Effect of MAPKis on cell numbers and single-cell transcriptome of different populations of iPSC-derived myelomonocytic cells. (A) Schematic of drug treatments on BRAF^V600E/WT and BRAF^WT/WT iPSCs derived cells: at day 7 of the protocol, culture medium is either switched to monocyte differentiation medium to generate CD14⁺ iMonocytes or kept as medium B to generate CD34⁺ iHPCs. For both culture conditions, cells are harvested at day 12 and either sorted for CD34⁺ expression (iHPCs) or into CD14⁺ and CD14⁻ fractions (iMonocytes). The separate fractions are cultured in 96-well plates with MAPKis or their respective DMSO/untreated controls for 48 hours. Cells are analyzed and counted via FACS. (B) Flow cytometry analysis of all living cell numbers for CD34⁺ BRAF^V600E/WT iHPCs normalized to DMSO after 48 hours of treatment with different MAPKis (n = 4 for all drugs except for trametinib and cobimetinib which was n = 3). Dotted line equaling 1 indicates DMSO. (C) Flow cytometry analysis of numbers of all living cells normalized to DMSO for BRAF^V600E/WT and BRAF^WT/WT cells sorted at day 12 of monocytic differentiation into CD14⁺ and CD14⁻ fractions then treated for 48 hours with different MAPKis (n = 6 for BRAF^V600E/WT CD14⁻ cells, n = 5 for BRAF^V600E/WT CD14⁺ cells, and n = 4 for all BRAF^WT/WT cells). Dotted line equaling 1 indicates DMSO. (D) CD14⁻ fractions from BRAF^V600E/WT samples were isolated at day 12 of monocytic differentiation and then treated for 48 hours with different MAPKis. Four different populations based on expression of CD14 or CD1C (same scheme as Figure 1H) were defined in flow cytometry and their numbers normalized to DMSO are shown (n = 6). Dotted line equaling 1 indicates DMSO. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. Panels B-C include replicates from patient-derived iPSCs as well as ShiPS-MIFF3 iPSCs cell line. For the statistical analysis of panels B-D, log-transformed ratios of cell numbers in drugs/DMSO were used. A mixed multivariate model was used with different inhibitors (belvarafenib, tovorafenib, encorafenib, dabrafenib, trametinib, and cobimetinib) mutation status (V600E or WT) and fraction (CD14⁺, CD14⁻, or CD34⁺) as fixed effects. Pairwise post-hoc testing was carried out using the Tukey method. (E) UMAP of integrated scRNA-seq data set comprising 16 502 cells generated from fraction sorted as shown in supplemental Figure 8A. For the annotation, the same labels as in Figure 2 were used. (F-G) Bar plots showing the distribution of cell clusters and cell cycle phases among treated samples. (H) Dot plots showing DEGs (adjusted P value <.05, log₂-fold change of less than −0.5 or >0.5) between V600E vs WT samples (left, data set from Figure 2), belvarafenib- vs DMSO-treated cells (center), or dabrafenib- vs DMSO-treated cells (right) among the same clusters. A positive log₂-fold change indicates higher expression in V600E vs WT or in treatment vs DMSO. Transcripts that are upregulated in BRAF^V600E/WT vs BRAF^WT/WT cells are downregulated upon treatment and vice versa. Dots for gene/cluster combinations, which are not significant, are not shown. (I) GSEA shows pathways that are positively (NES > 0) or negatively enriched (NES < 0) in treated vs DMSO cells. Some ontology terms have a similar score between dabrafenib- and belvarafenib-treated cells whereas others are only enriched in 1 of the 2 drugs or are negatively enriched for 1 inhibitor but positively enriched for the other. The letter in brackets indicates the database of each ontology/pathway of the heat map: Reactome_2022 (R), GO_Biological_Process_2021 (G), MSigDB_Hallmark_2020 (M), KEGG_2021_Human (K), TRRUST_Transcription_Factors_2019 (TR), and Azimuth_Cell_Types_2021 (A). (J) Heat map highlighting selected regulons, analyzed using the SCENIC⁴⁰ package, that are present in either belvarafenib-, dabrafenib-, or DMSO-treated samples.