Extended Data Fig. 9: Changing the therapy type to trametinib eliminates an additional resistant fate type present in the vemurafenib treatment.

a. UMAP where the resistant cells are colored by the associated therapy drug type, with dark blue representing vemurafenib (9,457 cells) and light blue representing trametinib (8,569 cells). Arrows represent UMAP regions that are present only in vemurafenib or trametinib. b. UMAP is split by each drug type, with colors representing clusters determined using Seurat’s FindClusters command(“Seurat clusters, resolution = 0.5”). Arrows represent UMAP regions that are present only in vemurafenib or trametinib. c. Painting of singletons and colonies onto the UMAP, colored by the condition, demonstrated that singletons largely belong to vemurafenib and are present predominantly in the MLANA-high cluster. Colonies are dispersed more across the UMAP with no particular region enriched for either condition except for the NGFR-high cluster. d. Imaging of nuclei (DAPI-stained) of resistant colonies emerging from treatment of WM989 A6-G3 cells to either vemurafenib or trametinib. The number of singletons in trametinib treated cells appear to be much less than those treated with vemurafenib, consistent with the sequencing data from FateMap. e. Quantification of the total number of colonies and singletons from each drug type of imaging data across biological replicates. This analysis demonstrated that while the total number of colonies are similar across the two drug types, there is a relative increase (~2.45-fold; n = 3 biological replicates) in the number of singletons in the case of vemurafenib. Error bars represent standard error of the mean. f. UMAPs are recolored for each cell by its expression for gene MLANA, which is a marker for cluster 3 relatively enriched in vemurafenib (as shown with arrows in A and B). g. A pie chart to demonstrate that of all the clones (barcodes) present in vemurafenib-treated split in cluster 3, only 4.8% were also present in the trametinib-treated split. h. A cumulative density contour plot capturing the types of fate switches that the MLANA-high cluster 3 clones from the vemurafenib-treated split adopt in the trametinib-treated split. i. Three representative examples of UMAP regions where twins from the MLANA-high cluster 3 in the vemurafenib-treated split adopt in the trametinib-treated split. j. UMAPs are recolored for each cell by its expression for gene NGFR, which is a marker for cluster 4 relatively enriched in trametinib (as shown with arrows in A and B). k. Composition of clones of different sizes within NGFR-high cluster 4 for both trametinib- and vemurafenib-treated splits. l. A pie chart to demonstrate that of all the clones (barcodes) present in the trametinib-treated split in cluster 4, 20.7% were also present in the vemurafenib-treated split. m. A cumulative density contour plot capturing the types of fate switches that the NGFR-high cluster 4 clones from the vemurafenib-treated split adopt in the trametinib-treated split. n. Two representative examples of UMAP regions where twins from the NGFR-high cluster 4 in trametinib-treated split adopt in the vemurafenib-treated split. o. UMAP for combined vemurafenib and trametinib treatment conditions recolored for each cell by its expression of the gene VCAM1, which is enriched in cluster 6. p. Painting of singletons and colonies onto the UMAP for the NGFR-high cluster 4, colored by the condition, showing a relative enrichment of cells from trametinib as compared to vemurafenib. This panel also demonstrates that both singletons and colonies occupy cluster 4 from each of the two conditions. q. We performed antibody stainings for NGFR on colonies emerging from treatment of the same number of starting melanoma cells with either vemurafenib or trametinib. Consistent with FateMap, we found an increased number of NGFR-positive resistant cells in trametinib treated cells as compared to the vemurafenib treatment. r. UMAP split by each drug condition (trametinib (8,569 cells) or vemurafenib and trametinib (7,023 cells)), with colors representing clusters determined using Seurat’s FindClusters command at a resolution of 0.5 (i.e. “Seurat clusters, resolution = 0.5”). s. UMAP recolored for combined resistant cells from trametinib (light blue) and vemurafenib and trametinib (dark blue). The cells from two conditions are interspersed into each other on the UMAP.