Figure 1: Adaptation and learning in WM989 melanoma cells.

(A) Schematic comparison of resistance mechanisms in WM989 melanoma cells. Upper panel: Pure selection process where primed cells survive targeted therapy without altering their state. Lower panel: Adaptive learning process in which primed cells modify their cellular state in response to treatment, resulting in a molecularly and phenotypically distinct resistant population.

(B) Transcriptional differences between non-primed, primed, and fully resistant cells measured by RNA-seq. Selected examples include previously identified priming marker genes. Resistant cells are those remaining after treatment with 1 μM vemurafenib for 21 days. Primed cells refer to cells identified as twins of those in the naive population that will eventually become resistant, with RNA FISH probes targeting barcodes identified from the resistant cell population. Non-primed cells refer to the rest of the therapy-naive population; in this case, those without matching resistant cell barcodes.

(C) Venn diagram showing 3,017 differentially expressed genes in resistant cells (as compared to therapy-naive, non-primed cells) and 190 differentially expressed genes in therapy-naive primed cells. 152 of the 190 differentially expressed genes in primed cells are also present in the set of differentially expressed genes in resistant cells (c.f. Emert et al., 2021)¹⁸.

(D) Heatmap of top variable genes from RNA-seq analysis in the global analysis of non-primed, primed, and fully resistant cells (as defined in panel B).

(E) Heatmap of chromVAR analysis of ATAC-seq data. Primed cells in this experiment were identified by the expression of the surrogate marker EGFR. Top 0.02–0.2% of live cells staining for EGFR were designated as EGFR-high. (left to right) Two biological replicates for EGFR-negative (non-primed) cells never exposed to the therapy, EGFR-high (primed) cells never exposed to the therapy, EGFR-high (primed) cells exposed to 7 days of 1 μM vemurafenib, and EGFR-high (primed) cells exposed to 28 days of 1 μM vemurafenib.