Transcriptomic regulation of inflammation and cell differentiation induced by BRAFV600E is cell type–specific and begins in myeloid progenitors. (A) Uniform manifold approximation and projection (UMAP) of 64 167 single living cells sequenced: 6 samples (3 × BRAF^V600E/WT and 3 × BRAF^WT/WT) were harvested at day 14, and 2 samples at day 11 (1 × BRAF^V600E/WT and 1 × BRAF^WT/WT) of differentiation toward iMonocytes. The main clusters discussed in this paper are highlighted as Myeloid Progenitors, Monocytes, and MΦs/DCs. (B) Frequencies of cell types between mutated and WT samples at day 14: BRAF^V600E/WT samples show fewer Myeloid Progenitors and Promonocytes than BRAF^WT/WT, consistent with a monocytic skewing induced by the mutation. (C) Pseudotime analysis using Monocle 3³⁹ of selected clusters showing that BRAF^V600E/WT cells have higher density at later pseudotimes, supportive of their ability to differentiate faster than their WT counterpart. (D) Dot plots showing DEGs (adjusted P value <.05, positive fold change means higher in BRAF^V600E/WT) associated with LCH and (E) inflammatory mediators. Dots for gene/cluster combinations, which are not significant, are not shown. (F) Gene set enrichment analysis (GSEA) shows that some pathways were uniquely enriched (adjusted P value <.05) for each cluster whereas others displayed similar normalized enrichment scores (NESs) among several clusters. A NES of >0 indicates positive enrichment in BRAF^V600E/WT clusters. (G) Dot plot of differentially expressed TFs and modulators between clusters: most DEGs are in the Myeloid Progenitors and Monocyte clusters. Dots for gene/cluster combinations, which are not significant, are not shown. (H) Enrichr⁶⁴ analysis of downregulated DEGs in mutated vs WT samples (adjusted P value <.05) with enrichment scores shown as negative log₁₀ of their adjusted P value. The letter in brackets in panels F,H indicates the database of each ontology/pathway of the heat map: Reactome_2022 (R), Transcription_Factor_PPIs (T), GO_Biological_Process_2021 (G), Rare_Diseases_GeneRIF_ARCHS4_Predictions (D), MSigDB_Hallmark_2020 (M), and KEGG_2021_Human (K). (I) Dot plot of DEGs at day 11 of differentiation involved in myelomonocytic differentiation. BRAF^V600E/WT induces downregulation of key genes associated with granulopoiesis (CEBPE, PRTN3, MPO, ELANE, and AZU1) and upregulation of monocyte-specific markers and TFs (MAFB, CD14, and S100A12). Dots for gene/cell type combinations, which are not significant, are not shown. (J) Regulon analysis showing the top 5 scoring regulons for Monos LYZ High, Myeloid Progenitors, and Promonocytes clusters in mutated and WT samples. (K) Heat map highlighting regulons that are uniquely present in either WT or mutated Monos LYZ High, Myeloid Progenitors, and Promonocytes clusters by showing their binary SCENIC area under the curve scores.