BRAFV600E induces key features of LCH in iPSCs with cell type–specific phenotypes and drug responses

Langerhans cell histiocytosis (LCH) is a clonal hematopoietic disorder defined by tumorous lesions containing CD1a+/CD207+ cells. Two severe complications of LCH are systemic hyperinflammation and progressive neurodegeneration. The scarcity of primary samples and lack of appropriate models limit our mechanistic understanding of LCH pathogenesis and affect patient care. We generated a human in vitro model for LCH using induced pluripotent stem cells (iPSCs) harboring the BRAFV600E mutation, the most common genetic driver of LCH. We show that BRAFV600E/WT iPSCs display myelo-monocytic skewing during hematopoiesis and spontaneously differentiate into CD1a+/CD207+ cells that are similar to lesional LCH cells and are derived from a CD14+ progenitor. BRAFV600E modulates the expression of key transcription factors regulating monocytic differentiation and leads to an upregulation of pro-inflammatory molecules and LCH marker genes early during myeloid differentiation. BRAFV600E-induced transcriptomic changes are reverted upon treatment with MAPK-pathway inhibitors (MAPKi). Importantly, MAPKi does not affect myeloid progenitors but reduces only the mature CD14+ cell population. Furthermore, iPSC-derived neurons (iNeurons) cocultured with BRAFV600E/WT microglia-like cells (iMGL), differentiated from iPSC-derived CD34+ progenitors, exhibit signs of neurodegeneration with neuronal damage and release of neurofilament light chain. In summary, our model provides a platform to study the effect of BRAFV600E in different hematopoietic cell types and provides a tool to compare and identify novel approaches for the treatment of BRAFV600E-driven diseases. Patient-derived iPSCs were created and analysed using single-cell RNA-seq. For iMonocyte differentiation 8 samples were processed: 2 are derived from iPSC_LCH_8 cell line (patient derived), one is WT, the other is V600E (the mutated one has a XYY karyotype). Both samples are harvested at D14 of Monocytic differentiation. 4 are derived from iPSC_LCH_15 cell line (patient derived) 2 are WT, 2 are V600E. All 4 samples are harvested at D14 of Monocytic differentiation. 2 are derived from iPSC_LCH_15 cell line (patient derived) one is WT, the other is V600E. These are the samples harvested at D11 of Monocytic differentiation; For iMonocyte treatment :3 samples in total, all derived from iPSC_LCH15 (patient derived) and all V600E; For iLCH: 2 samples in total, both derived from iPSC_LCH15 (patient derived) and both V600E. ***Submitters state that, using patient-derived samples of a rare disease in a small country, raw data could not be provided due to patient privacy concerns****