Defining the cancer-specific proteome in Calreticulin-mutant myeloproliferative neoplasms

费城阴性骨髓增生性肿瘤包括一组恶性血液病。骨髓增殖性肿瘤的遗传图谱已被很好地描述，钙调蛋白是继JAK2之后第二大最常见的突变基因。高达40%的骨髓增生性肿瘤患者存在钙网蛋白突变。此外，钙网蛋白和JAK2V617F突变是相互排斥的。所有钙网蛋白突变都集中在最后一个编码外显子上，并导致c端“KDEL”序列的缺失，该序列通常将野生型钙网蛋白保留在内质网中，在内质网中充当伴侣蛋白。两个最突出的突变（I型和II型）都导致了框架移位，产生了一个共同的突变特异性36个氨基酸的c端肽。在新的c端，带负电的氨基酸被带正电的氨基酸取代，主要是精氨酸和赖氨酸。最近，一种突变钙调蛋白致癌的机制已经被描述出来。Mullally博士的实验室表明，突变型钙网蛋白与血小板生成素受体发生致病性相互作用，后者以不依赖血小板生成素的方式激活下游信号通路。此外，已经证明突变体钙网蛋白c端的正静电电荷和突变体钙网蛋白的凝集素结合残基都是其致癌活性所必需的。在这里提出的工作中，我将描述突变钙调蛋白的新相互作用伙伴在骨髓增殖性肿瘤发展中的作用。此外，我将确定突变的钙网蛋白是如何异常地与其伴侣结合的，并评估这种结合在突变的钙网蛋白细胞增殖中的必要性。穆拉利博士的研究小组还发现，突变的钙钙蛋白失去了伴侣蛋白的活性。在第二种方法中，我将因此确定突变钙调蛋白是否会诱导蛋白质组变异，如细胞表面异常折叠的蛋白质。根据定义，这些非规范蛋白只存在于钙网蛋白突变的恶性细胞上，因此构成了未来免疫治疗的理想靶点。我期望通过突变钙钙蛋白的异常伴侣活性来识别非规范蛋白同种异构体。候选同种异构体将被确认并随后在原代细胞中验证。

Philadelphia-negative myeloproliferative neoplasms comprise a group of malignant hematological diseases. The genetic landscape of myeloproliferative neoplasms is very well described, with calreticulin being the second most common mutated gene after JAK2. Calreticulin mutations are found in up to 40% of patients with myeloproliferative neoplasms. Moreover, calreticulin and JAK2V617F mutations are mutually exclusive. All calreticulin mutations cluster in the last coding exon and result in deletion of the C-terminal “KDEL“ sequence, which normally retains wildtype calreticulin to the endoplasmic reticulum where it acts as a chaperone protein. The two most prominent mutations (Type I and II) both lead to frameshifts that generate a common mutant-specific 36-amino acid C-terminal peptide. In the new C-terminus, negatively charged amino acids are replaced by positively charged ones, mainly arginine and lysine. Recently, one mechanism by which mutant calreticulin is oncogenic has been delineated. Dr. Mullally’s lab showed that mutant calreticulin develops a pathogenic interaction with the thrombopoietin receptor, which activates the downstream signaling pathway in a thrombopoietin-independent manner. Moreover, it has been demonstrated that both the positive electrostatic charge of the mutant calreticulin C-terminus and the lectin binding residues of mutant calreticulin are required for its oncogenic activity. In work proposed here, I will characterise the role of novel interaction partners of mutant calreticulin in the development of myeloproliferative neoplasms. Moreover, I will determine how mutant calreticulin aberrantly binds to its partners and assess the necessity of this binding in mutant calreticulin cell proliferation. Dr. Mullaly’s group has also shown that mutant calreticulin loses its chaperone activity. In a second approach, I will therefore determine whether mutant calreticulin induces proteome variations such as aberrantly folded proteins at the cell surface. These non-canonical proteins would, by definition, only be present on calreticulin mutated, malignant cells and therefore constitute ideal targets of future immunological therapy. I anticipate identifying non-canonical protein isoforms due to the aberrant chaperone activity of mutant calreticulin. Candidate isoforms will be confirmed and subsequently validated in primary cells.