Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants
Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants
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DOI：
10.1182/blood-2015-11-681932
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发表时间：
2016-03-10
期刊：
影响因子：
20.3
通讯作者：
Constantinescu, Stefan N.
中科院分区：
文献类型：
--
作者：
Chachoua, Ilyas;Pecquet, Christian;Constantinescu, Stefan N.
钙网蛋白基因（CALR）的突变，以外显子9的缺失和插入为代表，诱导-1/+2移码，与很大一部分骨髓增生性肿瘤（mpn）有关。CALR突变体诱导MPN的机制尚不清楚。在这里，我们通过转录、增殖、生化和原代细胞分析表明，致病性CALR突变体特异性地激活了血小板生成素受体（TpoR/MPL）。I型和II型细胞因子受体未被检测到激活，除了粒细胞集落刺激因子受体，它只支持短暂和弱激活。CALR突变体通过TpoR诱导JAK2/STAT/磷酸基肌醇-3激酶（PI3-K）和丝裂原活化蛋白（MAP）激酶途径的配体非依赖性激活，并在Ba/F3细胞中自主生长。在这些转化的细胞中，没有观察到JAK2和PI3-K抑制剂之间在抑制细胞因子独立增殖方面的协同作用，因此与JAK2V617F细胞表现出很大的差异，后者的协同作用很强。TpoR的激活依赖于其胞外结构域和n -糖基化，特别是在N117位点。突变的CALR蛋白的聚糖结合位点和新的c端尾是激活TpoR所必需的。可溶性形式的TpoR能够阻止全长TpoR的激活，只要它是n -糖基化的。通过共聚焦显微镜和亚细胞分离，CALR突变体表现出与野生型CALR不同的细胞内定位。最后，在携带CALR突变的患者的巨核细胞祖细胞中，敲低MPL/TpoR或JAK2均可抑制细胞因子无关的巨核细胞集落形成。综上所述，我们的研究提供了一种新的信号范式，即突变的伴侣蛋白组成性地激活细胞因子受体信号。
Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.