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

使用患者来源的诱导多能干细胞 (iPSC) 模型和 CRISPR/Cas9 基因组编辑对与骨髓增生性肿瘤 (MPN) 相关的 CALR 突变进行功能研究

• 批准号: 394341827
• 负责人: Dr. Wei Wang, Ph.D.
• 金额: --
• 依托单位: Department of Oncological Sciences
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/394341827?language=en
• 关键词: Functional; studies; CALR; mutations; associated

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.

骨髓增生性肿瘤（mpn）是一种以一种或多种成熟血细胞过量产生为特征的克隆性造血疾病。mpn包括三个主要的临床实体：真性红细胞增多症（PV）、原发性血小板增多症（ET）和原发性骨髓纤维化（PMF）。近年来，随着Janus激酶2 （JAK2V617F）、血小板生成素受体（MPL）和钙网蛋白（CALR）获得性体细胞突变的发现，我们对mpn的分子发病机制有了很大的了解。根据这三种驱动因素的突变情况，MPN患者可分为三组：JAK2V617F阳性（70%的MPN，包括PV、ET和PMF）、MPL或CALR阳性（20%的MPN，主要是ET和PMF）和三阴性MPN（10%的MPN）。虽然JAK2和MPL突变的影响已被广泛研究，并且JAK2抑制剂已被引入临床，但最近发现的CALR突变在MPN病理生理中的作用目前尚不清楚，并且仍然没有靶向治疗。因此，本提案的主要目标是利用诱导多能干细胞（iPSC）模型和CRISPR/Cas9基因组编辑技术对来自MPN患者的细胞进行重编程，研究CALR突变的生物学后果，旨在发现致癌CALR突变的潜在治疗靶点。为了实现这一目标，我们将利用尖端技术，包括等基因iPSC系的衍生、大规模细胞术（CyTOF）、基于CRISPR/ cas9的精确基因组编辑、RNA-seq分析、基于CRISPR/ cas9的定制sgRNA文库筛选和大规模药物筛选。本提案的具体目标如下：具体目标1：利用患者细胞重编程和CRISPR/Cas9基因组编辑技术，建立calr突变MPN的等基因iPSC模型。具体目标2：在等基因CALR iPSC模型中进行CALR突变的分子表征。特异性目标3：执行基于CRISPR/ cas9的合成致死性筛选，以确定CALR突变mpn的新治疗靶点。拟议的研究将研究CALR突变在生理基因组背景下等基因条件下的人类细胞和iPSCs衍生的相关造血细胞类型中的致癌性，以更好地了解突变的CALR在mpn中的关键作用，并发现治疗机会。