Targeting the Epigenome for the Treatment and Prevention

表观遗传学与恶性转化之间的新关系为染色质重塑剂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。DNA去甲基化剂和组蛋白去乙酰化酶（HDAC）抑制剂对肺癌、食管癌和恶性胸膜间皮瘤（MPM）细胞基因表达的影响2 .暴露于染色质重塑剂后培养癌细胞和原发癌标本中基因表达谱的检测。DNA甲基转移酶（DNMT）和HDAC活性抑制剂介导胸部恶性肿瘤生长停滞和细胞凋亡的机制研究。此前，我们报道了DNA去甲基化剂5 - aza-2'脱氧胞苷（DAC）和HDAC抑制剂沉淀肽FK228 （DP）协同诱导细胞凋亡，并显著增强癌细胞中NY-ESO-1癌睾丸抗原（CTA）的表达，促进其被特异性的细胞溶解T淋巴细胞（CTL）识别。此外，我们观察到，在体内针对DAC诱导的CTA的过继免疫治疗减少了同基因小鼠肿瘤模型中的肺转移。这些临床前数据为NCI胸肿瘤科外科分会进行的几个方案提供了基本原理，这些方案试图在临床环境中概括药物暴露条件下介导培养的肺癌细胞凋亡和CTA诱导。在一项I期剂量递增研究中，34名患者在35天周期的第1-4天接受72小时DAC输注；在一项II期试验中，19名患者在21天周期的第1天和第7天以最大耐受剂量（MTD）输注4小时DP。最近，24名患者参加了一项评估顺序DAC/DP治疗的I期剂量递增研究，其中DAC在35天周期的第1-4天施用，DP在第4天和第10天施用。临床毒性和对治疗的反应分别使用标准CTCAE和RECIST标准进行评估。采用LC-MS和HPLC技术测定血浆DAC和DP水平。定量RT-PCR、甲基化特异性pcr、免疫组织化学和ELISA技术已被用于评估治疗前后肿瘤活检和血清中的各种分子终点。采用微阵列技术全面检测了20例患者FNAs激光捕获肿瘤细胞的基因表达谱，其中4例接受DAC输注，4例接受DP输注，12例接受DAC/DP序贯治疗。这些阵列的结果与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了比较，这些数据来自20名接受胸外科手术切除的肺癌患者。未观察到客观临床反应；然而，一些患者在DAC、DP或顺序DAC/DP治疗后表现出长期的疾病稳定。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。大约40- 50%接受DAC或DP输注的患者在肿瘤活检中表现出NY-ESO-1、p16或p21的表达诱导；一些个体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揭示了肺癌标本对DAC、DP和DAC/DP的复杂、异质性反应。有趣的是，DAC和DP似乎将基因表达从恶性谱转变为与正常支气管上皮细胞更密切相关的基因表达。尽管DAC和DP在体外具有明显的协同作用，但在体内对顺序DAC/DP的反应非常复杂，可能是由于肿瘤内药物浓度或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.