Functional studies of CALR mutations associated with Myeloproliferative Neoplasms (MPNs) using patient-derived induced pluripotent stem cell (iPSC) models and CRISPR/Cas9 genome editing

• Applicant: Dr. Wei Wang, Ph.D.
• Subject Area: Hematology, Oncology
• Term: from 2017 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 394341827

Myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoiesis characterized by the overproduction of one or more types of mature blood cells. MPNs include three main clinical entities: Polycythemia Vera (PV), Essential Thrombocytosis (ET) and Primary Myelofibrosis (PMF). Our understanding of the molecular pathogenesis of MPNs has been greatly advanced in recent years with the discoveries of acquired somatic mutations of Janus kinase 2 (JAK2V617F), thrombopoietin receptor (MPL) and calreticulin (CALR). Based on the mutation status of these three drivers, MPN patients can be grouped into three groups: JAK2V617F positive (70% of MPNs, including PV, ET and PMF), MPL or CALR positive (20% of MPNs, mainly ET and PMF) and triple negative MPNs (10% of MPNs). While the effects of JAK2 and MPL mutations have been extensively studied and JAK2 inhibitors have been introduced in the clinic, the role of the more recently identified CALR mutations in the pathophysiology of MPN is currently less understood and there is still no targeted therapy.Therefore, the main goal of this proposal is to investigate the biological consequences of CALR mutations using induced pluripotent stem cell (iPSC) models derived by reprogramming cells from MPN patients and CRISPR/Cas9 genome editing technology with the aim to uncover potential therapeutic targets for the oncogenic CALR mutations. To achieve this goal, we will utilize cutting-edge technologies, including derivation of isogenic iPSC lines, mass cytometry (CyTOF), precise CRISPR/Cas9-based genome editing, RNA-seq analysi CRISPR/Cas9-based custom sgRNA library screening and large-scale drug screening. The specific aims of this proposal are as follows:Specific Aim 1: To develop isogenic iPSC models of CALR-mutant MPN using reprogramming of patient cells and CRISPR/Cas9 genome editing.Specific Aim 2: To perform molecular characterization of CALR mutations in the isogenic CALR iPSC models.Specific Aim 3: To perform a CRISPR/Cas9-based synthetic lethality screen to identify new therapeutic targets for CALR mutated MPNs.The proposed studies will investigate the oncogenicity of CALR mutations in human cells in isogenic conditions in physiological genomic context and in relevant hematopoietic cell types derived from iPSCs to better understand the pivotal role of mutated CALR in MPNs and uncover therapeutic opportunities.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Dr. Eirini Papapetrou, Ph.D.