Functional Phosphoproteomics in Cancer Chemoresistance Using CRISPR-Mediated Base Editors

Area of Science:

• Oncology
• Molecular Biology
• Biochemistry

Background:

• Targeted protein kinase inhibitors are crucial for cancer therapy but face challenges from drug resistance.
• Tumor cells develop resistance by acquiring mutations or activating bypass pathways, necessitating identification of resistance mechanisms.
• Understanding downstream phospho-substrates is vital for developing effective combination therapies against malignancies.

Purpose of the Study:

• To identify functional phosphorylation sites contributing to 5-fluorouracil (5-FU) resistance in colorectal cancer.
• To explore novel therapeutic strategies by targeting key kinases involved in 5-FU resistance.
• To establish a functional phosphoproteomics screening approach for chemotherapy resistance.

Main Methods:

• Utilized CRISPR-mediated cytosine base editor (CBE) and adenine base editor (ABE) for large-scale functional screens.
• Developed two mutation libraries targeting 7779 and 10,149 phosphorylation sites.
• Analyzed enriched gRNAs to identify gain-of-function mutants and affected signaling pathways.

Main Results:

• Identified genes involved in cell cycle and post-translational modifications as critical in 5-FU resistance.
• Discovered RSK2 and PAK4 kinase substrates as key effectors in response to 5-FU chemotherapy.
• Demonstrated that combined 5-FU with RSK2 or PAK4 inhibitors significantly inhibits colorectal cancer cell growth and enhances apoptosis via a RSK2/TP53BP1/γ-H2AX axis.

Conclusions:

• The study identified a novel signaling axis (RSK2/TP53BP1/γ-H2AX) mediating 5-FU resistance in colorectal cancer.
• Combination therapy with 5-FU and RSK2 or PAK4 inhibitors shows promise for enhanced colorectal cancer treatment.
• The developed CRISPR-based screening approach is applicable for functional phosphoproteomics in various chemotherapy resistance contexts.