Manipulating the N-end Rule Protein Degradation Pathway to Build Bone and Decrease Tumor Growth in Multiple Myeloma Bone Disease

项目摘要/摘要 多发性骨髓瘤(MM)会造成毁灭性的骨破坏和长期的骨形成抑制， 显著增加骨折风险，影响死亡率，并导致MM细胞耐药。MM骨病变 大多数患者很少愈合，因此严重的骨痛和骨折风险持续存在。目前，还没有安全的骨骼 被批准用于MM骨病(MMBD)的合成代谢药。我们发现多结构域蛋白p62 封闭体-1通过其两个主要功能是MMBD的主要贡献者。它是多个 促进MM细胞生长、骨破坏和抑制MM的信号通路p62也介导了 作为泛素介导的自噬的货物受体，MM细胞具有促进生存的功能。P62-ZZ结构域 (p62-zz)，在这两个职能中都发挥着关键作用，也是本提案的重点。我们和同事们一起在 匹兹堡大学的一位科学家开发了XRK3F2(XRK)，这是一种结合p62-ZZ的小分子。我们发现XRK 诱导多发性骨髓瘤小鼠大量新骨形成，允许多发性骨髓瘤患者来源的骨髓基质 MM-BMSC向功能性OB分化，诱导MM细胞死亡并增强其抗MM作用 用于治疗多发性骨髓瘤的蛋白酶体抑制剂(PI)--波特佐米(BTZ)和卡菲佐米。然而，这些机制 对XRK在MM中的影响负责的人还不清楚。最近，我们的合作者发现p62-ZZ是一种高亲和力的 N-末端规则通路产生的N-末端精氨酸化蛋白质的N-识别蛋白，而XRK作为一个 高亲和力降解p62-ZZ，模拟内源性N端降解。N端规则通路(N-ERP)是一种 保护细胞免受胞浆中错误折叠蛋白(MPS)的蛋白毒性应激的蛋白分解系统 通常是针对内质网相关的降解，并将它们运送到蛋白酶体和自噬介导的 退化途径。过多的MPS触发内质网伴侣的释放，然后N-精氨酸化和 绑定下院议员。N-R蛋白复合体与N-识别蛋白的UBR盒结合，以用于随后的蛋白酶体 降解，或p62-ZZ用于自噬蛋白分解。我们最近发现，用XRK饱和p62-ZZ增加了 PI通过坏死性下垂诱导MM细胞死亡，即使在PI抵抗的细胞中也是如此。因此，p62-ZZ作为分子 MM细胞中的坏死性和凋亡性细胞死亡途径的切换。然而，XRK作为一个 多发性骨髓瘤或骨髓间充质干细胞中未见高亲和力N-降解子与p62-ZZ结合。我们将在体外和体内使用 活体方法确定；目标1：XRK缓解受抑制的OB的机制 MM细胞或肿瘤坏死因子诱导骨髓间充质干细胞分化及炎性细胞因子在其中的作用 进程。目的2：XRK对大鼠成骨细胞作用的机制及相对贡献 MMBD。目的3：坏死下垂介导的细胞死亡在多发性骨髓瘤中的重要性，以及p62-ZZ/N-1对其的调节作用。 事件相关蛋白和蛋白酶体通路。目的4：联合XRK靶向p62-ZZ+/-PI治疗的体内效应 关于MMBD和MM骨损伤的治疗。这些研究应该会提供关于p62的重要新信息- ZZ/N-ERP通路作为MMBD成骨和抑制肿瘤生长的治疗靶点。

Project Summary/Abstract Multiple Myeloma (MM) causes devastating bone destruction and protracted suppression of bone formation that markedly increases fracture risk, impacts mortality and contributes to MM cell drug resistance. MM bone lesions rarely heal in most patients, so that severe bone pain and fracture risk persist. Currently, there are no safe bone anabolic agents approved for MM bone disease (MMBD). We showed that the multi-domain protein p62 (sequestosome-1) is a major contributor to MMBD through its two major functions. It is a signaling hub for multiple signaling pathways that increase MM cell growth, bone destruction OB suppression in MM. p62 also mediates pro-survival functions in MM cells as a cargo receptor for ubiquitin-mediated autophagy. The p62-ZZ domain (p62-ZZ), plays a key role in both these functions, and is the focus of this proposal. We, with colleagues at University of Pittsburgh, developed XRK3F2 (XRK), a small molecule that binds p62-ZZ. We found that XRK induced dramatic new bone formation in MM-bearing mice, allowed MM patient-derived bone marrow stromal cells (MM-BMSC) to differentiate to functional OB, induced MM cell death and enhanced the anti-MM effects of Bortezomib (Btz) and Carfilzomib, proteasome inhibitors (PIs) used for MM treatment. However, the mechanisms responsible for XRK’s effects in MM are unclear. Recently, our collaborators found that p62-ZZ is a high-affinity N-recognin for N-terminal argininylated proteins generated by the N-end rule pathway, and that XRK acts as a high affinity degron for p62- ZZ, mimicking endogenous N-end degrons. The N-end rule pathway (N-ERP) is a proteolytic system that protects cells from the proteotoxic stress of misfolded proteins (MPs) in the cytosol that normally are targeted for ER-associated degradation, and shuttles them to proteasome and autophagy-mediated degradation pathways. Excess MPs trigger release of chaperones from the ER that are then N-arginylated and bind MPs. The N-R protein complexes bind either the UBR box of N-recognins for subsequent proteosomal degradation, or p62-ZZ for autophagic proteolysis. We recently found that saturating p62-ZZ with XRK increased PI-induced MM cell death via necroptosis, even in PI-resistant cells. Thus, p62-ZZ serves as the molecular switch for necroptotic vs. apoptotic cell death pathways in MM cells. However, how XRK’s actions as a high-affinity N-degron that binds p62-ZZ are uncharacterized in MM or BMSCs. We will use in vitro and in vivo approaches to determine; Aim 1: The mechanisms responsible for XRK’s relief of the suppressed OB differentiation in BMSC exposed to MM cells or TNF, and the contributions of inflammatory cytokines to this process. Aim 2: The mechanisms responsible and relative contributions of XRK’s effects on osteocytes in MMBD. Aim 3: The importance of necroptosis-mediated cell death in MM, and its regulation by the p62-ZZ/N- ERP and proteasome pathways. Aim 4: The in vivo effects of combining XRK targeting of p62-ZZ +/- PI treatment on MMBD and healing MM bone lesions. These studies should provide important new information on the p62- ZZ/N-ERP pathway as a therapeutic target to build bone and decrease tumor growth in MMBD.