CRISPR/Cas9 genome engineering to dissect MPN pathogenesis

Myeloproliferative neoplasms (MPNs) are a set of chronic hematopoietic neoplasms characterized by the aberrant proliferation of one or more myeloid lineages and progressive bone marrow fibrosis. Depending on the cell type affected, the disease is called polycythemia vera (PV, red blood cells), essential thrombocythemia (ET, platelets), or primary myelofibrosis (MF, various lineages affected with an additional increase in BM fibrosis). For the majority of patients with PV and half of those with ET and MF, a single point mutation in the gene JAK2 (Janus kinase 2) resulting in JAK2V617F could be identified as a common genetic basis. Physiologically, JAK2 is an integral part of signal transduction within cells. JAK2V617F is rendered constitutively active and results in cytokine-independent growth of mutant cells. Up to now, it is not known how a single mutation can cause several clinically distinct phenotypes. Although substantial efforts have been devoted towards the characterization of JAK2V617F mutations in transgenic murine pathophysiological processes are different between mouse and human hematopoietic stem cells, and therefor insights gained from mouse models can unfortunately not be directly translated to the clinics. Our proposal aims to elucidate the role of JAK2V617F in human hematopoietic cells in order to better understand its contribution to MPN development. We propose to use CRISPR-Cas9 technology to prospectively introduce the JAK2V617F-mutation into human hematopoietic stem and progenitor cells and investigate the functional consequences in vitro. Furthermore, we will employ our recently developed novel mouse xenotransplantation model (transplantation of human cells into immune-compromised mice) that allows for the first time to investigate genetically modified human hematopoietic cells in vivo. Our result will help to shed light on the role of JAK2V617F and the molecular mechanisms involved in MPN development. Our data will contribute to the development of novel therapeutic strategies for the treatment of MPN patients.

Our research project aimed to advance the understanding of myeloproliferative neoplasms (MPNs), a group of chronic blood cancers caused by genetic mutations in blood stem cells. These conditions lead to the overproduction of blood cells and can result in serious health issues such as blood clots, bleeding, and progression to acute leukemia. We focused on studying CALR (calreticulin) mutations, which are genetic driver lesions in certain types of MPNs like essential thrombocythemia (ET) and primary myelofibrosis (PMF). We employed cutting-edge CRISPR/Cas9 technology to introduce CALR mutations into healthy human blood stem cells. This approach allowed us to study how these mutations affect cell behavior and contribute to disease development. Our experiments revealed that CALR mutations enable blood stem cells to grow independently of normal growth signals, leading to an overproduction of blood cell types such as megakaryocytes and platelets. In animal models, these mutated cells demonstrated increased survival and growth, mimicking key features of MPNs such as enlarged spleens and bone marrow fibrosis. Additionally, we discovered that CALR mutations activate specific cellular pathways related to stress responses and protein processing. This insight opens new possibilities for targeted treatments by identifying vulnerabilities in these pathways. Our research suggests that existing drugs could potentially be repurposed to treat patients with CALR-mutant MPNs, offering new avenues for more effective therapies. Beyond these findings, our project contributed to broader scientific advancements. We published detailed protocols for our gene-editing techniques, enabling other researchers to replicate our methods. We also explored other genetic mutations linked to blood cancers, further expanding our understanding of these diseases. Overall, this project not only deepened scientific knowledge of MPNs but also paved the way for developing targeted treatments that could improve outcomes for patients affected by these challenging conditions. By focusing on CALR mutations, we have provided valuable insights into the mechanisms driving MPNs and highlighted potential therapeutic strategies that could benefit patients in the future.