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

骨髓增生性肿瘤(MPN)是一种以一种或多种成熟血细胞过度产生为特征的克隆性造血疾病。MPNS包括三种主要临床类型：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 kinase2(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MPNS分子发病机制的认识有了很大的提高。根据这三个基因的突变情况，MPN患者可分为三组：JAK2V617F阳性(占MPN的70%，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%)。虽然JAK2和MPL突变的作用已被广泛研究，JAK2抑制剂也已被引入临床，但最近发现的CALR突变在MPN病理生理学中的作用目前尚不清楚，仍然没有靶向的治疗方法。因此，本研究的主要目的是利用MPN患者细胞重编程获得的诱导多能干细胞(IPSC)模型和CRISPR/Cas9基因组编辑技术来研究CALR突变的生物学后果，以期发现癌基因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系的派生、质量细胞术(CyTOF)、基于CRISPR/Cas9的精确基因组编辑、基于RNA-seq分析的CRISPR/Cas9定制sgRNA文库筛选和大规模药物筛选。具体目标1：通过患者细胞重编程和CRISPR/Cas9基因组编辑，建立CALR突变MPN的等基因IPSC模型。特定目标2：对等基因CALR突变IPSC模型中CALR突变进行分子表征。特定目标3：进行基于CRISPR/Cas9的合成致死性筛选，以确定CALR突变MPN的新治疗靶点。本研究将在生理基因组环境和来自IPSC的相关造血细胞类型中，研究同基因条件下CALR突变在人类细胞中的致癌性，以更好地了解突变的CALR在MPN中的关键作用和发现治疗机会。

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.