Targeting oncoprotein stability for cancer therapy

泛素蛋白酶体系统控制着大多数细胞蛋白质的丰度。这个系统包括一组酶，如泛素连接酶和去泛素酶，将泛素转移到蛋白质底物上并将其从蛋白质底物中移除。泛素化的底物被一个大型的蛋白分解复合体--蛋白酶体降解。蛋白酶体抑制可以抑制癌细胞的存活，并已成功地用于多发性骨髓瘤的治疗。然而，蛋白酶体抑制的细胞毒性机制仍然不清楚，这突显了更多选择性治疗的必要性。对维持癌蛋白稳定性的通路进行特异性靶向可能是一种替代的抗肿瘤策略。癌蛋白在动态平衡过程中驱动关键的生物学过程，在肿瘤发生过程中常常成为细胞生存所必需的。癌蛋白的丰度可以由特定的泛素依赖途径非冗余地控制，其中包括泛素特定的蛋白酶。例如，在小鼠生殖系中去除USP7去泛素酶会破坏致癌泛素连接酶MDM2的稳定，过度激活P53，并导致胚胎死亡，表明维持癌蛋白的稳定性是一个重要的细胞过程。泛素特异的蛋白水解酶容易受到小分子的抑制，因此是很有希望的治疗靶点。USP7的抑制剂可以消除小鼠的肿瘤形成，但可以被健康的成人组织耐受，它表明靶向癌蛋白稳定性可以创造一个重要的治疗窗口。一个控制包括Myc、Notch和Cyclin E在内的多种癌蛋白降解的关键途径是由SCF(Fbw7)泛素连接酶驱动的。Fbw7对于组织动态平衡是必不可少的，它的功能丧失促进了小鼠模型的肿瘤发生。我们最近发现，Fbw7介导的底物降解可以被Usp28脱泛素酶拮抗。肿瘤细胞系中Usp28的耗尽或小鼠中Usp28的遗传消融会破坏Fbw7底物的稳定性。重要的是，Usp28在肠道中的缺失对组织稳态只有轻微的影响，但会强烈地抑制肠道肿瘤的发展，因此可能为实体肿瘤提供治疗窗口。有趣的是，我们发现Usp28的催化活性丧失在促进癌蛋白降解方面比完全基因敲除更有效。因此，我们推测小分子抑制USP28的催化功能可能在肿瘤抑制中特别有效。在本项目中，我们将追求以下目标：1)通过在小鼠模型中表达催化失活的Usp28来模拟Usp28在动态平衡和肿瘤发生过程中的小分子抑制作用；2)阐明Usp28去泛素化的结构基础并设计针对Usp28的小分子抑制剂；3)识别通过Usp28/Fbw7途径促进癌蛋白降解的信号事件。

The ubiquitin proteasome system controls the abundance of most cellular proteins. This system includes a set of enzymes, such as ubiquitin ligases and deubiquitinases, which transfer ubiquitin onto and remove it from protein substrates. Ubiquitinated substrates are degraded by a large proteolytic complex, the proteasome. Proteasome inhibition can be instrumental in suppressing cancer cell survival and has been successfully used in treatment of multiple myeloma. However, the mechanisms of cytotoxicity of proteasome inhibition remain elusive, highlighting the need for more selective therapies. Specific targeting of pathways, which maintain stability of oncoproteins may present an alternative antineoplastic strategy. Oncoproteins drive key biological processes during homeostasis, and often become indispensable for cell viability during tumorigenesis. The abundance of oncoproteins can be non-redundantly controlled by specific ubiquitin-dependent pathways, which include ubiquitin-specific proteases. For example, ablation of the Usp7 deubiquitinase in mouse germline destabilizes the oncogenic ubiquitin ligase Mdm2, hyperactivates p53, and leads to embryonic lethality, demonstrating that maintenance of oncoprotein stability is an essential cellular process. Ubiquitin-specific proteases are amenable to small molecule inhibition and therefore are promising therapeutic targets. Inhibitors of Usp7, which abrogate tumorigenesis in mice but are tolerated by healthy adult tissues, show that targeting oncoprotein stability creates a significant therapeutic window.A critical pathway, which controls degradation of multiple oncoproteins including Myc, Notch and cyclin E, is driven by the SCF(Fbw7) ubiquitin ligase. Fbw7 is essential for tissue homeostasis and its loss-of-function promotes tumorigenesis in mouse models. We have recently shown that Fbw7-mediated substrate degradation is antagonized by the Usp28 deubiquitinase. Depletion of Usp28 in tumor cell lines or genetic ablation of Usp28 in mice destabilizes Fbw7 substrates. Importantly, deletion of Usp28 in the intestine has only subtle effects on tissue homeostasis but strongly attenuates intestinal tumor development, and may thus provide a therapeutic window for solid tumors. Intriguingly, we find that loss of the catalytic activity of Usp28 is more efficient in promoting oncoprotein degradation than the complete gene knockout. Therefore, we speculate that inhibition of the catalytic function of Usp28 by small molecules might be particularly effective in tumor suppression. In this project, we will pursue the following aims: 1) model small molecule inhibition of Usp28 during homeostasis and tumorigenesis via expression of catalytically inactive Usp28 in mouse models, 2) elucidate the structural basis of deubiquitination by Usp28 and design small molecule inhibitors targeting Usp28, and 3) identify signaling events that promote oncoprotein degradation via the Usp28/Fbw7 pathway.