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。骨髓增生性肿瘤的遗传景观是非常好的描述，钙网蛋白是JAK 2之后第二个最常见的突变基因。在高达40%的骨髓增生性肿瘤患者中发现钙网蛋白突变。此外，钙网蛋白和JAK 2 V617 F突变是相互排斥的。所有钙网蛋白突变都聚集在最后一个编码外显子中，并导致C-末端“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，在内质网中其充当伴侣蛋白。两个最突出的突变（I型和II型）都导致移码，产生一个共同的突变特异性的36个氨基酸的C-末端肽。在新的C-末端，带负电荷的氨基酸被带正电荷的氨基酸取代，主要是精氨酸和赖氨酸。最近，突变钙网蛋白致癌的一种机制已被阐明。Mullally博士的实验室表明，突变钙网蛋白与血小板生成素受体发生致病性相互作用，后者以血小板生成素非依赖性方式激活下游信号通路。此外，已经证明突变钙网蛋白C-末端的正静电电荷和突变钙网蛋白的凝集素结合残基都是其致癌活性所需的。在这里提出的工作中，我将阐明突变钙网蛋白的新的相互作用伙伴在骨髓增生性肿瘤的发展中的作用。此外，我将确定如何突变钙网蛋白aberrantly结合到它的合作伙伴，并评估这种结合在突变钙网蛋白细胞增殖的必要性。Mullaly博士的研究小组还表明，突变的钙网蛋白失去了它的伴侣活性。因此，在第二种方法中，我将确定突变钙网蛋白是否诱导蛋白质组变异，如细胞表面的异常折叠蛋白质。根据定义，这些非典型蛋白质将仅存在于钙网蛋白突变的恶性细胞上，因此构成未来免疫治疗的理想靶点。我预计识别非典型的蛋白质亚型，由于异常的伴侣活性突变钙网蛋白。候选亚型将在原代细胞中得到确认并随后得到验证。

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.