efficacy as a new non-invasive PET-based imaging probe to identify crizotinib-avid tumors with high MET/RON activity. Together, these studies may provide novel mechanistic insights into RTK cross-talk and provide a positive predictive biomarker for precision therapy in colorectal cancer. Citation Format: Vivian T. Jones, Ramona Graves-Deal, KyuOK Jeon, Adam J. Rosenberg, Zheng Cao, Galina T. Bogatcheva, Sarah J. Harmych, James N. Higginbotham, Kwangho Kim, James W. Janetka, Gary A. Sulikowski, Bhuminder Singh. Role of MET signaling in cetuximab resistance and generation of an imaging biomarker to identify MET-driven cetuximab resistance 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 2562.