KRAS inhibitors (Sunshine Lake)

Therapy-induced acquired resistance limits the clinical effectiveness of mutation-specific KRAS inhibitors in colorectal cancer (CRC). Here, we investigated whether broad-spectrum, active-state RAS inhibitors meet similar limitations. We found that KRAS-mutant CRC cell lines were sensitive to the RAS(ON) multiselective RAS inhibitor RMC-7977, given that treatment resulted in RAS-RAF-MEK-ERK pathway inhibition; halted proliferation; and, in some cases, induced apoptosis. RMC-7977 initially reduced the activity of a compartment-specific, dual-color reporter of ERK activity, with reporter reactivation emerging after long-term dose escalation. These drug-resistant cell populations exhibited distinct patterns of phospho-protein abundance, transcriptional activities, and genomic mutations, including a Y71H mutation in KRAS and an S257L mutation in RAF1. Transgenic expression of KRAS G13D, Y71H or RAF1 S257L in drug-sensitive CRC cells induced resistance to RMC-7977. CRC cells that were resistant to RMC-7977 and harboring RAF1 S257L exhibited synergistic sensitivity to concurrent inhibition of RAS and RAF. Our findings demonstrate the power of reporter-assisted screening together with single-cell analyses for dissecting the complex landscape of therapy resistance. The strategy offers opportunities to develop clinically relevant combinatorial treatments to counteract the emergence of resistant cancer cells.

Alternative splicing (AS) dysregulation is increasingly recognized as a critical factor in cancer progression and drug response. However, precisely detecting and characterizing complex splicing events, particularly those involving microexons, remains technically challenging. Ribosomal Protein 24 (RPS24), which contains three microexons (3, 18, and 22 bp), serves as an ideal model for studying complex AS regulation in cancer. We developed a high-resolution detection method for RPS24 microexon variations and investigate their relationship with KRAS proto-oncogene, GTPase (KRAS) inhibition in lung adenocarcinoma (LUAD) to identify potential biomarkers for KRAS-targeted inhibitors. We established an integrated methodological approach combining RNA-seq analysis with fragment analysis to detect RPS24 AS patterns. Using this method, we analyzed RPS24 AS across a panel of lung cancer cell lines and examined AS changes in KRAS-mutant cell lines following treatment with KRAS inhibitors. Our method successfully characterized distinct RPS24 AS isoform compositions across lung cancer cell lines, demonstrating high accuracy in detecting 3 bp variations. In KRASmutant cell lines, we observed a consistent upregulation of the 3 bp-containing isoform following KRAS inhibition, indicating a specific correlation with treatment response. This study provides a robust methodology for analyzing complex AS events and supports the RPS24 3 bp-containing isoform as a potential biomarker for KRAS inhibitor response in LUAD. These findings offer new insights into the molecular mechanisms of KRAS inhibitor therapy and strategies for monitoring treatment response. [BMB Reports 2025; 58(6): 244-249].