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

项目总结 许多癌症含有c-Myc原基因的易位、扩增或激活突变- 致癌基因。MYC调节细胞内的许多程序，包括生长、增殖和凋亡。 此外，主要是通过激活P53。然而，Myc的去调控和P53的失活 通常在B细胞淋巴瘤中并存，如Burkitt淋巴瘤(BL)；因此，化疗对 病人并不总是治愈的。研究表明，非常高水平的致癌Myc是 因此，改进当前治疗方法的一个策略是暂时地 增加Myc的表达和稳定性。Gsk3β通过磷酸化Myc调节Myc蛋白稳定性 在Thr58残基，标志着它的泛素化和降解。Gsk3β可以成为目标 使用特定的抑制剂，使其成为调节Myc水平的一个有吸引力的靶点。 初步数据显示，CHIR99021通过抑制Gsk3β瞬时上调Myc 联合化疗药物可促进B细胞淋巴瘤的细胞凋亡 外在的细胞凋亡途径对于这种增强的细胞凋亡是重要的。外源性细胞凋亡是 通过与细胞表面死亡受体(Fas、TRAIL、Fas、TRAIL)结合而激活 TNFR1、DR4、DR5)。这项提案的目标将是确定反腐败的机制 GSK3β辅助治疗可促进B细胞淋巴瘤细胞的凋亡，并探讨MYC-1和MYC-2在B细胞淋巴瘤中的作用。 依赖和独立的机制。我的第一个目标将是研究Myc和其他 GSK3β靶点用于抗GSK3β辅助治疗。为了评估Myc的作用，我将比较诱导 WT与突变体Thr58Myc的细胞凋亡比较，其中Myc不能稳定在Gsk3β上 抑制力。我将研究PTEN作为另一个Gsk3β靶点，并验证Gsk3β抑制的效果 对PTEN蛋白水平、稳定性、Thr366磷酸化(Gsk3β靶点)和活性的影响。我 然后将比较用WT和Thr366突变体PTEN改造的BL细胞对抗- GSK3型β辅助治疗。我的第二个目标是研究外源性细胞凋亡的作用 利用CRISPR/Cas9缺失BL细胞上的死亡受体并辅以Flip-Long调控 表达，外在途径的负调控；我将测试这些修饰的反应 细胞给予抗GSK3β辅助治疗。最后，我将研究外在途径在体内的作用 通过比较完整和受扰外源细胞的异种皮下移植反应 抗GSK_3β辅助治疗及GSK_3β抑制+外源性配体的细胞凋亡信号 小路。这些拟议的实验将为抑制Gsk3β提供理论基础和机制，如 Myc驱动的B细胞淋巴瘤的辅助治疗。

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.