Targeting the Epigenome for Lung Cancer Therapy

针对肺癌治疗的表观基因组

• 批准号: 8763700
• 负责人: DAVID SCHRUMP
• 金额: $ 54.47万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763700
• 关键词: Aberrant DNA Methylation; Acute Erythroblastic Leukemia; Adjuvant; Adoptive Transfer; Aftercare; Aliquot; Allogenic; Antigens; Apoptosis; Aspirate substance; Attenuated; Award; Back; Biological Assay; Biopsy; CTAG1 gene; Cancer Patient; Cancer Vaccines; Cell Cycle Progression; Cell Line; Cell Proliferation; Cells; Chest; Chromatin Structure; Chromosome Mapping; Clinical; Clinical Protocols; Continuous Infusion; Cyclophosphamide; DNA; Data; Decitabine; Depsipeptides; Derivation procedure; Disease; Dose; Drug Kinetics; Endogenous Retroviruses; Epithelium; Erythema; Evaluable Disease; Evaluation; Exanthema; Excision; Exhibits; Freezing; Funding; Gene Expression; Genes; Genomic Instability; Germ Cells; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Growth; H1299; Histologic; Histology; Histone Deacetylase Inhibitor; Histones; Immune response; Immunocompetent; Immunologics; Infusion procedures; Injection of therapeutic agent; Institutes; K-562; K562 Cells; Laboratories; Lasers; Liquid substance; Lung; MAGEA3 gene; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of esophagus; Malignant neoplasm of lung; Malignant neoplasm of testis; Mediastinal; Mediating; Memorial Sloan-Kettering Cancer Center; Mesothelioma; Metastatic to; Modeling; Modification; Molecular; Molecular Profiling; Mus; Non-Small-Cell Lung Carcinoma; Oncogenes; Oral; Patients; Pattern; Peritoneal Fluid; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Phase II Clinical Trials; Pleural; Polycomb; Positioning Attribute; Primary Neoplasm; Process; Proteins; Publishing; RNA; Recombinants; Regulatory T-Lymphocyte; Relative (related person); Repression; Resected; Series; Serological; Serum; Signal Transduction; Site; Source; Specimen; Staging; Structure of respiratory epithelium; T-Lymphocyte; Techniques; Thoracic Neoplasms; Thoracic Oncology; Thymoma; Time; Tissues; Tobacco; Toxic effect; Transport Process; Tumor Suppressor Genes; Tumor Tissue; Universities; Up-Regulation; Vaccination; Vaccine Adjuvant; Vaccines; X Chromosome; bench to bedside; bronchial epithelium; cancer cell; cancer immunotherapy; cancer therapy; celecoxib; cell bank; cell growth; chemotherapy; chromatin remodeling; cohort; cytokine; demethylation; epigenome; established cell line; flavopiridol; functional group; gene induction; genome-wide; immunogenic; immunogenicity; imprint; in vivo; neoplastic cell; novel; nucleic acid metabolism; pre-clinical; preclinical study; research study; response; senescence; therapeutic vaccine; treatment duration; tumor

During the past decade, a series of clinical protocols were initiated to determine toxicities and clinical responses in thoracic oncology patients receiving DNA demethylating agents, and HDAC inhibitors. In a phase I trial, 35 patients received escalating doses of DAC administered via continuous infusion on days 1 through 3 of a 35 day cycle. Although no objective responses were observed, two lung cancer patients exhibited prolonged stabilization of disease (> 1 yr). Nearly one quarter of all patients exhibited increased expression of p16, MAGE-3, or NY-ESO-1 in tumor tissues. Serologic responses to NY-ESO-1 were observed in several patients receiving DAC for more than six months. In a subsequent phase II trial, 19 lung cancer patients were treated with DP at the MTD administered as a 4h infusion on days 1 and 7 of a 21 day cycle. Intratumoral levels of H3Ac and p21 were increased in approximately 50% of patients following DP therapy. In addition, several patients exhibited enhanced expression of NY-ESO-1 and MAGE-A3 in tumor biopsies following DP infusions. In a more recent phase I trial, 31 patients with thoracic neoplasms (including 20 lung cancer, 4 esophageal cancer and 3 MPM patients) received sequential DAC/DP infusions. DAC was administered at the MTD (75mg/m2) as a continuous 72h infusion commencing on day 1 of a 35 day treatment cycle. DP was administered as a 4h infusion immediately following DAC, and on day 10; the dose of DP was increased in sequential cohorts to the previously established MTD (18mg/m2). Median steady state DAC and DP levels were 13.4 ng/ml (range: 4.7-78 ng/ml; 5-80nM), and 323 ng/ml (range: 136-923 ng/ml), respectively. Whereas no objective tumor regressions were observed, several patients exhibited stabilization of disease lasting 4-12 months. Micro-array techniques were used to comprehensively examine gene expression profiles in RNA amplified from laser-captured tumor cells from pre- and post treatment biopsies from eight lung cancer patients; results of these arrays were compared to data derived from analysis of laser-captured tumor cells and adjacent, histologically normal bronchial epithelia from 20 patients undergoing definitive lung cancer resections. Approximately 1230 genes were significantly repressed, whereas 500 genes were up-regulated in lung cancer cells following DAC/DP exposure. These findings were consistent with additional array data demonstrating repression of 1002 genes and up-regulation of 383 genes in cultured lung cancer cells treated with DAC/DP under conditions mimicking exposures in clinical settings. Genes that were up-regulated by DAC/DP were repressed in lung cancers relative to adjacent nomal lung epithelia, whereas genes that were repressed by DAC/DP were up-regulated in lung cancers compared to normal lung epithelia. Enriched functional groups modulated by DAC/DP in vivo included cell signaling, molecular transport, nucleic acid metabolism, cell growth and proliferation, and cancer. In an ongoing phase I dose-escalation trial, 24 patients with thoracic malignancies including 7 lung cancer, 4 esophageal cancer, 3 MPM, and 2 thymoma patients, have received sequential 4h DP/72h Flavopiridol infusions under exposure conditions corresponding to those used in our preclinical experiments. Whereas no objective responses have been observed, seven patients have exhibited disease stabilization lasting from 4 to >12 months. Despite these encouraging results, this trial will close soon due to poor accrual. Analyses of pharmacokinetics and molecular endpoints for this trial have not been performed as yet. A series of laboratory experiments were conducted to examine the potential feasibility of using epigenetically modified tumor cells for cancer vaccines. Briefly, tissues/fluids from patients with thoracic malignancies of various histologies were processed for primary culture. Sources of tissue included pleural and peritoneal fluid, pleural as well as mediastinal biopsies via endoscopic techniques, CT-guided FNAs, and resected primary specimens. Cell lines were established from 10 of 38 patients (26%) including 3 of 14 NSCLC, 1 of 2 SCLC, 2 of 6 esophageal cancer, and 4 of 6 mesothelioma patients. Average time from initial culture of tumor digests/aspirate to cell lines, including frozen aliquot back-up and >1x107 cells ready for drug manipulation, was approximately 2-3 months. Several of these cancer lines were treated with DAC (1-3mcM x 6 days) with or without DP (10-25ng/ml x 6-24h). qRT-PCR analysis revealed heterogeneous CT-X gene expression in untreated cell lines; patterns and magnitude of gene induction varied considerably in these lines following drug treatment. Cytokine release assays using allogeneic PBL expressing recombinant TCRs for NY-ESO-1 and MAGE-A3 confirmed HLA-restricted recognition of tumor targets treated with DAC or sequential DAC/DP. DP did not consistently enhance DAC-mediated gene induction or CTL recognition under these exposure conditions. The aforementioned studies provided the preclinical rationale for evaluation of epigenetically-modified tumor cells as adjuvant vaccines in patients with primary thoracic malignancies, as well as patients with extra-thoracic malignancies metastatic to the chest, who are rendered NED by standard therapy. The vaccines are to be administered with Iscomatrix, a proprietary adjuvant that has been shown to elicit potent immunologic responses to purified CTA vaccines; additionally, oral celecoxib will be used to inhibit activity of Tregs that have been shown to attenuate immune responses to therapeutic vaccines in cancer patients. To date, 25 patients have been accrued to these trials. Despite preclinical studies demonstrating feasibility of this approach, reliable derivation of cell lines from primary tumors has been a significant challenge, in part due the histologies of the resected malignancies. In additional studies, we have identified several established cell lines including K562 erythroleukemia, and H1299 lung cancer cells that exhibit high level CT-X gene expression without pharmacologic manipulation, which we have sought to evaluate as adjuvant vaccines. To date, 15 patients have been accrued to trials involving administration of K562 cells constitutively expressing GM-CSF (K562-GM) in conjunction with oral metronomic cyclophosphamide and celecoxib to inhibit Treg activity. These cells were expanded from a master cell bank at Johns Hopkins University using funds from a previous Bench-to-Bedside Award. The vaccines are administered subcutaneously q 28days x 6, followed by evaluation of immune response one month later. Standard staging studies are obtained at baseline, prior to the fourth vaccine, and during treatment evaluation. Virtually all patients have tolerated metronomic chemotherapy, and there have been no vaccine-related toxicities. Sera from all evaluable patients will be comprehensively assessed at the Ludwig Cancer Institute at MSKCC using ELISAs capable of detecting responses to more than 30 CTAs. Eight patients have developed erythema and rash at their vaccination sites, suggesting response to some component of the K562-GM injections.

在过去的十年中，启动了一系列临床方案来确定接受 DNA 去甲基化药物和 HDAC 抑制剂的胸部肿瘤患者的毒性和临床反应。在一项 I 期试验中，35 名患者在 35 天周期的第 1 天至第 3 天通过连续输注接受递增剂量的 DAC。尽管没有观察到客观反应，但两名肺癌患者表现出疾病长期稳定（> 1 年）。近四分之一的患者肿瘤组织中 p16、MAGE-3 或 NY-ESO-1 的表达增加。在接受 DAC 六个月以上的几名患者中观察到对 NY-ESO-1 的血清学反应。在随后的 II 期试验中，19 名肺癌患者按照 MTD 接受 DP 治疗，在 21 天周期的第 1 天和第 7 天输注 4 小时。 DP 治疗后，约 50% 的患者瘤内 H3Ac 和 p21 水平升高。此外，一些患者在 DP 输注后的肿瘤活检中表现出 NY-ESO-1 和 MAGE-A3 表达增强。在最近的一项 I 期试验中，31 名胸部肿瘤患者（包括 20 名肺癌患者、4 名食管癌患者和 3 名 MPM 患者）接受了序贯 DAC/DP 输注。 DAC 以 MTD（75mg/m2）作为连续 72 小时输注，从 35 天治疗周期的第一天开始。 DP 在 DAC 后立即输注 4 小时，并在第 10 天进行；连续队列中 DP 的剂量增加至先前建立的 MTD（18mg/m2）。中位稳态 DAC 和 DP 水平分别为 13.4 ng/ml（范围：4.7-78 ng/ml；5-80nM）和 323 ng/ml（范围：136-923 ng/ml）。虽然没有观察到客观的肿瘤消退，但一些患者表现出疾病持续 4-12 个月的稳定。使用微阵列技术全面检查 RNA 中的基因表达谱，这些 RNA 是从 8 名肺癌患者的治疗前和治疗后活组织检查中激光捕获的肿瘤细胞中扩增出来的；这些阵列的结果与来自 20 名接受肺癌根治性切除术的患者的激光捕获肿瘤细胞和邻近组织学正常支气管上皮的分析数据进行了比较。接触 DAC/DP 后，肺癌细胞中大约 1230 个基因被显着抑制，而 500 个基因被上调。这些发现与其他阵列数据一致，这些数据表明在模拟临床环境暴露的条件下，用 DAC/DP 处理的培养肺癌细胞中 1002 个基因被抑制，383 个基因被上调。与邻近的正常肺上皮相比，在肺癌中被DAC/DP上调的基因受到抑制，而与正常肺上皮相比，在肺癌中被DAC/DP抑制的基因被上调。体内 DAC/DP 调节的丰富功能组包括细胞信号传导、分子运输、核酸代谢、细胞生长和增殖以及癌症。在一项正在进行的 I 期剂量递增试验中，24 名胸部恶性肿瘤患者，包括 7 名肺癌患者、4 名食管癌患者、3 名 MPM 患者和 2 名胸腺瘤患者，在与我们所用暴露条件相对应的暴露条件下接受了连续 4 小时 DP/72 小时 Flavopiridol 输注。临床前实验。尽管尚未观察到客观反应，但 7 名患者的疾病已表现出持续 4 至 12 个月以上的稳定状态。尽管取得了这些令人鼓舞的结果，但由于应计效果不佳，该试验将很快结束。该试验的药代动力学和分子终点分析尚未进行。进行了一系列实验室实验，以检验使用表观遗传修饰的肿瘤细胞用于癌症疫苗的潜在可行性。简而言之，对来自不同组织学的胸部恶性肿瘤患者的组织/液体进行原代培养。组织来源包括胸膜和腹膜液、通过内窥镜技术进行的胸膜和纵隔活检、CT 引导下的 FNA 和切除的原发标本。细胞系是从 38 名患者中的 10 名 (26%) 建立的，其中包括 14 名 NSCLC 患者中的 3 名、2 名 SCLC 患者中的 1 名、6 名食管癌患者中的 2 名和 6 名间皮瘤患者中的 4 名。从肿瘤消化物/抽吸物的初始培养到细胞系（包括冷冻等分备用和准备用于药物操作的 >1x107 个细胞）的平均时间约为 2-3 个月。其中一些癌症系用 DAC（1-3mcM x 6 天）联合或不联合 DP（10-25ng/ml x 6-24 小时）进行治疗。 qRT-PCR 分析揭示了未经处理的细胞系中 CT-X 基因表达的异质性；药物治疗后，这些品系中基因诱导的模式和程度差异很大。使用表达 NY-ESO-1 和 MAGE-A3 重组 TCR 的同种异体 PBL 进行细胞因子释放测定，证实了对 DAC 或连续 DAC/DP 治疗的肿瘤靶点的 HLA 限制性识别。在这些暴露条件下，DP 并没有始终如一地增强 DAC 介导的基因诱导或 CTL 识别。上述研究为评估表观遗传修饰肿瘤细胞作为辅助疫苗用于原发性胸部恶性肿瘤患者以及通过标准治疗导致 NED 的胸外恶性肿瘤转移至胸部的患者提供了临床前依据。这些疫苗将与 Iscomatrix 一起施用，Iscomatrix 是一种专有佐剂，已被证明可引发对纯化 CTA 疫苗的有效免疫反应；此外，口服塞来昔布将用于抑制Tregs的活性，这些Tregs已被证明可以减弱癌症患者对治疗性疫苗的免疫反应。迄今为止，已有 25 名患者参加了这些试验。尽管临床前研究证明了这种方法的可行性，但从原发性肿瘤中可靠地衍生细胞系一直是一个重大挑战，部分原因是切除的恶性肿瘤的组织学。在其他研究中，我们已经鉴定了几种已建立的细胞系，包括 K562 红白血病细胞和 H1299 肺癌细胞，它们无需药理学操作即可表现出高水平 CT-X 基因表达，我们试图将其作为佐剂疫苗进行评估。迄今为止，已有 15 名患者接受了组成型表达 GM-CSF (K562-GM) 的 K562 细胞联合口服节拍环磷酰胺和塞来昔布来抑制 Treg 活性的试验。这些细胞是利用之前的实验室到临床奖的资金从约翰·霍普金斯大学的主细胞库中扩增而来的。疫苗每 28 天 x 6 皮下注射一次，一个月后评估免疫反应。标准分期研究是在第四种疫苗接种之前和治疗评估期间的基线时获得的。几乎所有患者都能耐受节拍化疗，并且没有出现与疫苗相关的毒性。所有可评估患者的血清将在 MSKCC 路德维希癌症研究所使用能够检测对 30 多种 CTA 反应的 ELISA 进行全面评估。八名患者的疫苗接种部位出现红斑和皮疹，表明对 K562-GM 注射的某些成分有反应。