Targeting the unfolded protein response in multiple myeloma with monoclonal antibodies to overcome drug resistance

多发性骨髓瘤(MM)是一种罕见的血液病，起源于骨髓中转化的浆细胞。过度合成和分泌单抗免疫球蛋白(Ig)或不完整的分子Ig轻链是MM的标志之一。这种分泌活动导致内质网(ER)应激水平显著增加，并经常导致错误或错误折叠的蛋白质积累。为了避免不必要的甚至有害的蛋白质生物合成，这种积累可以触发一种称为未折叠蛋白反应(UPR)的高度保守的机制，最终导致细胞凋亡。一类新的治疗学使用这一机制，通过抑制细胞降解系统-蛋白酶体-导致严重的内质网应激。蛋白酶体抑制剂，如Bortezomib，是当今最强的浆细胞耗尽剂，在MM的治疗中非常成功地使用。错误折叠Ig的积累另一方面增加了参与蛋白质组装和UPR调节的伴侣分子的数量。UPR的一个关键传感器是伴侣分子葡萄糖调节蛋白(GRP)78，它主要位于内质网，但也存在于肿瘤细胞表面或作为分泌突变体。在体外，后一种变异体似乎与PI耐药性有关。然而，GRP78与泛素-蛋白酶体途径之间的联系却鲜为人知。我们已经开发了一种治疗性抗GRP78抗体，并在临床试验中对该产品进行了测试。单药活性中等，但与PI联合使用时，即使在PI难治性疾病患者中也能看到客观反应。在这项建议中，我的目标是从床边回到工作台，在体外阐明GRP78诱导的PI耐药，并研究抗GRP78抗体治疗是否可以克服PI耐药。我将利用ELISA法和Western印迹技术探讨不同疾病阶段骨髓瘤患者分泌型GRP78的频率和功能作用。此外，我还将比较PI敏感和PI耐药的MM患者血清GRP78水平。此外，还将通过细胞内GRP78的过表达和敲除来研究其对PI易感性的影响。将重组表达的GRP78与PI共孵育几个细胞毒试验，最后将不同的抗GRP78抗体与PI在敏感和难治细胞系和患者材料中进行联合评估。阳性组合将在MM的实验动物模型中得到验证。该研究的目的是了解GRP78介导的耐药的分子发病机制，另一方面开发一种基于抗体的治疗策略，以克服难治性MM患者的PI耐药。

Multiple myeloma (MM) is a rare hematological cancer originating from transformed plasma cells in the bone marrow. Excessive synthesis and secretion of monoclonal immunoglobulins (Ig) or incomplete molecules, the Ig light chains, is one hallmark of MM. This secretory activity leads to a significant increased level of stress in the endoplasmatic reticulum (ER) and results frequently in the accumulation of incorrect or missfolded proteins. To circumvent unnecessary or even harmful protein biosynthesis this accumulation can trigger a highly conserved mechanism called the Unfolded Protein Response (UPR), which finally results in the apoptosis of the cell. A novel class of therapeutics uses this mechanism by inhibiting the cellular degradation system- the proteasome- which causes severe ER stress. Proteasome inhibitors such as bortezomib represent the strongest plasma cell depleting agents of today and are used very successfully in the treatment of MM. Accumulation of missfolded Ig on the other hand increases the number of chaperone molecules which are involved in protein assembly as well as in the regulation of the UPR. A key sensor of the UPR is chaperone molecule Glucose regulated protein (GRP) 78 which is mainly located in the ER but was also found on the surface of tumor cells or as secreted variant. The latter variant seems to contribute to PI resistance in vitro. However, the connection of GRP78 and the ubiquitin-proteasome pathway is barely understood. We have developed a therapeutic anti-GRP78 antibody and tested the product in clinical trials. Single-agent activity was moderate, but in combination with PI objective responses were seen even in a patient with PI refractory disease. In this proposal I aim to go back from bedside to the bench to elucidate GRP78 induced PI resistance in vitro and to investigate whether treatment with anti-GRP78 antibodies can overcome PI resistance. I will explore the frequency and functional role of secreted GRP78 in myeloma patients at different stages of their disease using ELISA and Western blot techniques. In addition, I will compare GRP78 serum levels from PI sensitive to PI resistant MM patients. Furthermore, intracellular overexpression as well as knock down of GRP78 will be performed to study its impact on susceptibility to PI. Recombinant expressed GRP78 will be co-incubated with PI in several cytotoxicity assays and finally different anti-GRP78 antibodies will be evaluated in combination with PI in sensitive and refractory cell lines and patients material. Positive combinations will be validated in an experimental animal model of MM. The aim of the study is to obtain insights in the molecular pathogenesis of GRP78 mediated resistance and on the other hand to develop an antibody based treatment strategy to overcome PI resistance in refractory MM patients.