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: 10355454
• 负责人: Garson DAVID ROODMAN
• 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/10355454
• 关键词: Affinity; Anabolic Agents; Apoptosis; Apoptotic; Autophagocytosis; Binding; Bone Diseases; Bone Marrow; Bone Pain; Bortezomib; Caspase; Cell Death; Cells; Clinical; Collaborations; 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; TNF gene; Tertiary Protein Structure; Therapeutic Agents; Ubiquitin; Universities; antitumor effect; base; bisphosphonate; bone; cell growth; cytokine; fracture risk; healing; improved; in vivo; inhibitor; misfolded protein; mortality; multicatalytic endopeptidase complex; novel; osteoblast differentiation; 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.

项目概要/摘要 多发性骨髓瘤 (MM) 会导致毁灭性的骨质破坏和骨形成的长期抑制， 显着增加骨折风险、影响死亡率并导致 MM 细胞耐药。多发性骨髓瘤骨病变 大多数患者很难治愈，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头 批准用于治疗 MM 骨病 (MMBD) 的合成代谢药物。我们发现多结构域蛋白 p62 (sequestosome-1) 通过其两个主要功能是 MMBD 的主要贡献者。它是多个信号的枢纽 增加 MM 细胞生长、抑制 MM 骨破坏 OB 的信号通路。 p62 也介导 MM 细胞中作为泛素介导的自噬的货物受体发挥促生存功能。 p62-ZZ 结构域 (p62-ZZ)，在这两个功能中都发挥着关键作用，也是本提案的重点。我们与同事们在 匹兹堡大学开发了 XRK3F2 (XRK)，一种结合 p62-ZZ 的小分子。我们发现XRK 在携带 MM 的小鼠中诱导显着的新骨形成，允许 MM 患者来源的骨髓基质 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，蛋白酶体抑制剂 (PI)，用于多发性骨髓瘤治疗。然而，这些机制 XRK 对 MM 的影响尚不清楚。最近，我们的合作者发现p62-ZZ是一种高亲和力的 N 端规则途径生成的 N 端精氨酸化蛋白的 N 识别蛋白，XRK 充当 p62-ZZ 的高亲和力降解决定子，模仿内源性 N 端降解决定子。 N端规则路径（N-ERP）是一个 保护细胞免受胞质溶胶中错误折叠蛋白 (MP) 的蛋白毒性应激的蛋白水解系统 通常是内质网相关降解的目标，并将它们运送到蛋白酶体和自噬介导的 降解途径。过量的 MP 会触发 ER 中分子伴侣的释放，然后分子伴侣被 N-精酰化并 绑定国会议员。 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 降解，或用于自噬蛋白水解的 p62-ZZ。我们最近发现用 XRK 饱和 p62-ZZ 会增加 PI 通过坏死性凋亡诱导 MM 细胞死亡，甚至在 PI 抗性细胞中也是如此。因此，p62-ZZ 作为分子 MM 细胞中坏死性凋亡与凋亡细胞死亡途径的转换。然而，XRK 的行为如何作为 结合 p62-ZZ 的高亲和力 N-降解决定子在 MM 或 BMSC 中尚未表征。我们将在体外和体内使用 体内方法确定；目标 1：XRK 解除受抑制 OB 的机制 暴露于 MM 细胞或 TNFα 的 BMSC 的分化，以及炎症细胞因子对此的贡献 过程。目标 2：XRK 对骨细胞影响的机制和相对贡献 MMBD。目标 3：坏死性凋亡介导的细胞死亡在 MM 中的重要性及其受 p62-ZZ/N- 的调节 ERP 和蛋白酶体途径。目标 4：联合 XRK 靶向 p62-ZZ +/- PI 治疗的体内效果 MMBD 和愈合 MM 骨病变。这些研究应该提供有关 p62- 的重要新信息。 ZZ/N-ERP 通路作为 MMBD 中骨构建和减少肿瘤生长的治疗靶点。