Identification and Functional Characterization of Regulators of Compartmentalized ERK1/2 Activity in Colorectal Cancer

• Applicants: Professor Dr. Michael Böttcher; Dr. Markus Morkel
• Subject Area: Gastroenterology ; Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 444691702

The project focuses on investigating compartment-specific roles of extracellular signal-related kinases ERK1 and ERK2 (ERK1/2), which are crucial in the MAPK signaling pathway, influencing cellular processes such as proliferation, motility, differentiation, and survival. In the previous DFG-funded project “ERK as a proxy for colorectal cancer targeted therapy resistance”, we have developed an advanced ERK1/2 reporter toolkit, termed FIREX, which can simultaneously report on cytoplasmic and nuclear ERK1/2 activities in cells. This new reporter system improves upon previous versions by enhancing the promoter, localization signals, and fluorescent proteins, allowing for more precise tracking of ERK1/2 activity. The present follow-up application aims to explore the regulators of subcellular activity of ERK1/2 in colorectal cancer (CRC) cells, especially in the context of resistance to targeted therapies. The project's primary objectives include characterizing mutations that lead to subcellular reactivation of ERK1/2 in RAS-inhibitor resistant CRC cells, conducting kinome-wide CRISPR screens to identify new regulators of ERK1/2. We will also explore genetic redundancies among candidate kinases using combinatorial CRISPR screens, which could reveal critical interactions that may be important for developing new therapeutic strategies. By understanding these redundancies, the team aims to improve the effectiveness of combination therapies targeting compensatory pathways in CRC. The CRISPR screens will be followed by functional characterization of the hits in CRC cell lines, including the assessment of signalling networks with single cell resolution. For this, we will use advanced techniques like CyTOF and single-cell RNA sequencing to probe signaling networks and assess the potential of specific kinases or pathways as predictive markers for targeted therapies. We plan to perturb relevant signalling networks by CRISPR-mediated knockouts, followed by functional analysis, to identify potential modulators of ERK1/2 subcellular localization. This work is expected to shed light on the mechanisms underlying therapy resistance in CRC and suggest potential therapeutic targets. Finally, the project will investigate the clinical relevance of compartment-specific ERK1/2 activity by analyzing publicly available databases for patterns of misexpression or mutations in candidate regulators. The ultimate goal is to provide insights that could guide the development of more effective treatments for CRC, particularly for patients who exhibit resistance to existing targeted therapies.

• DFG Programme: Research Grants