DNA Repair, Cell Cycle Checkpoints and Apoptosis as Targets for Anticancer Drugs

由于大多数癌症在细胞周期检查点途径（p53, pRb）和细胞周期机制（细胞周期蛋白，细胞周期蛋白依赖性激酶抑制剂如p16）中都有改变，我们正在探索细胞周期检查点抑制剂作为新型抗癌药物。我们正在研究Chk2在癌细胞细胞周期检查点反应中的作用。我们已经将Chk2作为重组蛋白表达，并建立了高通量筛选来发现Chk2抑制剂(与dr。Shoemaker and Scudiero， DTP， NCI)。我们发现了一个新的Chk2抑制剂家族，双胍基腙，并表明它们作为竞争性ATP抑制剂对抗Chk2。合成并选择了类似物。目前正在开发细胞测定方法，以测量Chk2在细胞中的抑制作用，并确定Chk2抑制剂是否可用于与Top1抑制剂和其他现有化疗药物协同作用。DNA修复缺陷易导致癌症（例如着色性干皮病和共济失调毛细血管扩张症），并且可能在癌症对靶向DNA和染色质治疗的反应中发挥重要作用。我们建立了酪氨酸-DNA磷酸二酯酶（Tdp1）抑制剂的高通量筛选，Tdp1是碱基切除修复（BER）途径中的一种酶，参与拓扑异构酶介导的DNA损伤的修复。我们已经确定了第一个Tdp1抑制剂，我们正在寻找具有治疗潜力的新抑制剂。Tdp1抑制剂与Top1抑制剂联合应具有协同作用。我们正在研究几种临床前和早期临床开发的新药，包括nci -发育治疗项目（DTP）的药物。我们专注于改变染色质和细胞周期进程的药物，如氨基黄酮。氨基黄酮已经开始临床试验，我们发现氨基黄酮诱导复制双链断裂和组蛋白H2AX磷酸化（γ -H2AX）。因此，γ - h2ax可作为监测肿瘤样品中氨基黄酮活性的生物标志物。利用NCI 60细胞系数据库，我们发现硫转移酶的表达与氨基黄酮活性高度相关，可以用来选择应该从氨基黄酮中获益的患者。最近，我们发现氨基黄酮介导细胞角蛋白和RNA之间的交联。这项研究表明，细胞角蛋白（构成细胞微丝网络的主要组成部分）可能作为细胞质RNA的支架并可能用于翻译。我们继续对trabectedin的分子药理学进行研究，trabectedin最近在欧洲被批准用于治疗软组织肉瘤。我们之前发现trabectedin与其他临床使用的抗癌药物不同，因为它在DNA次要凹槽中的特定鸟嘌呤上形成共价加合物，并且因为它选择性地捕获转录偶联的NER （TC-NER）。我们现在发现γ - h2ax可以作为trabectedin的药效学生物标志物。γ - h2ax的激活使我们发现trabectedin捕获TC-NER可诱导Mre11和转录依赖性DNA双链断裂的形成。我们对细胞凋亡的研究主要集中在染色质修饰上。我们发现凋亡的早期事件之一是诱导凋亡的Top1-DNA复合物。凋亡的Top1-DNA复合物可由多种凋亡刺激诱导：三氧化二砷、依托泊苷、喜树碱、铂衍生物、紫杉醇和长春花素。我们的工作假设是，这些凋亡的Top1-DNA复合物是由基因组DNA的氧化损伤产生的，它将Top1束缚在染色质上。凋亡的Top1-DNA复合体反过来激活额外的凋亡反应/途径，并可能代表一个不可逆的凋亡激活环。为了进一步阐明凋亡程序诱导的分子事件，我们将重点放在临床试验中TRAIL产生的核改变上。

Because most cancers have alterations in the cell cycle checkpoint pathways (p53, pRb) and cell cycle machinery (cyclins, cyclin-dependent kinase inhibitors such as p16), we are exploring inhibitors of cell cycle checkpoints as novel anticancer agents. We are investigating the role of Chk2 in cell cycle checkpoint response in cancer cells. We have expressed Chk2 as a recombinant protein and set up a high throughput screen to discover Chk2 inhibitors (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI). We have discovered a novel family of Chk2 inhibitors, the bis-guanidylhydrazones and shown they act as competitive ATP inhibitors against Chk2. Analogs have been synthesized and selected. Cellular assays are being developed to measure Chk2 inhibition in cells and to determine whether Chk2 inhibitors can be used to synergize with Top1 inhibitors and other currently available chemotherapeutic agents. It is also well established that DNA repair defects predispose to cancers (for instance Xeroderma Pigmentosum and ataxia telangiectasia) and may play an important role in the response of cancers to treatments that target DNA and chromatin. We have set up high-throughput screens for inhibitors of tyrosyl-DNA phosphodiesterase (Tdp1), an enzyme of the base excision repair (BER) pathways involved in the repair of topoisomerase-mediated DNA damage. We have identified the first Tdp1 inhibitors, and we are searching for new inhibitors with therapeutic potential. Tdp1 inhibitors should be synergistic in combination with Top1 inhibitors. We are studying several new drugs in preclinical and early clinical development including agents from the NCI-Developmental Therapeutics Program (DTP). We are focusing on drugs that alter chromatin and cell cycle progression such as aminoflavone. Aminoflavone is beginning clinical trials and we found that aminoflavone induces replication double-strand breaks and histone H2AX phosphorylation (gamma-H2AX). Hence, gamma-H2AX can be used as a biomarker to monitor aminoflavone activity in tumor samples. Using the NCI 60 cell line database we found that sulfotransferase expression is highly correlated with aminoflavone activity and can be used to select patients who should benefit from aminoflavone. More recently, we found that aminoflavone mediates crosslinking between cytokeratins and RNA. This study suggests the possibility that cytokeratins (which constitute a major component of the cellular microfilament network) could serve as a scaffold for cytoplasmic RNA and potentially for translation. We have continued our studies on the molecular pharmacology of trabectedin, which has recently been approved for the treatment soft tissue sarcomas in Europe. We previously found that trabectedin differs from other clinically used anticancer agents because it forms covalent adducts at specific guanines in the DNA minor groove and because it selectively traps the transcription-coupled NER (TC-NER). We have now found that gamma-H2AX could serve as a pharmacodynamic biomarker for trabectedin. The activation of gamma-H2AX led us to show that the trapping of TC-NER by trabectedin induces the formation of Mre11- and transcription-dependent DNA double-strand breaks. Our studies on apoptosis are focused on chromatin modifications. We found that one of the early events in apoptosis is the induction of apoptotic Top1-DNA complexes. The apoptotic Top1-DNA complexes are induced by a variety of apoptotic stimuli: arsenic trioxide, etoposide, camptothecin, platinum derivatives, taxol, and vinblastin. Our working hypothesis that these apoptotic Top1-DNA complexes are produced by oxidative lesion of genomic DNA, which trap Top1 bound to chromatin. Apoptotic Top1-DNA complexes in turn activate additional apoptotic responses/pathways and might represent an irreversible apoptotic activation loop. To further elucidate the molecular events induced by the apoptotic program, we are focusing on nuclear alterations produced by TRAIL, which is in clinical trials.