钙网蛋白（CALR）突变对骨髓增殖性肿瘤中巨核细胞异常增殖分化的作用及机制研究

钙网蛋白（CALR）突变对MPN发生发展起重要作用，具体机制不清。研究发现CALR突变与MPN中巨核细胞（Mk）异常有关，但对Mk分化和功能的作用机制尚未阐明，且缺乏有效分化模型。利用iPSC多向分化潜能，我们已建立了一例CALR突变患者来源的iPSC模型，发现CALR纯合突变iPSC来源的造血干细胞向Mk分化比例升高，同时Mk分化关键转录因子FLI1上调、表面受体MPL和表面标志物表达升高。我们曾证明CALR突变蛋白能结合MPL激活下游信号通路导致细胞异常增殖。据此我们推测：CALR突变蛋白可能通过上调FLI1促进造血干细胞偏向Mk分化，通过激活MPL及下游通路促进Mk生成血小板等功能。本项目中我们拟扩建患者来源iPSC模型，诱导iPSC向造血干细胞及髓系分化，探讨CALR突变对Mk生成分化和功能的影响。本研究旨在通过iPSC模型阐明CALR突变对Mk的作用机制，为MPN治疗探索新靶点。

Calreticulin (CALR) mutations play an important role in the pathogenesis of MPNs, but the mechanism of action remains unclear. It is shown that CALR mutations are associated with abnormal megakaryocytes (MK) proliferation and differentiation in MPN patients. However, the exact impacts of CALR mutations on Mk differentiation have not been elucidated yet. Besides, an effective cell differentiation model which could further solve this problem is not available at the moment. Taking advantage of multilineage differentiation capacity of iPSCs, we established an iPSC model derived from a patient carrying CALR mutation. We found that HSPCs derived from homozygouly mutated iPSCs could generated more Mks and obtain upregulated level of MPL as well as MK differentiation-related transcription factor FLI1. Our previous work also showed that CALR mutant proteins could bind to MPL and activate the downstream signaling, which enhances the proliferation of tumor cells. Accordingly, we hypothesize that mutant CALR could promote Mk differentiation by upregulating Mk-specific transcription factors FLI1 at the HSPC stage, while enhance platelet production via activating MPL downstream pathways in Mk. In this project, we will continue establishing CALR mutant MPN patient-derived iPSCs as effective disease models. To evaluate the effects of CALR mutations on Mk differentiation and functions, we will differentiate iPSCs into hematopoietic stem cells and further into myeloid subsets. This project aims to elucidate the impact of CALR mutations on Mk differentiation through the effective iPSC model, so as to further explore novel therapy targets for MPNs.