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 许多癌症都有易位，扩增或激活C-Myc原始的突变 癌基因。 MYC调节细胞中的许多程序，包括生长和增殖以及凋亡 同样，主要通过p53的激活。但是，MYC放松管制和p53失活 经常在B细胞淋巴瘤（例如Burkitt的淋巴瘤（BL））中共同发生；因此，化学疗法 患者并不总是治愈的。研究表明，高水平的致癌MYC是 需要参与凋亡，因此改善当前疗法的一种策略是瞬时 增加MYC的表达和稳定性。 GSK3β通过磷酸化MYC来调节MYC蛋白稳定性 在THR58住宅中，标记为泛素化和退化。 GSK3β可以针对 在药理上具有特定抑制剂，使其成为调节MYC水平的吸引力。 初步数据表明，通过用CHIR99021抑制GSK3β的瞬时MYC上调 当与化学治疗药物结合使用时，增强B细胞淋巴瘤的凋亡并建议 外部凋亡途径对于这种增强的凋亡至关重要。 通过结合配体（FASL，TNF，TRAIL）与细胞表面上的死亡受体的结合（FAS，FAS，FAS） TNFR1，DR4，DR5）。该提案的目的是确定反抗的机制 GSK3β调整疗法增强了B细胞淋巴瘤的凋亡，并研究了Myc- 依赖和独立的机制。我的第一个目的是检查MYC和其他 抗GSK3β调整治疗中的GSK3β靶标。为了评估MYC的作用，我将比较 WT和突变体THR58 MYC的BL细胞中的凋亡，在GSK3β上无法稳定MYC 抑制。我将研究PTEN作为另一个GSK3β靶标，并验证GSK3β的效果 在PTEN蛋白水平上，稳定性，THR366磷酸化（由GSK3β靶向）和活性。我 然后，将比较用WT与Thr366突变体PTEN对抗抗 - GSK3β调整疗法。我的第二个目标将调查外部凋亡的贡献 使用CRISPR/CAS9通过调制长时间删除BL细胞上的死亡受体 表达，外部途径的负调节剂；我将测试这些修改的响应 细胞进行抗GSK3β调整治疗。最后，我将研究体内外部途径的作用 通过比较BL细胞的皮下Xenographothic semagromenthogrotion vs. 对抗GSK3β调整疗法以及GSK3β抑制作用 +外部配体的凋亡信号传导 踪迹。这些提出的实验将为GSK3β提供基本原理和机制 MYC驱动的B细胞淋巴瘤的辅助治疗。