Fig. 2 |. Expression of the BRAF^V600E mutation in multipotent hematopoietic progenitors enforces their differentiation toward the MNP lineage.

a–c, BRAF-V600E^Scl+ mice were generated as described in Fig. 1a, and, in these mice, Rosa^YFP+ cells mark BRAF-V600E⁺ cells, while Rosa^YFP− cells mark BRAF-V600E⁻ cells. In BRAF-WT^Scl+ control littermates, Rosa^YFP+ cells mark those that underwent a Cre recombination as well. a, Graphs show the percentage of HSCs, MPPs, common myeloid progenitors, granulocytic myeloid progenitors and megakaryocytic erythroid progenitors among Rosa^YFP+ cells in the BM of BRAF-V600E^Scl+ mice and BRAF-WT^Scl+ control littermates (n = 6–7 mice). b, Percentages of neutrophils, Ly6C^hi monocytes (mono), macrophages and dendritic cells among Rosa^YFP+ cells in the BM of BRAF-V600E^Scl+ mice and BRAF-WT^Scl+ control littermates (n = 4–6 mice). c, CFU assays were performed on purified lineage-negative BM cells isolated from BRAF-V600E^Scl+ mice or BRAF-WT^Scl+ control littermates (n = 3 mice) cultured in the presence of methylcellulose, with representative phase contrast photomicrographs of colonies. GEMM, granulocytes, erythrocytes, megakaryocytes, monocytes, megakaryocytes. d–f, Purified CD34⁺ HPCs transduced with BRAF^V600E (BRAF-V600E^hu) or NGFR control (NGFR^hu) lentiviral vectors were cultured in stem cell medium for 8 d. d, Graphs show the percentages of GMPs (defined as CD34⁺CD38⁺CD10⁻CD123^hi/intCD45RA^+/−)³⁴ and the percentages of MNPs (defined as CD66b⁻CD11c⁺MHCII⁺) among live cells (n = 3 donors). e, CFU assays in methylcellulose performed on purified BRAF-V600E⁻GFP⁻ and BRAF-V600E⁺GFP⁺ cells isolated from HPCs transduced with the BRAF^V600E lentiviral vector or purified NGFR⁺GFP⁺ or NGFR⁻GFP⁻ cells isolated from HPCs transduced with the NGFR lentiviral vector as described in Extended Data Fig. 3 (n = 6 donors). Representative phase contrast photomicrographs of colonies are shown. f, Human CD34⁺ HPCs transduced with BRAF^V600E or the control NGFR lentiviral vector were cultured for 7 d in stem cell medium and analyzed using microarray sequencing. Heatmap representation of genes involved in granulopoiesis and the macrophage–dendritic cell (DC) lineage is shown (n = 4 independent donors per group). g, Purified CD34⁺ BM cells were isolated from a healthy pediatric donor (in duplicate) and from three patients with LCH and BM mononuclear cells known to be BRAF-V600E⁺, and these cells were analyzed using bulk RNA-seq. Heatmap representation of genes involved in granulopoiesis and the macrophage–dendritic cell lineage is shown. h,i, BRAF-V600E^Scl+ mice and BRAF-WT^Scl+ control littermates were generated as described in Fig. 1a. The percentages of GMPs (h), neutrophils, Ly6C^hi monocytes, macrophages and dendritic cells (i) among BRAF^WT;Rosa^YFP− cells in the BM of BRAF-V600E^Scl+ mice and BRAF-WT^Scl+ control littermates is shown. j, Human CD34⁺ HPCs were transduced with BRAF^V600E or NGFR lentiviral constructs and cultured in stem cell medium. Seven days later, the percentages of CD11c⁺ and/or CD14⁺ MNP cells among BRAF^V600E (GFP⁺) or BRAF^WT (GFP⁻) cells were measured by flow cytometry. Because the transduction efficiency was around 40% for both vectors, GFP⁺ cells mark BRAF-V600E⁺ or NGFR⁺ cells, while GFP⁻ cells mark BRAF^WT or NGFR⁻ cells among HPCs transduced with the BRAF^V600E or NGFR lentiviral construct, respectively (n = 8 independent donors). k, Healthy human CD34⁺ cord blood HPCs were cultured in stem cell medium (concentration is represented by the white triangle) in addition to supernatant isolated from HPCs transduced with the BRAF^V600E lentiviral construct or the NGFR control (concentration is represented by the black triangle). The graph shows the percentage of HPC differentiation into CD14⁺ MNPs analyzed by flow cytometry after 5 d of culture (n = 4 independent donors), analyzed by paired t-tests. Data are represented as mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired two-sided t-tests, two-way ANOVA test for d and paired two-sided t-tests for j,k). Data are representative of at least three independent experiments.