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

操纵 N 端规则蛋白降解途径来构建骨并减少多发性骨髓瘤骨病中的肿瘤生长

• 批准号: 10582710
• 负责人: JOHN M CHIRGWIN
• 金额: $ 32.58万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-04 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582710
• 关键词: Affinity; Anabolic Agents; Apoptosis; Apoptotic; Autophagocytosis; Binding; Bone Diseases; Bone Marrow; Bone Pain; Bortezomib; Caspase; Cell Death; Cells; Clinical; Collaborations; Cytoprotection; Cytosol; Degradation Pathway; Drug resistance; Exposure to; Fracture; GRP78 gene; GTF2H1 gene; Goals; In Vitro; In complete remission; Inflammatory; Lesion; Malignant Neoplasms; Mediating; Molecular; Molecular Chaperones; Multiple Myeloma; Mus; N-terminal; New Agents; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Pathway interactions; Patients; Play; Process; Proteasome Inhibitor; Protein Fragment; Proteins; Proteolysis; RIPK1 gene; Regulation; Resistance; Resistance development; Signal Pathway; Signal Transduction; Stress; Stromal Cells; System; Tertiary Protein Structure; Therapeutic Agents; Ubiquitin; Universities; antitumor effect; bisphosphonate; bone; bone healing; cell growth; cytokine; fracture risk; healing; improved; in vivo; inhibitor; misfolded protein; mortality; multicatalytic endopeptidase complex; novel; osteoblast differentiation; p38 Mitogen Activated Protein Kinase; prevent; pro-apoptotic protein; protein complex; protein degradation; proteotoxicity; receptor; recognins; small molecule; therapeutic target; tumor growth

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.

项目总结/摘要 多发性骨髓瘤（MM）会导致破坏性的骨破坏和骨形成的长期抑制， 显著增加骨折风险，影响死亡率并导致MM细胞耐药性。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，没有安全的骨骼 批准用于MM骨病（MMBD）的合成代谢药物。我们发现多结构域蛋白p62 （多价螯合体-1）通过其两个主要功能是MMBD的主要贡献者。它是一个信号枢纽， 增加MM细胞生长的信号通路，MM中的骨破坏OB抑制。 在MM细胞中，促存活功能作为泛素介导的自噬的货物受体。p62-ZZ结构域 （p62-ZZ），在这两个功能中起着关键作用，是本提案的重点。我们与同事在 匹兹堡大学开发了XRK 3F 2（XRK），一种结合p62-ZZ的小分子。我们发现XRK 诱导MM荷瘤小鼠显著的新骨形成，使MM患者来源的骨髓基质细胞 细胞（MM-BMSC）分化为功能性OB，诱导MM细胞死亡，并增强 硼替佐米（Btz）和卡非佐米，用于MM治疗的蛋白酶体抑制剂（PI）。然而，机制 负责XRK在MM中的作用尚不清楚。最近，我们的合作者发现p62-ZZ是一种高亲和力的 N-recognin是由N-末端规则途径产生的N-末端精氨酸化蛋白的N-识别蛋白，XRK作为一种免疫调节剂， 对p62- ZZ的高亲和力降解决定子，模拟内源性N-末端降解决定子。N-end rule pathway（N-ERP） 蛋白水解系统，保护细胞免受胞质溶胶中错误折叠蛋白（MP）的蛋白毒性应激， 通常是ER相关降解的靶点，并将其穿梭于蛋白酶体和自噬介导的 降解途径过量的MP触发从ER释放分子伴侣，然后将其N-酰基化， 约束议员。N-R蛋白复合物结合N-识别蛋白的UBR盒，用于随后的蛋白体结合。 降解，或p62-ZZ用于自噬蛋白水解。我们最近发现用XRK饱和p62-ZZ增加了 PI通过坏死性凋亡诱导MM细胞死亡，即使在PI耐药细胞中。因此，p62-ZZ作为分子 MM细胞中坏死性凋亡与凋亡性细胞死亡途径开关。然而，XRK作为一个 结合p62-ZZ的高亲和力N-降解决定子在MM或BMSC中未被表征。我们将在体外和体内 体内方法来确定;目的1：XRK缓解受抑制OB的机制 暴露于MM细胞或TNF α的BMSC的分化，以及炎性细胞因子对此的贡献。 过程目的2：探讨XRK对成骨细胞的作用机制及其相对贡献。 MMBD。目的3：研究MM中坏死性凋亡介导的细胞死亡的重要性，以及p62-ZZ/N- ERP和蛋白酶体途径。目的4：结合XRK靶向p62-ZZ +/- PI治疗的体内效应 MMBD和愈合MM骨病变。这些研究将提供关于p62的重要新信息。 ZZ/N-ERP通路作为MMBD中构建骨和减少肿瘤生长的治疗靶点。