TDG as a novel target to enhance gemcitabine killing of pancreatic cancer cells

 描述(申请人提供)：胰腺癌(PCA)是美国第四大致死性癌症，平均预期寿命不到一年。核苷类似物吉西他滨(Gem)通常用作一线治疗，但与其他单一药物或联合治疗相比，只能将患者的生存时间延长几个月。Gem+NaB-紫杉醇的组合最近被证明比单独使用Gem可延长生存期约2个月。FOLFIRINOX方案是一种多种药物的配方，已经显示出希望将PCA患者的存活率提高一倍(~4-6个月)，而不是仅使用GEM；然而，其高毒性可能会限制能够耐受该方案的患者。了解和靶向化疗耐药的分子机制是改善治疗结果的合理途径。抑制这种途径应该会改善药物反应，从而延长患者的生存时间。重要的是，化学增敏剂可以减少化疗的有效剂量，从而限制药物毒性。癌细胞严重依赖生存途径，使它们能够耐受一系列外在和内在压力，这些压力通常会阻止细胞生长。Gem、紫杉醇和FOLFIRINOX等药物可以抑制DNA复制和染色体分离，这两个过程在癌细胞中已经不稳定。癌细胞进化为耐受基因组不稳定的生存途径也是使它们能够在药物治疗中存活的原因。这些途径包括那些处于DNA修复和细胞周期检查点的途径，因为它们保护癌症基因组不会积累有害突变或诱导细胞死亡。然而，表观基因组在促进癌细胞在内在和外在压力下存活的作用尚不清楚；然而，DNA甲基化模式的改变可以对全球基因表达产生深远的影响，这可能使肿瘤细胞对细胞压力做出快速反应。我们最近报道胸腺嘧啶脱氧核糖基酶(TDG)是一种T：G错配修复酶，也是胞嘧啶去甲基化的主要酶。因此，TDG直接参与表观基因组的DNA修复和维持。事实上，肿瘤细胞中TDG的敲除会导致S期停滞，5-羧基胞嘧啶(胞嘧啶去甲基化的中间产物)的积累，以及TDG缺失的小鼠胚胎成纤维细胞对Gem高度敏感。因此，靶向TDG应该同时阻止癌细胞用来存活Gem的DNA修复和表观基因组途径。为了实现这一目标，我们已经确定了一系列FDA批准的化合物，在体外可以有效地抑制TDG。在这个R21的应用中，我们建议通过使用患者来源的PCA细胞和异种移植来验证TDG作为药物靶点与Gem的结合。这是一个令人兴奋的R21提议，因为它通过双重靶向DNA修复和表观基因组途径而具有很高的翻译潜力。

 DESCRIPTION (provided by applicant): Pancreatic cancer (PCa) is the 4th deadliest cancer in the US, with median life expectancy of less than a year. The nucleoside analog Gemcitabine (Gem) is commonly used as frontline treatment, but only extends patient survival by several months over other single agent or combination therapies. The combination of Gem + nab- paclitaxel was recently shown to extend survival by ~2 months over Gem alone. The FOLFIRINOX regimen is a formulation of multiple drugs that has shown promise by doubling (~4-6 months) survival of PCa patients over Gem alone; however, its high toxicity may limit the patients that can tolerate this regimen. Understanding and targeting the molecular mechanism responsible for chemoresistance is a rational approach to improve treatment outcomes. Inhibiting such pathways should improve drug response, and thus extend patient survival. Importantly, chemosensitizers may reduce the effective dose of chemotherapy and thus limit drug toxicity. Cancer cells rely critically on survival pathways that allow them to tolerate a broa range of extrinsic and intrinsic stresses that would normally block cell growth. Drugs such as Gem, paclitaxel, and FOLFIRINOX, inhibit DNA replication and chromosome segregation, two processes that are already destabilized in cancer cells. The survival pathways that cancer cells have evolved to tolerate genome instability are also responsible for allowing them to survive drug treatment. These pathways include those in DNA repair and cell cycle checkpoints, as they protect the cancer genome from accumulating deleterious mutations or inducing cell death. Less understood is the role of the epigenome in promoting survival of cancer cells to intrinsic and extrinsic stress; however alterations of DNA methylation patterns can have profound effects on global gene expression that may allow the tumor cell to rapidly respond to cellular stresses. We recently reported that Thymine DNA Glycosylase (TDG), a T:G mismatch repair enzyme, is also a major enzyme for active cytosine demethylation. TDG therefore is directly involved in DNA repair and maintenance of the epigenome. Indeed knockdown of TDG in tumor cells leads to S phase arrest, and accumulation of 5- carboxylcytosine (an intermediate in cytosine demethylation), and TDG-null mouse embryo fibroblasts are hypersensitive to Gem. Targeting TDG should therefore block both DNA repair and epigenome pathways that cancer cells use to survive Gem. Towards this goal, we have identified a collection of FDA-approved compounds that potently inhibit TDG in vitro. We propose in this R21 application to validate TDG as a drug target in combination with Gem, by using patient-derived PCa cells and xenografts. This is an exciting R21 proposal because of its high translational potential by dually targeting DNA repair and epigenome pathways.