Whole-genome CRISPR screening identifies N-glycosylation as an essential pathway and a potential novel therapeutic target in CALR-mutant MPN (Pooled CRISPR Screen).. Whole-genome CRISPR screening identifies N-glycosylation as an essential pathway and a potential novel therapeutic target in CALR-mutant MPN (Pooled CRISPR Screen).

Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPN). Although the biological mechanism by which CALR mutations cause MPN has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPN. To identify unique genetic dependencies in CALR-mutant MPN, we performed a whole-genome CRISPR knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (amongst others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells in vitro. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor, 2-deoxy-glucose (2-DG), and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared to wild-type cells, and a normalization of key MPN disease features. These findings advance the development of clonally selective treatments for CALR-mutant MPN. Overall design: CRISPR pooled screen, BA/F3-MPL cells, n=4 biological replicates per condition, empty vector cells and CALR DEL52-expressing cells, grown in the presence or absence of interleukin 3 (IL3). Genes of special interest included genes related to N-glycosylation, protein secretion, unfolded protein response, protein arginine methylation and top hits from our whole-genome screen. 1 positive control (EV#2 -_Day 21_without IL3) was included in order to corroborate that most of BA/F3-MPL cells are unviable without IL3.

钙网蛋白（Calreticulin, CALR）突变是骨髓增殖性肿瘤（myeloproliferative neoplasms, MPN）中常见的疾病始发事件。尽管CALR突变诱发MPN的生物学机制已被阐明，但目前尚无针对CALR突变型MPN的克隆选择性治疗方案。为明确CALR突变型MPN的独特遗传依赖特征，我们在经CALR突变转化的造血细胞中开展了全基因组CRISPR敲除耗竭筛选。我们发现，与对照细胞相比，经CALR突变转化的细胞中，N-糖基化（N-glycosylation）通路等相关基因呈现差异性耗竭。针对CRISPR筛选中发现的独特易感靶点开展聚焦型药物筛选后，我们发现N-糖基化的化学抑制可在体外削弱经CALR突变转化的细胞的增殖能力。我们使用N-糖基化抑制剂2-脱氧葡萄糖（2-deoxy-glucose, 2-DG）处理CALR突变敲入小鼠，发现与野生型细胞相比，CALR突变细胞对2-DG具有优先敏感性，且关键MPN疾病特征得以正常化。本研究结果推动了CALR突变型MPN克隆选择性治疗方案的开发进程。
实验整体设计：采用CRISPR混合筛选体系，使用BA/F3-MPL细胞，每组设置4次生物学重复；实验分组为空载载体细胞与表达CALR DEL52的细胞，分别在白细胞介素3（interleukin 3, IL3）存在或缺失的条件下培养。重点关注的基因包括与N-糖基化、蛋白质分泌、未折叠蛋白反应、蛋白质精氨酸甲基化相关的基因，以及全基因组筛选中的显著命中靶点。本研究设置1个阳性对照（EV#2 - 第21天，无IL3），用于验证多数BA/F3-MPL细胞在缺乏IL3时无法存活。