UnVEiL

Langerhans cell histiocytosis (LCH) is a rare disease of the hematopoietic system characterized by CD1a and CD207 positive cells in tumor-like lesions. The spectrum of the disease ranges from spontaneous healing of individual lesions to tumors in multiple organs and systemic inflammation, which requires treatment with chemotherapeutics. What they all share, however, is the activation of the MAPK signaling pathway, which is triggered by a mutation in the BRAF gene (BRAFV600E) in 50-60% of cases. Aside from these specific mutations that activate the MAPK signaling pathway, no other genetic alterations are present. On the one hand, this enables the disease to be modeled relatively easily, as only one mutation needs to be inserted, but on the other hand, this has proven to be a very difficult undertaking. A fundamental problem is that the origin or cell of origin is still unknown. The question therefore arises as to which cell the mutation must be inserted into. The models used so far are almost exclusively mouse models, which fulfill individual aspects of the disease, but do not match the patients` symptoms in essential features. With this project we are not only aiming to clarify the origin of the LCH cells, but also develop a new model system for LCH and other BRAFV600E mutated histiocytoses, such as Erdheim Chester or Rosai Dorfman syndrome. To do this, we will use induced pluripotent stem cells (iPSCs), in which we will heterozygously insert the BRAF mutation using the CRISPR Cas9 method. We will differentiate these cells along the hematopoietic line and analyze the effects of the mutation in the different cell types and understand the developmental path of the hematopoietic cells towards LCH cells. We will also use a combination of two sequencing methods to investigate the influence of the mutation in different cells from lesions or the blood of patients. Using 10x single cell sequencing we will analyze the gene expression of individual cells. We will sequence a part of the cDNA obtained, using long read Oxford Nanopore sequencing. This will allow us to distinguish cells with or without the mutation and compare the respective gene expression patterns. The overall goal of this project is to explore the fundamental mechanisms of how BRAFV600E controls the development of cells towards LCH and to study the interaction of LCH cells with immune cells.