Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro 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, through a reduction in MPL cell surface expression. 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 with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.

中文翻译：

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全基因组 CRISPR 筛选将 N-糖基化确定为 CALR 突变 MPN 的遗传和治疗脆弱性。


钙网蛋白 (CALR) 突变是骨髓增生性肿瘤 (MPN) 中常见的疾病引发事件。尽管CALR突变导致MPN的生物学机制已被阐明，但目前还没有针对CALR突变MPN的克隆选择性疗法。为了识别 CALR 突变 MPN 中独特的遗传依赖性，我们在突变 CALR 转化的造血细胞中进行了全基因组聚类规则间隔短回文重复 (CRISPR) 敲除筛选。我们发现，与对照细胞相比，突变型 CALR 转化细胞中 N-糖基化途径（以及其他）中的基因存在差异性缺失。使用针对 CRISPR 筛选中发现的独特漏洞的集中药理学体外筛选，我们发现 N-糖基化的化学抑制通过减少 MPL 细胞表面表达而损害突变 CALR 转化细胞的生长。我们用 N-糖基化抑制剂 2-脱氧葡萄糖 (2-DG) 治疗 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，并且关键 MPN 疾病正常化特征。为了验证我们在原代人类细胞中的发现，我们进行了巨核细胞集落形成单位（CFU-MK）测定。我们发现，与来自健康供体的骨髓相比，N-糖基化抑制显着减少了患者来源的 CALR 突变骨髓中 CFU-MK 的形成。总的来说，我们的研究结果推动了 CALR 突变 MPN 克隆选择性治疗的发展。