FIGURE 6 from Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2

MDSC differentiation arrest is a result of a combination of cell cycle and myeloid lineage alteration. A and B, Relative frequency of cells in each Seurat cluster from scRNA-seq data of MDSCs ± 1.5 µmol/L DAB. Relative levels were calculated on the basis of the standardized residuals from using Pearson χ2 overlaid on the UMAP plot of integrated samples (A) and grouped by functional annotation (B). Positive standardized residuals (yellow) for each cluster indicates increased frequency in the DAB-treated sample whereas a negative number (purple) indicates increased frequency in the DMSO control sample. A dotted line indicates standardized residual values of ±3. C, UMAP plot of the integrated scRNA-seq data from MDSCs ± 1.5 µmol/L DAB, showing the trajectory branches calculated by Monocle3 and represented over the 22 clusters defined by Seurat. Most relevant nodes at the end of the branches (black dots) and at intersections (red dots) are shown. Nodes used as starting (a) or end (b and c) points for regulon analysis across the branch are labeled. D, Heat map of the regulons significantly regulated by DAB calculated on AUCell (q 2FC| >0.01) for each Seurat cluster. Color represents regulons significantly enriched in cells treated with DAB (red), those enriched in control samples (blue) and the ones that do not show significant changes (light yellow). E, Heat map of the regulons significantly regulated during differentiation in the direction from node a to node b in C, which spans from cluster 6 (immature granulocytes) to cluster 9 (intermediate granulocytes) in DMSO control cell cultures. Scale represents z-score of the normalized average AUCell score per regulon. F, Heat map of the regulons significantly regulated during differentiation in the direction from node a to node c in C, which spans from cluster 6 (immature granulocytes) to cluster 5 (mature PMN) in DMSO control cell cultures. Scale represents z-score of the normalized average AUCell score per regulon. DAB = dabrafenib.