Epigenetic therapy with 3-deazaneplanocin A and gemcitabine for pancreatic cancer

描述(申请人提供)：尽管吉西他滨对早期胰腺肿瘤具有良好的细胞毒性，但由于肿瘤细胞的侵袭性生长特性和抗凋亡能力，对晚期肿瘤实际上无效。文献证据表明，表观遗传改变(如组蛋白和DNA高甲基化)可能在沉默肿瘤抑制基因方面发挥重要作用，使肿瘤细胞克隆性扩张并抵抗细胞毒作用。我们的长期目标是改善胰腺癌的化疗管理。R03拨款申请的总体目标是测试表观遗传沉默的肿瘤抑制基因的重新激活可以增强胰腺癌的化疗敏感性这一概念。具体地说，我们的中心假设是，基于机制的新型组蛋白去甲基化药物3‘去氮杂环素-A(DZnep)与吉西他滨联合使用将在胰腺癌细胞中产生更好的细胞毒反应。这一假说是基于我们的初步数据，这些数据表明DZnep对培养的胰腺癌细胞的吉西他滨化疗增敏作用具有运输依赖性。这项拟议研究的基本原理是，合成去甲基化药物改善吉西他滨化疗敏感性的证据将为继续研究胰腺癌表观遗传治疗的可能性提供实验基础。核心假设将通过追求三个具体目标来检验。特异性靶点1将确定DZnep在胰腺癌中的细胞转运和激活机制。目标1的工作假设是，亲水性DZnep的去甲基化和化学增敏能力将受到载体介导的运输过程的速率限制。这一假说是基于我们的初步数据，这些数据表明，当核苷转运活性被抑制时，DZnep诱导的反应显著减少。特效目标2将在体外产生和测试一组DZnep酰基衍生物。Aim 2的工作假设是，N4取代的脂肪酸酰胺衍生物将增加DZnep的亲脂性，使其绕过细胞内激活所需的运输。我们的假设是基于我们的初步研究表明，另一种核苷(曲沙他滨)的酰基前药绕过了运输要求。具体目标3将确定DZnep前药-吉西他滨联合的体内抗癌效果。我们的工作假设是，与吉西他滨单独治疗相比，DZnep前药与吉西他滨联合治疗携带胰腺癌异种移植瘤的小鼠将显示出更好的化疗效果。这一假设是基于在细胞培养中使用DZnep和吉西他滨联合方案所获得的初步体外结果的推断。这些研究的贡献将是巨大的，因为成功地证明了一种优越的表观遗传-化疗方案可能会取代效果较差的吉西他滨单一疗法。这项拟议的工作具有创新性，因为它首次解决了胰腺癌基于核苷的表观遗传-化疗疗法的可能性。 与公共健康相关：这项拟议的研究与公共健康相关，因为胰腺癌在美国是一个可怕的问题，估计每年约有3.5万人死亡。由于吉西他滨单一疗法不是很有效，表观遗传剂与标准化疗护理相结合将成为提高胰腺癌患者存活率所必需的一种更好的治疗方案。

DESCRIPTION (provided by applicant): Despite superior cytotoxicity against early-stage pancreatic tumors, gemcitabine is practically ineffective in late-stage tumors due to the invasive growth characteristics and apoptotic resistance of tumor cells. Evidence in literature suggest that epigenetic alterations (e.g. histone and DNA hypermethylation) may play vital roles in silencing tumor suppressor genes, allowing tumor cells to clonally expand and resist cytotoxicity. Our long-term goal is to improve the chemotherapeutic management of pancreatic cancer. The overall objective of this R03 grant application is to test the concept that the reactivation of epigenetically silenced tumor suppressor genes can augment chemosensitization in pancreatic cancer. Specifically, it is our central hypothesis that a mechanism-based structure optimization of a novel histone-demethylation agent, 3'deazaneplanocin-A (DZnep), in combination with gemcitabine will enact a superior cytotoxic response in pancreatic cancer cells. This hypothesis is based on our preliminary data that show transport-dependent augmentation of gemcitabine chemosensitization by DZnep in cultured pancreatic cancer cells. The rationale underlying the proposed research is that proof of synthetic demethylating agents improving gemcitabine chemosensitivity will provide an experimental basis to continue investigating the potential of epigenetic therapy for pancreatic cancer. The central hypothesis will be tested by pursuing three specific aims. Specific aim 1 will determine the cellular transport and activation mechanisms of DZnep in pancreatic cancer. The working hypothesis for aim 1 is that the demethylating and chemosensitizing abilities of hydrophilic DZnep will be rate-limited by a carrier-mediated transport process. This hypothesis is based on our preliminary data that show a significant reduction in DZnep-induced responses when nucleoside transport activity was inhibited. Specific aim 2 will generate and test a battery of DZnep acyl derivatives in vitro. The working hypothesis for aim 2 is that N4-substituted fatty acid amide derivatives will increase the lipophilicity of DZnep and allow it to bypass the transport requirement for intracellular activation. Our hypothesis is based on our preliminary studies showing the acyl prodrugs of another nucleoside (troxacitabine) bypassing the transport requirement. Specific aim 3 will determine the in vivo anti-cancer efficacies of DZnep prodrug-gemcitabine combinations. Our working hypothesis is that the combined treatment of a DZnep prodrug with gemcitabine in mice carrying pancreatic cancer xenografts will exhibit superior chemotherapeutic efficacy compared with gemcitabine treatment alone. This hypothesis is based on the extrapolation of preliminary in vitro results obtained with DZnep and gemcitabine combination schedules in cell culture. The contribution from these studies will be significant because successful demonstration of a superior epigenetic-chemotherapeutic regimen is likely to replace the less efficacious gemcitabine monotherapy practice. The proposed work is innovative because it addresses for the first time the possibility of a nucleoside-based epigenetic-chemotherapeutic therapy in pancreatic cancer. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because pancreatic cancer is a dire problem in the US with an estimated death of about 35,000 people annually. Since gemcitabine monotherapy is not very effective, epigenetic agents in combination with standard chemotherapeutic care will act as a superior treatment regimen necessary for improving survival in pancreatic cancer patients.