GSK3 inhibition as an adjuvant therapy for Burkitt's lymphoma

描述（由申请人提供）：c-Myc原癌基因是b细胞稳态和b淋巴瘤形成的关键因素。它的转化潜力很大程度上取决于它驱动细胞增殖的能力。同时，Myc还可以通过诱导p53或不依赖p53的方式诱导细胞凋亡。然而，到目前为止，将Myc的促凋亡活性用于抗癌治疗的目标被证明是难以实现的，主要是因为暂时增加Myc水平的方法尚不存在。我们最近的研究表明，增强Myc表达可提高p53并增加对硼替佐米的敏感性。该研究的一个局限性是依赖于p53通路，而p53在人类b型淋巴瘤中经常缺失。为了确定在真正的治疗环境（CHOP治疗）中Myc如何促进p53非依赖性细胞凋亡，我们现在使用从p53ERTAM敲入小鼠骨髓中分离的细胞，随后用表达Myc的逆转录病毒转导。这种诱导系统使我们能够区分p53依赖性（用他莫昔芬处理的细胞）和独立型（不给他莫昔芬）细胞死亡。此外，我们实验室最近的数据显示GSK-3¿积极参与b细胞中Myc蛋白降解的调节，并且用CHIR99021抑制GSK-3¿会急剧增加Myc水平。使用这些试剂，我们观察到GSK-3¿抑制剂对Myc的药理学稳定性强烈增强了p53非依赖性阿霉素诱导的细胞凋亡。最重要的是，即使在p53突变的Ramos Burkitt淋巴瘤细胞中，Myc稳定导致对阿霉素的反应增加。这些结果完全支持了我们的创新假设，即即使p53缺失或MDM2扩增，短暂上调Myc也可能是Myc驱动的肿瘤的可行辅助治疗方法。我们将在以下两个目标中追求这一假设。1)研究myc依赖性和非依赖性事件驱动p53非依赖性细胞凋亡对阿霉素+GSK3抑制剂的反应。具体来说，我们将确定遗传或药理学抑制Myc是否会消除CHIR99021的促凋亡作用，或者其他GSK-3靶点如BCL2L12是否有助于化学致敏。2)探讨GSK3¿抑制在急性和慢性治疗模型中如何影响伯基特淋巴瘤对阿霉素的反应。基于我们的体外数据，我们将测试GSK-3¿抑制是否增强了基于阿霉素的化疗对小鼠同基因移植物和人类异种移植物的作用，从而产生更强的凋亡反应（急性模型）和延长无事件生存期（慢性模型）。在完成这项工作后，我们将更好地了解GSK3¿及其靶点如Myc和BCL2L12调控的凋亡途径。此外，我们将验证GSK3¿抑制剂作为辅助疗法治疗myc驱动的B细胞淋巴瘤突变p53或MDM2扩增。

DESCRIPTION (provided by applicant): The c-Myc proto-oncogene is a key factor in both B-cell homeostasis and B-lymphomagenesis. Its transforming potential is based largely on its ability to drive cell proliferation. At the same time, Myc can also induce apoptosis, either throug induction of p53 or in a p53-independent manner. Yet the goal of channeling the pro-apoptotic activity of Myc toward anti-cancer therapies has so far proven elusive, primarily because methods to transiently increased Myc levels didn't exist. Our recent research showed that boosting Myc expression elevates p53 and increases sensitivity to bortezomib. One limitation of that study was the reliance on the p53 pathway, which is frequently lost in human B-lymphomas. To determine how Myc contributes to p53-independent apoptosis in a bona fide therapeutic setting (CHOP therapy), we now used cells isolated from bone marrows of p53ERTAM knock-in mice and subsequently transduced with a Myc-expressing retrovirus. This inducible system allowed us to distinguish between p53-dependent (cells treated with tamoxifen) and independent (no tamoxifen given) cell deaths. Additionally, very recent data from our lab show that GSK-3¿ is actively involved in the regulation of Myc protein degradation in B-cells, and that inhibition of GSK-3¿ with CHIR99021 sharply increases Myc levels. Using these reagents, we observed that pharmacological stabilization of Myc with GSK-3¿ inhibitors strongly enhanced p53-independent doxorubicin-induced apoptosis. Most importantly, even in p53-mutated Ramos Burkitt's lymphoma cells, Myc stabilization resulted in increased responses to doxorubicin. These results fully support our innovative hypothesis that transient up-regulation of Myc could be a viable adjuvant therapy for Myc-driven tumors even with p53 loss or MDM2 amplification. We will pursue this hypothesis in the following two aims. 1) To investigate Myc-dependent and - independent events that drive p53-independent apoptosis in response to doxorubicin+GSK3¿ inhibitors. Specifically, we will determine whether genetic or pharmacological inhibition of Myc abolishes pro- apoptotic effects of CHIR99021 or whether other GSK-3¿ targets such as BCL2L12 contribute to chemosensitization. 2) To investigate how GSK3¿ inhibition affects Burkitt's lymphoma response to doxorubicin in acute and chronic treatment models. On the strength of our in vitro data, we will test if GSK-3¿ inhibition potentiates doxorubicin-based chemotherapy against murine syngeneic grafts and human xenografts, resulting in a more robust apoptotic response (acute model) and prolonged event- free survival (chronic model). Upon completion of this work we will have a better understanding of the apoptotic pathways regulated by GSK3¿ and its targets such as Myc and BCL2L12. Also, we will validate GSK3¿ inhibitors as adjuvant therapeutics to treat Myc-driven B cell lymphomas with mutant p53 or MDM2 amplification.