According to our current understanding, dysplastic megakaryocytes, pathognomonic for MPN and PMF (primary myelofibrosis), stimulate Gli1+ stroma cells and transform them into myofibroblasts, which then induce and maintain myelofibrosis. Strategies to specifically target dysplastic megakaryocytes hold the promise of antagonizing fibrosis progression. In addition, decreased secretion of pathogenic cytokines by the aberrant megakaryocytes might alleviate the often debilitating symptoms and thromboembolic complications of MPN patients. We have generated a panel of patient-specific JAK2V617F or CALR mutated iPS cells and the respective unmutated controls. We have differentiated these cells into megakaryocytes and set up systems to study the impact of diseased megakaryocytes on bone marrow mesenchymal stromal cells (MSCs). This subproject´s aims are to
- dissect the steps of megakaryocyte differentiation on a heterozygous and homozygous JAK2 or CALR mutated background,
- evaluate the transcriptome, secretome and proteome of JAK2 or CALR mutated megakaryocytes, and
- determine their role in MSC to myofibroblast transition.
Furthermore, we are using data obtained to perform gain-of-function and loss-of-function studies in JAK2 or CALR mutated megakaryocytes using gene overexpression by lentiviral gene transfer and gene inactivation by CRISPR/Cas9 editing. In summary, we expect to identify critical regulators and mechanisms of fibrotic tissue formation, which will support the development of novel therapeutics for treatment of myelofibrotic progression in MPN.