CALR基因突变是JAK2野生型（WT）髓增殖性肿瘤（MPN），包括骨髓纤维化和必需性血小板增多症中主要致癌驱动因素，其中CALR突变被越来越认为是一种适合特异性药物靶标。然而，我们对其作用机制的了解来自于小鼠模型或免疫细胞系，这些跨物种差异、异位过度表达和缺乏疾病穿透力的因素阻碍了转化研究。在此，我们使用CRISPR/Cas9和腺相关病毒载体介导的敲入策略在原代人类造血干细胞和祖细胞中建立了CALR MUT MPN的第一个人类基因工程模型，以便在体内和异种移植的小鼠中建立可重复和可追踪的表型。我们的人类模型重现了许多疾病特征：独立于血小板生成素的巨核母细胞生成、骨髓系列偏倚、脾脏增大、骨髓纤维化以及CD41+的巨核细胞前体细胞的扩增。引入CALR突变显著促进了人类造血干细胞的早期重编程和内质网应激响应。观察到的分子伴侣补偿性上调显示出偏好敏感性CALR突变细胞抑制BiP分子伴侣和蛋白酶体。总体而言，我们的人化模型改善了纯小鼠模型，并为测试新的治疗策略提供了一个可使用的基础。© 2023. 作者。

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.© 2023. The Author(s).