Novel CTD inhibitors with synthetic lethality to oncogenic Ras for cancer therapy

描述(由申请人提供)：致癌的RAS蛋白在癌症表型的发展和维持中起关键作用，并作为抗癌治疗的重要靶点。然而，致癌的RAS靶向治疗药物尚未问世。因此，开发能有效消除RAS突变癌细胞的抗癌药物迫在眉睫。我们推测，在表达致癌基因RAS基因的癌细胞中诱导合成杀伤力而不是在正常同基因细胞中诱导合成杀伤力的药物将是开发RAS靶向抗癌疗法的有价值的原型。在寻找这类药物的过程中，我们筛选了一个化学库，并确定了一种化合物(指定为oncrasin-1)，它可以杀死表达致癌基因K-RAS的永生化和致癌的人卵巢上皮细胞，但不能杀死它们的同基因正常细胞。Oncrasin-1可有效杀伤多种K-RAS突变的肺癌细胞。这些细胞毒作用与化合物诱导细胞凋亡有关，并可被K-RAS siRNA或PKCiota(PKCiota)siRNA阻断，提示RAS和/或PKCiota活性是肿瘤抑素诱导细胞凋亡所必需的。用oncrasin-1、-60或-231处理敏感的癌细胞后，RNA聚合酶II最大亚单位的C末端结构域(CTD)的磷酸化受到抑制，这与先前的报道一致，即RNA聚合酶II的持续活性是防止癌基因诱导转化细胞凋亡所必需的，并且随着酵母中RAS活性水平的提高，损害CTD功能的突变是合成致命的。Oncrasin-1处理还导致PKCiota和剪接因子在巨倍体中的共聚集，并破坏了PKCiota与CDK9/Cyclin T1复合体之间的相互作用，从而使CTD磷酸化。因此，我们推测，oncrasin化合物是一类具有选择性抗癌活性的新型CTD抑制剂。体内应用oncrasin-1可抑制人肺癌移植瘤生长70%，延长荷瘤裸鼠的存活时间，且不会引起明显的毒性反应。其中一些类似物在NCI-60细胞系上的测试表明，oncrasin对来自肺癌、结肠癌、乳腺癌、卵巢癌和肾癌的几种细胞株具有活性，oncrasin-60不属于充分研究的抗肿瘤药物类别，表明其新的抗癌机制。因此，oncrasin化合物可能是一类新型的CTD抑制剂，具有增强RAS/PKCiota活性的合成致死癌症的作用。然而，在临床评估之前，有必要对Oncrasin化合物的药代动力学、抗肿瘤疗效和安全性进行体内评估。本研究的目的是通过评估具有或不具有RAS基因突变的几种肺癌细胞株的体外活性、药代动力学、体内活性和毒性，来确定最具活性的类似物的抗肺癌活性。拟议的研究将为可能的oncrasin化合物的临床评估提供坚实的临床前数据，并可能导致对肺癌治疗有用的新治疗剂的开发。 与公共卫生相关：我们已经确定了一组新的药物，它们对RAS活性增加的癌细胞具有选择性毒性，但对正常细胞没有影响。分子表征表明，这些化合物抑制了RNA聚合酶II最大亚单位C-末端结构域的磷酸化和功能。本研究的目的是在体外和体内对它们在肺癌细胞中的抗肿瘤活性进行临床前研究，这是未来临床试验所需的。

DESCRIPTION (provided by applicant): The oncogenic Ras proteins play critical roles in the development and maintenance of cancer phenotypes and serve as important targets for anticancer treatment. However, oncogenic Ras-targeted therapeutic agents are not yet available. Therefore, it is urgent to develop anticancer agents that can effectively eliminate Ras-mutant cancer cells. We hypothesize that agents that induce synthetic lethality in cancer cells expressing oncogenic Ras genes but not in normal isogenic cells will be valuable prototypes for developing Ras-targeted anticancer therapeutics. In searching for such agents, we screened a chemical library and identified a compound (designated oncrasin-1) that kills immortalized and tumorigenic human ovarian epithelial cells expressing oncogenic K-Ras but not their isogenic normal counterparts. Oncrasin-1 can effectively kill various lung cancer cells with K-Ras mutations. The cytotoxic effects correlated with apoptosis induction by the compounds and could be blocked by K-Ras siRNA or protein kinase C iota (PKCiota) siRNA, suggesting that Ras and/or PKCiota activities are required for oncrasin-induced apoptosis. Treatment of sensitive cancer cells with oncrasin-1, -60, or -231 led to suppression of the phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II, whish is consistent with previous reports that the continuous activity of RNA polymerase II is required to prevent oncogene-induced apoptosis in transformed cells and that mutations compromising CTD function is synthetically lethal with elevated levels of Ras activity in yeast. Treatment with oncrasin-1 also led to co-aggregation of PKCiota and splicing factors in megaspliceosomes and to disruption of the interaction between PKCiota and CDK9/cyclin T1 complex, which phosphorylates the CTD. Thus, we hypothesized that oncrasin compounds are a novel class of CTD inhibitors with selective anticancer activity. The in vivo administration of oncrasin-1 suppressed the growth of human lung tumor xenografts by >70% and prolonged the survival of tumor-bearing nude mice without causing detectable toxicity. Testing some of those analogues on NCI-60 cell lines showed that oncrasins are active against several cell lines derived from lung, colon, breast, ovary, and kidney cancers and oncrasin-60 lies outside the category of adequately studied classes of antitumor agents, indicating its novel anticancer mechanisms. Thus, oncrasin compounds are potentially a novel class of CTD inhibitors that are synthetically lethal to cancers with increased Ras/PKCiota activity. However, in vivo evaluation of the pharmacokinetics, antitumor efficacy, and safety of oncrasin compounds is necessary before they can be evaluated clinically. The goal of this proposal is to determine anti- lung cancer activity of the most active analogues by evaluating their in vitro activities in several lung cancer cell lines with or without Ras gene mutations, their pharmacokinetics, and their in vivo activity and toxicity. The proposed studies will provide solid preclinical data for possible clinical evaluation of oncrasin compounds and may lead to the development of new therapeutic agents that are useful for the treatment of lung cancers. PUBLIC HEALTH RELEVANCE: We have identified a group of new agents that are selectively toxic to cancer cells with increased Ras activity but not to normal cells. The molecular characterization revealed those compounds inhibit the phosphorylation and function of C-terminal domain of the largest subunit of RNA polymerase II. The goal of this proposal is to perform pre-clinical studies on their antitumor activities in lung cancer cells in vitro and in vivo that are required for future clinical trials.