Ion-Channel-Mediated Drug Repurposing Opportunities Validated by Single-Cell Perturbation in Colorectal Cancer

Colorectal cancer (CRC) remains a leading cause of cancer mortality, yet no systematic effort has linked druggable CRC driver genes to downstream ion channel effectors. We integrated differential expression analysis, weighted gene co-expression network analysis (WGCNA), and protein-protein interaction (PPI) network pharmacology to identify CRC hub genes and their ion channel connections, validated by dual single-cell perturbation approaches: variational graph autoencoder-based virtual knockout (VGAE-KO) and experimental HCT116 CRISPRi Perturb-seq (6 genes, 8445 cells). WGCNA identified 100 hub genes spanning three functional programs. Ribosomal proteins link to K⁺ channels (<i>RPS21</i> → <i>KCNQ2</i>, targetable by EMA-approved ataluren, passed dual validation at 97.8th-98.7th percentile). RNA processing genes connect to Cl^- channels (<i>LSM7</i> → <i>CLIC1</i>, strongest signal at 99.8th-99.4th percentile). Immune checkpoint receptors (<i>LAG3</i>, <i>CD27</i>) connect via PPI intermediates to Ca2+ and K⁺ channels, targetable by relatlimab (FDA-approved) and varlilumab (Phase 2). This work maps previously unknown links between CRC driver genes and ion channel regulation, with the ataluren-<i>RPS21</i>-<i>KCNQ2</i> axis ready for pharmacological testing.