Extended Data Fig. 10: Inhibition of histone methyltransferase DOT1L results in the emergence of additional resistant proliferative clones and a reduction in singletons.

a. For each barcode identified by sequencing, we plotted its abundance in corresponding splits A (DMSO control) and B (DOT1L inhibition). Those present in both control and DOT1L splits are colored in dark blue, and those present only in either A (control) and B (DOT1L) are colored in cyan. Those present in both (dark blue; 171) exhibited a strong correlation, suggesting that their ability to survive and become resistant is invariant of drug dose. For those present only in either (cyan), we found them to be much more abundant in DOT1L (B, 43 barcodes) than DMSO control (A, 7 barcodes), suggesting that new barcodes, otherwise unable to survive in the control condition, become drug-resistant in the DOT1L inhibited condition. A total of one biological replicate. b. (left) Combined resistant cells in the control (9,343 cells) and DOT1L (7,044 cells) conditions obtained from UMAP applied to the first 50 principal components. Cells are colored by clusters determined using Seurat’s FindClusters command(“Seurat clusters, resolution = 0.8”). (right) UMAP is split by each condition. c. UMAP where the resistant cells are colored by the associated condition (control vs DOT1L). The arrow represents the UMAP region present predominantly in the control region and missing from the DOT1L-associated UMAP region. d. Quantification of singletons and colonies showed that while the number of resistant colonies is higher in DOT1L, it is accompanied by a reduced number of singletons cells compared to control. e. Painting of singletons and colonies onto the UMAP, colored by the condition, demonstrated that singletons largely belong to the control condition and are present predominantly in cluster 2 (MLANA-high). Colonies are dispersed more across the UMAP with no particular region enriched for either condition. f. Imaging of the nuclei (DAPI-stained) of resistant colonies emerging from vemurafenib treatment of WM989 A6-G3 cells, either for control or cells lacking DOT1L. g. Quantification of the total number of colonies and singletons from each fate type across n = 3 biological replicates demonstrated a relative increase (3.65-fold; n = 3 biological replicates) in total colonies and reduction in total singletons in the DOT1L and control conditions, respectively. Error bars represent standard error of the mean. h. UMAP is recolored for each cell by its expression for the gene MLANA, a marker for cluster 2, which is relatively enriched in control (as shown with an arrow). i. A pie chart to demonstrate that of all the clones (barcodes) present in the control condition split, only 3.1% were also present in the DOT1L inhibitor pretreatment split. j. Two representative examples of UMAP regions where twins from the MLANA-high cluster in the control condition go in the DOT1L condition. A cumulative density contour plot capturing the types of fate switches that MLANA-high cluster clones from control adopt in the DOT1L inhibitor-pretreated condition. k. A cumulative density contour plot capturing the types of fate switches that the MLANA-high cluster 2 clones from the control condition split adopt in the DOT1L inhibitor pretreatment split. l. Distribution of cells across clusters for control (top) and DOT1L inhibitor-pretreated (bottom) conditions for clone size>2.