Elucidating the mechanism of anti-GSK3 adjuvant therapy for Myc-driven lymphomas

阐明抗 GSK3 辅助治疗 Myc 驱动的淋巴瘤的机制

• 批准号: 9328735
• 负责人: Colleen T Harrington
• 金额: $ 4.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9328735
• 关键词: Adjuvant Therapy; Apoptosis; Apoptotic; B lymphoid malignancy; B-Cell Lymphomas; Biology; Burkitt Lymphoma; CASP8 and FADD-like apoptosis regulating protein; CRISPR/Cas technology; Cell Cycle Arrest; Cell Death; Cell Line; Cell surface; Cells; Cellular Stress; Cessation of life; Chromosomal translocation; Complement; Data; Doxorubicin; Event; FDA approved; Family; Genetic Transcription; Goals; Growth; In Vitro; Individual; Induction of Apoptosis; Laboratories; Ligand Binding; Ligands; Light; Lithium; Lymphoma; MDM2 gene; MYC Family Protein; Malignant Neoplasms; Mediating; Messenger RNA; Metabolism; Mitochondria; Modeling; Mutation; Non-Hodgkin' s Lymphoma; Oncogenic; PMAIP1 gene; PTEN gene; PTEN protein; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Phosphorylation Site; Protein Overexpression; Proteins; Proto-Oncogenes; Refractory; Relapse; Reporting; Role; Serum; Signal Transduction; Small Interfering RNA; TNF gene; TNFRSF10A gene; TNFRSF10B gene; TNFRSF1A gene; TNFRSF6 gene; TNFSF10 gene; TP53 gene; Technology; Testing; Tumor Suppressor Proteins; Up-Regulation; Work; Xenograft procedure; c-myc Proto-Oncogenes; cancer therapy; cell growth; cellular engineering; chemotherapy; experimental study; genome editing; glycogen synthase kinase 3 beta inhibitor; improved; in vivo; inhibitor/antagonist; knock-down; loss of function mutation; mutant; overexpression; programs; receptor; reconstitution; response; small hairpin RNA; subcutaneous; tumor

PROJECT SUMMARY Many cancers harbor translocations, amplifications, or activating mutations of the c-Myc proto- oncogene. Myc regulates many programs in the cell including growth and proliferation, and apoptosis as well, predominantly through the activation of p53. However, Myc deregulation and p53 inactivation frequently co-occur in B-cell lymphomas such as Burkitt's lymphoma (BL); thus, chemotherapies for patients are not always curative. Studies have shown that very high levels of oncogenic Myc are required to engage apoptosis, thus one strategy to improve current therapies would be to transiently increase Myc expression and stability. GSK3β regulates Myc protein stability by phosphorylating Myc at the Thr58 residue, marking it for ubiquitylation and degradation. GSK3β can be targeted pharmacologically with specific inhibitors, making it an attractive target to modulate Myc levels. Preliminary data show that transient up-regulation of Myc by inhibiting GSK3β with CHIR99021 enhances apoptosis in B-cell lymphomas when combined with chemotherapeutic drugs and suggest that the extrinsic apoptotic pathway is important for this enhanced apoptosis. Extrinsic apoptosis is activated by binding of ligands (FasL, TNF, TRAIL) to death receptors on the cell surface (Fas, TNFR1, DR4, DR5). The goal of this proposal will be to determine the mechanism by which anti- GSK3β adjuvant therapy enhances apoptosis in B-cell lymphomas and to investigate both Myc- dependent and independent mechanisms. My first aim will be to examine the role of Myc and other GSK3β targets in anti-GSK3β adjuvant therapy. To assess the role of Myc, I will compare induction of apoptosis in BL cells with WT vs. mutant Thr58 Myc, where Myc cannot be stabilized upon GSK3β inhibition. I will investigate PTEN as another GSK3β target and validate the effect of GSK3β inhibition on PTEN protein level, stability, Thr366 phosphorylation (the site targeted by GSK3β) and activity. I will then compare the response of BL cells engineered with WT vs. Thr366 mutant PTEN to anti- GSK3β adjuvant therapy. My second aim will investigate the contribution of extrinsic apoptosis by using CRISPR/Cas9 to delete death receptors on BL cells complemented by modulation of FLIP-long expression, the negative regulator of the extrinsic pathway; I will test the response of these modified cells to anti-GSK3β adjuvant therapy. Finally I will investigate the role of the extrinsic pathway in vivo by comparing the response of subcutaneous xenografts of BL cells with intact vs. perturbed extrinsic apoptotic signaling to anti-GSK3β adjuvant therapy as well as GSK3β inhibition + the extrinsic ligand TRAIL. These proposed experiments will provide rationale and mechanism for GSK3β inhibition as adjuvant therapy for Myc-driven B-cell lymphomas.

项目摘要 许多癌症具有c-Myc原核表达系统的易位、扩增或激活突变。 癌基因Myc调控着细胞的生长、增殖和凋亡等多个过程 也主要是通过激活p53。然而，Myc失调和p53失活 B细胞淋巴瘤，如伯基特淋巴瘤（BL）经常共同发生;因此， 病人并不总是治愈的。研究表明，非常高水平的致癌性Myc是 因此，改善当前疗法的一种策略将是瞬时地 增加Myc表达和稳定性。GSK 3 β通过磷酸化Myc调节Myc蛋白稳定性 在Thr 58残基，标记它的泛素化和降解。GSK 3 β可以靶向 与特异性抑制剂结合，使其成为调节Myc水平的有吸引力的靶点。 初步数据显示，通过用CHIR 99021抑制GSK 3 β瞬时上调Myc 与化疗药物联合使用时可增强B细胞淋巴瘤的凋亡， 外源性凋亡途径对于这种增强的凋亡是重要的。外源性凋亡是 通过配体（FasL，TNF，TRAIL）与细胞表面上的死亡受体（Fas， TNFR1、DR4、DR5）。该提案的目标是确定反- GSK 3 β辅助治疗增强B细胞淋巴瘤的细胞凋亡，并研究Myc- 依赖和独立机制。我的第一个目标将是检查Myc和其他 抗GSK 3 β辅助治疗中的GSK 3 β靶点。为了评估Myc的作用，我将比较 WT与突变型Thr 58 Myc的BL细胞凋亡，其中Myc不能在GSK 3 β上稳定 抑制作用我将研究PTEN作为另一个GSK 3 β靶点，并验证GSK 3 β抑制剂的作用 对PTEN蛋白水平、稳定性、Thr 366磷酸化（GSK 3 β靶向位点）和活性的影响。我 然后将比较用WT与Thr 366突变体PTEN工程化的BL细胞对抗- GSK 3 β辅助治疗。我的第二个目标是研究外源性凋亡的贡献， 使用CRISPR/Cas9删除BL细胞上的死亡受体，并通过调节FLIP-长 表达，外在途径的负调节;我将测试这些修饰的反应。 抗GSK 3 β辅助治疗。最后，我将研究外源性途径在体内的作用 通过比较BL细胞的皮下异种移植物与完整的与扰动的外源性 抗GSK 3 β辅助治疗以及GSK 3 β抑制+外源性配体的凋亡信号传导 小道。这些拟议的实验将为GSK 3 β抑制提供理论基础和机制， Myc驱动的B细胞淋巴瘤的辅助治疗。