Investigation of ERK Activity as a Proxy for Targeted Therapy Resistance in KRAS-mutant Colorectal Cancer

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The EGFR-RAS-ERK signaling cascade has fundamental relevance for colorectal cancer (CRC) biology and therapy response. However, no targeted therapy options are available for patients with KRAS-mutant CRC. This is because these cancers are intrinsically resistant to EGFR inhibition, as well as therapeutic inhibition of downstream signaling nodes. Therapies targeting the RAS-ERK network generally result in ERK reactivation through dynamic changes in the signaling network. We have shown previously that activity of the signaling network converging on ERK is intrinsically coupled to cell differentiation in CRC.In this application, we hypothesize that ERK activity is a suitable read-out to detect resistant CRC cell states emerging during targeted therapy. To test this hypothesis, we will analyze heterocellular CRC organoids with single-cell methodologies established in our lab: CyTOF and single-cell RNA sequencing.In a first step, we will combine these single-cell technologies with fluorescent reporters to read-out ERK activity and subcellular localization in KRAS-mutant and control organoid cells.In a second step, we will evaluate the impact of targeted therapies on the CRC organoids. Combining CyTOF and single-cell RNA-seq, we will record cell differentiation states, activities of key nodes of the signaling network converging on ERK, and the different levels of ERK regulation. For the planned experiments, we have shortlisted several single agent and combinatorial therapies that are currently under clinical investigation in CRC, mainly comprising combinations of RAF, MEK, ERK, and EGFR inhibitors. We will also test the new direct KRAS(G12C) inhibitor AMG510 in KRAS(G12C)-mutant organoid lines.In a third step, we will use the data to improve the therapeutic approaches, and these experiments will be guided and facilitated by the fine-grained data on ERK activity that we have gathered in the previous experiments. In collaboration with the group of Nils Blüthgen, we will employ the single cell-resolved network activity data converging on ERK for quantitative modeling. This approach aims at identifying and exploiting novel vulnerabilities of signaling network states that confer resistance to targeted therapy. In a parallel approach, we will seek to pre-treat the organoids in order to bring the cells to a more uniform (cell differentiation) state that is drug-sensitive rather than resistant before experimental therapy. In summary, the planned experiments aim at unraveling the connections between cancer cell differentiation, ERK re-activation, and resistance to targeted inhibitors, in order to test new ideas on how to provide targeted treatment options to the many KRAS-mutant CRC patients that are currently without targeted treatment options.