Figure 2 from A Plastic EMP1+ to LGR5+ Cell State Conversion as a Bypass to KRASG12D Pharmacologic Inhibition in Metastatic Colorectal Cancer

Plasticity between Lgr5+ CSC to Emp1+ HRC states by KRAS activity across models. A, Growth (CellTiter-Glo) of AKTP MTOs treated with vehicle or RMC-9945 for 9 days normalized to day 0 of treatment. Mean ± SEM. P values between vehicle day 2 and RMC-9945 day 4, 6, or 9 are derived from ANOVA followed by Šídák multiple comparisons test. B, Gene set enrichment analysis of AKTP MTOs upon vehicle versus RMC-9945 treatment. C, Relative mRNA expression of indicated marker genes in vehicle-treated and RMC-9945–treated AKTP MTOs for 48 hours assessed by RT-qPCR. Mean ± SEM. P values are derived from ANOVA followed by Šídák multiple comparisons test. D, CRISPR-Cas9 targeting strategy to introduce APK mutations in normal mucosa (NM) organoids derived from C57BL/6J mice. E and F, Boxplots depicting the normalized and z-scored expression of Lgr5+ ISC gene signature (E) and EpiHR signature (F) in APK CTOs treated with either vehicle or RMC-9945 for 48 hours. t test was used to assess median differences. G, Gene set enrichment analysis (GSEA) of APK CTOs upon vehicle and RMC-9945 treatment. H, Growth (CellTiter-Glo) of PDO131 treated with vehicle or RMC-9945 for 10 days normalized to day 0 of treatment. I, Differential gene expression analysis of CSC and HRC key genes for PDO 131. J, GSEA of PDO131 in vehicle versus RMC-9945 treatment. K, Growth (CellTiter-Glo) of PDO C31M treated with vehicle or RMC-9945 for 10 days normalized to day 0 of treatment. L, Differential gene expression analysis of CSC and HRC key genes for PDO C31M. M, GSEA of PDO C31M in vehicle versus RMC-9945 treatment. PDOs were treated with RMC-9945 for 4 days and subjected to RNA-seq analysis. Dusp6 gene is included as a reference for KRAS/MAPK pathway inhibition. Log2 fold change (FC) and adjusted P value are shown. For GSEA, selected Hallmark, GOCC, Kyoto Encyclopedia of Genes and Genomes, and custom gene signatures are shown. NES, normalized enrichment score. P value < 0.05 for all gene sets, color gradient represents significance as shown. For the growth curves, P values are derived from multiple unpaired t test.