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

• BRAFV600E/WT iPSCs show monocytic skewing, develop into CD1a+/CD207+ LCH-like cells and microglia-like cells, which cause neurodegeneration

• MAPKi target mature monocytic cells and revert transcriptomic changes induced by BRAFV600E but do not eradicate progenitors

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. We show that 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. In vitro drug testing revealed that BRAFV600E-induced transcriptomic changes are reverted upon treatment with MAPK-pathway inhibitors (MAPKi). Importantly, MAPKi do not affect myeloid progenitors but reduces only the mature CD14+ cell population. Furthermore, iPSC-derived neurons (iNeurons) co-cultured with BRAFV600E/WT iPSC-derived 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, the iPSC-based model described here provides a platform to investigate the effects of BRAFV600E in different hematopoietic cell types and provides a tool to compare and identify novel approaches for the treatment of BRAFV600E-driven diseases.