Targeting the Epigenome for the Treatment and Prevention

表观遗传学与恶性转化之间新出现的关系为染色质重塑药物在肺癌治疗中的应用提供了动力。在过去的一年里，我的研究继续集中在：1.评估DNA去甲基化药物和组蛋白脱乙酰基酶(HDAC)抑制剂介导的肺癌、食道癌和恶性胸膜间皮瘤(MPM)细胞的基因表达；2.检测染色质重建剂作用后培养的癌细胞和原发肿瘤标本中的基因表达谱；3.探讨DNA甲基转移酶(DNMT)和HDAC活性抑制物介导胸部恶性肿瘤生长停滞和凋亡的机制。此前，我们曾报道DNA去甲基化试剂5-氮-2‘-脱氧胞苷(5-aza-2’deoxcytidine，DAC)和脱氧核糖核酸酶抑制剂FK228(DP)协同诱导细胞凋亡，并显著增强NY-ESO-1肿瘤-睾丸抗原(CTA)在癌细胞中的优先表达，促进该CTA特异的细胞溶解T淋巴细胞(CTL)识别。此外，我们还观察到，在体内，以DAC诱导的CTA为靶点的过继免疫疗法可以减少同基因小鼠肿瘤模型中的肺转移。这些临床前数据为NCI外科分部胸部肿瘤科进行的几项方案提供了理论基础，试图在临床环境中总结介导培养的肺癌细胞凋亡和CTA诱导的药物暴露条件。在I期剂量-逐步递增研究中，34名患者在35天周期的第1-4天接受72小时DAC输注；在II期试验中，19名患者在21天周期的第1天和第7天以最大耐受量(MTD)给予4h DP输注。最近，24名患者参加了一项评估DAC/DP顺序治疗的I期剂量递增研究，在该研究中，DAC在第1-4天给药，DP在35天周期的第4天和第10天给药。临床毒性和治疗反应分别使用标准的CTCAE和RECIST标准进行评估。用LC-MS和高效液相色谱技术检测血浆DAC和DP水平。定量逆转录聚合酶链式反应、甲基化特异性聚合酶链式反应、免疫组织化学和酶联免疫吸附试验技术已用于评估治疗前和治疗后肿瘤活检组织和血清中的各种分子终点。微阵列技术已经被用来全面检测激光捕获的来自20名患者FNA的肿瘤细胞的基因表达谱，其中4名患者接受DAC输注，4名接受DP输注，12名接受DAC/DP序贯治疗。将这些阵列的结果与来自20名接受胸部外科手术的肺癌切除患者的激光捕获的肿瘤细胞和邻近组织学正常的支气管上皮的阵列数据进行比较。没有观察到客观的临床反应；然而，有几名患者在接受DAC、DP或顺序的DAC/DP治疗后表现出了疾病的长期稳定。在培养的肺癌细胞中，血浆DAC和DP浓度通常超过基因诱导和凋亡的阈值水平。在接受DAC或DP输注的患者中，大约40%-50%的患者在肿瘤活检组织中表现出NY-ESO-1、p16或p21的诱导表达；几个人在药物治疗后产生了针对NY-ESO-1的抗体。长寡核苷酸阵列分析显示，肺癌标本中对DAC、DP和DAC/DP的反应复杂、不同。有趣的是，DAC和DP似乎将基因表达从恶性转变为与正常支气管上皮细胞关系更密切的基因。尽管DAC和DP在体外有明显的协同作用，但在体内对顺序DAC/DP的反应是高度复杂的，可能是由于肿瘤内的药物浓度，或者基质元素对癌细胞基因表达的影响。

The emerging relationships between epigenetics and malignant transformation provide impetus for the use of chromatin remodeling agents for lung cancer therapy. During the past year my research has continued to focus on:1.Evaluation of gene expression in lung and esophageal cancer and malignant pleural mesothelioma (MPM) cells mediated by DNA demethylating agents and histone deacetylase (HDAC) inhibitors.2.Examination of gene expression profiles in cultured cancer cells and primary cancer specimens following exposure to chromatin remodeling agents.3.Investigation of the mechanisms by which inhibitors of DNA methyltransferase (DNMT) and HDAC activity mediate growth arrest and apoptosis in thoracic malignancies. Previously, we reported that the DNA demethylating agent, 5 aza-2'deoxycytidine (DAC) and the HDAC inhibitor, Depsipeptide FK228 (DP) synergistically induce apoptosis, and markedly enhance expression of the NY-ESO-1 cancer-testis antigen (CTA) preferentially in cancer cells, facilitating their recognition by cytolytic T lymphocytes (CTL) specific for this CTA. Furthermore, we have observed that adoptive immunotherapy targeting a CTA induced by DAC in vivo diminishes pulmonary metastases in a syngeneic murine tumor model. These preclinical data provided the rationale for several protocols have been conducted in the Thoracic Oncology Section, Surgery Branch, NCI in an attempt to recapitulate in clinical settings drug exposure conditions that mediate apoptosis and CTA induction in cultured lung cancer cells.In a phase I dose-escalation study, 34 patients received 72h DAC infusions administered on days 1-4 of a 35 day cycle; in a phase II trial, 19 patients received 4h DP infusions administered at the maximum tolerated dose (MTD) on days 1 and 7 of a 21 day cycle. Most recently, 24 patients have been enrolled on a phase I dose-escalation study evaluating sequential DAC/DP therapy, in which DAC is administered on days 1-4, and DP is administered on days 4 and 10 of a 35 day cycle. Clinical toxicities, and response to therapy have been assessed using standard CTCAE and RECIST criteria, respectively. Plasma DAC and DP levels have been evaluated by LC-MS and HPLC techniques. Quantitative RT-PCR, methylation-specific-PCR, immunohistochemistry, and ELISA techniques have been used to assess a variety of molecular endpoints in pre-and post-treatment tumor biopsies and sera. Micro-array techniques have been used to comprehensively examine gene expression profiles in laser-captured tumor cells from FNAs of 20 patients, including 4 individuals receiving DAC infusions, 4 receiving DP infusions, and 12 receiving sequential DAC/DP therapy. Results of these arrays have been compared to array data from laser-captured tumor cells and adjacent histologically normal bronchial epithelia from 20 patients undergoing lung cancer resections on the Thoracic Surgery Service.No objective clinical responses have been observed; however, several patients have exhibited prolonged stabilization of disease following DAC, DP, or sequential DAC/DP therapy. Plasma DAC and DP concentrations typically exceeded threshold levels for gene induction and apoptosis in cultured lung cancer cells. Approximately 40- 50% of patients receiving DAC or DP infusions exhibited induction of NY-ESO-1, p16, or p21 expression in tumor biopsies; several individuals developed antibodies against NY-ESO-1 following drug treatment. Long-oligo array analysis revealed complex, heterogeneous responses to DAC, DP, and DAC/DP in lung cancer specimens. Interestingly, DAC as well as DP appeared to shift gene expression from a malignant profile to one more closely related to normal bronchial epithelial cells. Despite the apparent synergy of DAC and DP in vitro, the response to sequential DAC/DP in vivo was highly complex, possibly due to intra-tumoral drug concentrations, or influence of stromal elements on cancer cell gene expression.