Gene Set Enrichment Analysis

Paired gene set enrichment analyses between off- versus on-drug conditions or between two different cell lines were performed as described previously(1). We computed differential gene set enrichments of the gene sets in the C2 CGP, C6 and Hallmark subsets from the Molecular Signature Database of the Broad Institute using the following steps: i) Calculating log₂ fold changes (log₂ FC) of mRNA expression of each gene in M249 DDR5 compared to SKMEL28 DDR1 at all three treatment conditions, i.e., on-drug 6 h, off-drug 6 and 24 h, ii) Based on the log₂ FC values, we computed the differential enrichment of each gene set between M249 DDR5 and SKMEL28 DDR1 in all three treatment conditions (cutoff for differential enrichment, Wilcoxon rank-sum test between genes within the gene set and the rest of the genes; p ≤ 0.05; median of up-expression across all genes in the gene set ≥ 25%, i.e, median log₂ FC ≥ 0.322), iii) To exclude differential enrichment already present between on-drug condition, for each of the gene sets meeting the cutoffs in step 2, we required that the difference between the median log₂ FC in either off-drug condition to be higher than the median log₂ FC in the on-drug condition by at least 0.322 (1.25 fold higher), and iv) For visualization, we computed the single sample enrichment GSVA scores (33) of the selected gene sets from step 3 using log₂ CPM values as input.