RAS相关肿瘤靶点鉴定及其化学干预

RAS proteins are small GTPases that act as molecular switches, transducing signals from many activated receptors that regulate cell proliferation, survival, and differentiation. Hyperactivation of RAS is common in human cancers. Since the enzymatic activity of RAS is used to turn itself off and is inactive in oncogenic RAS, RAS proteins are considered to be "non-targetable" for developing cancer therapies. Identification of alternative targets that block RAS signaling is critical to develop therapies for RAS-related cancer. . The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Protein palmitoylation regulates the membrane targeting, subcellular trafficking, and functions of proteins. We have previously examined the importance of PTMs in NRAS leukemogenesis and found for the first time that palmitoylation is essential for NRAS leukemogenesis. These studies suggest that targeting RAS palmitoylation may be an effective therapy for cancers involving N- and HRAS, which rely on palmitoylation for plasma membrane binding. In addition, although KRAS4B is the more abundant splice variant and does not undergo palmitoylation, a recent study showed that KRAS4A, which relies on palmitoylation for plasma membrane localization, plays an essential role in the development of KRAS-driven lung cancer. This study suggests that targeting palmitoylation may impact on cancers with KRAS mutations as well.. Therapeutic intervention of RAS palmitoylation requires targeting enzymes that mediate RAS palmitoylation. The reaction of RAS palmitoylation is catalyzed by palmitoyl acyltransferases (PATs). Thus far, 24 mammallian PATs, each exhibiting a high degree of enzyme-substrate specificity, have been identified. Yet the physiological role of PATS and their role in tumorigenesis are largely unknown. We hypothesize that RAS-PATs play an important role in RAS transformation and can serve as therapeutic targets for RAS-related cancers. In this proposal, we will examine the role of RAS-PATs in normal physiology and tumorigenesis by using gene knockout and RNAi techenologies; such studies would help to reveal the potential of RAS-PATs as targets for cancer therapies. Like most enzymes, RAS-PATs are likely to have multiple substrates. We will systematically identify substrates of RAS-PATs using proteomic approaches. These studies would help to develop high efficacy and low toxicity therapies. In addition, we have screened a focused library and identified several novel anti-RAS (AR) transformation compounds. We will study the mechanism of these AR compounds in killing RAS transformed cells and identify chemical inhibitors for RAS palmitoylation. These studies would lead to novel therapies, as well as provide tools to investigate the role of palmitoylation in cellular processes.

RAS蛋白是具有分子开关作用的小分子GTP酶，在调控细胞的生长、分化、凋亡等重要生命活动中起信号传导作用,RAS异常激活与恶性肿瘤的发生发展密切相关。我们发现RAS棕榈酰化对于其诱导白血病发生是必需的，揭示针对RAS棕榈酰化修饰的靶向干预可能在相关肿瘤治疗方面有效。本课题应用基因敲除及RNA干扰技术研究RAS棕榈酰转移酶（PATs) 的正常生理功能及其在肿瘤发生过程中的作用，揭示RAS-PATs作为疾病治疗靶点的潜能，预测靶向蛋白质棕榈酰化修饰治疗对机体的作用；系统性鉴定RAS-PATs底物，为靶向药物研发策略提供依据；前期研究中，我们还发现了能够选择性杀伤RAS转化细胞的一批具有不同化学核心结构的新小分子化合物。本课题将进一步研究这些小分子化合物对蛋白质棕榈酰化修饰的作用，探索其成为攻克RAS相关肿瘤的可能，为肿瘤的防治提供新的药物作用靶点和新的先导结构，从而为创新药物的发现奠定基础。

RAS异常激活与恶性肿瘤的发生发展密切相关。由于RAS蛋白本身难以成为抗肿瘤治疗的理想“靶点”，寻找其他可阻抑RAS信号通路的靶点对于RAS相关肿瘤的治疗至关重要。RAS蛋白只有通过翻译后修饰才能正确锚定在细胞膜内侧并发挥其生物学功能，因此抑制RAS蛋白的翻译后修饰可能是阻抑RAS致癌通路的有效方法之一。.我们通过基因敲除的方法揭示DHHC9是RAS棕榈酰转移酶之一；本研究首次在体揭示棕榈酰转移酶在肿瘤发生发展中的作用，提示zDHHC9并不是催化NRAS的唯一棕榈酰转移酶，但在NRAS诱导的白血病发病过程中是起重要作用的，可作为抗RAS肿瘤的靶点，为研发有效抗白血病及其他肿瘤的靶向小分子新药打下理论基础。.KRAS4A在肿瘤发生发展过程中发挥重要的作用，在白血病细胞中也表达。我们首次证明了KRAS4A的白血病转化能力与NRAS和KRAS4B相当，证实棕榈酰化修饰是NRAS白血病发病过程的必要条件；而对于KRAS4A引起的白血病发病过程，棕榈酰化修饰也非常重要，但不是必要条件。通过序列对比和功能分析，我们发现了KRAS4A的KIKK序列作为一种细胞膜靶向模体，在这种差异中发挥了主要作用。.我们成功建立RAS靶向细胞毒性的药物筛选方法，筛选并获得了一系列具有抗RAS肿瘤活性的具有不同化学核心结构的小分子化合物。由于RAS蛋白的生物活性依赖于脂修饰，且RAS调节肿瘤细胞中的脂类代谢，我们筛选了一个活性脂分子化合物库并发现内源性大麻素／内源性辣椒素N-花生四烯酸多巴胺（N-arachidonoyl dopamine , NADA）可以有效杀死RAS转化的细胞。在生物活性－结构分析中，进一步证明NADA的抗RAS活性是结构特异性的，且与它本身的激活大麻素受体和辣椒素受体的生理活性无关。