Targeting the Epigenome for Lung Cancer Therapy

针对肺癌治疗的表观基因组

基本信息

批准号：
8350071
负责人：
DAVID SCHRUMP
金额：
$ 51.44万
依托单位：
DIVISION OF CLINICAL SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8350071
关键词：
Aberrant DNA Methylation Acute Erythroblastic Leukemia Adjuvant Adoptive Transfer Aftercare Allogenic American Antigens Apoptosis Autologous Autologous Tumor Cell Biopsy Blood Circulation CTAG1 gene Cancer Patient Cell Cycle Progression Cell Line Cells Chest Chromatin Structure Chromosome Mapping Chronic Clinical Common Terminology Criteria for Adverse Events Complex Cyclophosphamide DNA Data Decitabine Depsipeptides Disease Dose Drug Exposure Endogenous Retroviruses Enzyme-Linked Immunosorbent Assay Epigenetic Process Evaluation Excision Exhibits Gene Expression Genes Genomic Instability Germ Cells Goals Granulocyte-Macrophage Colony-Stimulating Factor Growth H1299 HLA A*0201 antigen High Pressure Liquid Chromatography Histologic Histology Histone Deacetylase Inhibitor Histone H3 Histones Immune Immune response Immunity Immunocompetent Immunohistochemistry Immunosuppressive Agents Individual Induction of Apoptosis Infusion procedures Institutional Review Boards International K-562 K562 Cells Lasers Liquid substance Malignant - descriptor Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of testis Manuscripts Mediastinal Mediating Metastatic to Methylation Modeling Modification Molecular Molecular Profiling Mus Oncogenes Oral Patients Peritoneal Fluid Pharmaceutical Preparations Plasma Pleural Polycomb Positioning Attribute Process Proteins Protocols documentation Publications Publishing Regimen Regulatory T-Lymphocyte Reporting Repression Reverse Transcriptase Polymerase Chain Reaction Schedule Secure Serum Site Source Structure of respiratory epithelium T-Cell Receptor T-Lymphocyte Techniques Testis Thoracic Neoplasms Thoracic Oncology Thoracic Surgical Procedures Tissues Tobacco Toxic effect Tumor Suppressor Genes Up-Regulation Vaccination Vaccines X Chromosome bronchial epithelium cancer cell cancer immunotherapy cancer regression cancer therapy celecoxib cell bank chromatin remodeling clinically relevant combinatorial demethylation experience flavopiridol gene induction genome-wide immunogenic immunogenicity imprint in vivo inhibitor/antagonist meetings neoplastic cell novel posters pre-clinical preclinical study research study response senescence symposium tumor

项目摘要

To date, nearly 120 patients with thoracic malignancies (mostly lung cancers) have received DAC), DP, or sequential DAC/DP infusions on protocols initiated in the Thoracic Oncology Section. Clinical toxicities and response to therapy have been assessed by 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 pre- and post treatment biopsies from 21 individuals receiving DAC, DP, or sequential DAC/DP infusions. Results of these arrays have been 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. Whereas no objective clinical responses have been observed, several patients have exhibited prolonged stabilization of disease following DAC, DP, or sequential DAC/DP infusions. Plasma DAC and DP concentrations have approximated threshold levels for gene induction and apoptosis in cultured lung cancer cells. Approximately 30% of patients receiving DAC or DP infusions have exhibited enhanced expression of NY-ESO-1, p16, p21, or acetylated histones H3 and H4 in tumor biopsies indicative of molecular response to therapy. Micro-array analyses have revealed complex, heterogeneous responses to DAC, DP, and DAC/DP in laser-captured lung cancer cells, with a shift of gene expression profiles toward those observed in histologically normal bronchial epithelia. These findings suggest that more prolonged exposures may be required to mediate cancer regressions. In additional studies, we have demonstrated that the cdk inhibitor, Flavopiridol (FLA), significantly enhances apoptosis mediated by DP, in part by abrogating HDAC-inhibitor-mediated up-regulation of p21. These findings provided the preclinical rationale for a protocol evaluating the toxicities and potential efficacy of sequential DP/FLA infusions in patients with thoracic malignancies. To date, 21 patients have received these infusions with acceptable toxicities. Although no tumor regressions have been noted as yet in this dose-escalation study, 7 patients have exhibited stabilization of disease lasting 4-12 months. These finding suggest a molecular response to therapy. The study will close after 4-6 additional patients have been treated. Collectively, this experience warrants further analysis of chromatin remodeling agents for treatment of thoracic malignancies, using schedules that enable chronic drug exposures. Although CT-X genes are coordinately de-repressed in lung cancers, immune responses to CT-X antigens are exceedingly rare in patients with these malignancies, due in part to levels of antigen expression that are below the threshold for immune recognition, as well as the presence of immunosuppressive T regulatory cells within the tumor and systemic circulation of these individuals. As such, we have sought to develop combinatorial regimens that will enhance immune responses to CT-X antigens in lung cancer patients. One potential strategy involves the use of epigenetically-modified autologous tumor cells to immunize lung cancer patients against a variety of CT-X antigens that potentially can be up-regulated in their respective primary cancers by systemic gene induction regimens. To date, no such efforts have been reported. To examine the potential feasibility of this approach, tissues/fluids from patients with thoracic tumors of various histologies were processed for primary culture. Sources of tumor cells included peritoneal fluid, endoscopic pleural or mediastinal biopsies, as well as CT-guided FNAs. To date, 26 cell lines have been established, several of which have been exposed to DAC+/-DP under exposure conditions exceeding those achievable in clinical settings. These experiments revealed robust, heterogeneous CT-X gene induction; following drug treatment, primary HLA-A*0201 cancer lines were recognized by allogeneic PBL expressing HLA-A*0201 T cell receptors for NY-ESO-1 and MAGE-A3. These studies provided the preclinical rationale for two IRB-approved protocols evaluating the use of autologous epigenetically-modified tumor cell vaccines as a means to broadly immunize thoracic oncology patients against multiple CT-X antigens following complete resection of their malignancies. Various aspects of the preclinical studies pertaining to these trials have been presented in oral or poster format at the Annual Meeting of the American Association of Thoracic Surgery, as well as an international cancer epigenetics conference. A manuscript describing this experience is being prepared for publication. An alternative, and perhaps less expensive strategy involves utilization of allogeneic cancer cells, which express high levels of numerous clinically relevant CT-X antigens without pharmacologic manipulation. In ongoing experiments, we have observed that K562 erythroleukemia cells broadly express CT-X antigens. Furthermore, H1299 lung cancer cells express even more CT-X antigens at higher levels than K562; both cell lines express CT-X antigens at levels significantly higher than testes controls. Two protocols have been initiated to ascertain the feasibility of using K562 cells constitutively expressing GM-CSF (K562-GM) to induce immunity to CT-X antigens in patients undergoing complete resection of primary thoracic malignancies or those with extra thoracic neoplasms metastatic to the chest. These vaccines are administered monthly in conjunction with metronomic oral cyclophosphamide and celecoxib to inhibit/deplete immunosuppressive T regulatory cells. To date 6 patients have received vaccinations on these recently initiated protocols. Additional vaccine efforts utilizing H1299 cells, administered either as whole cells or lysates in Iscomatrix (a potent adjuvant) will commence as soon as the master cell bank has been established and IND secured.

迄今为止，已有近 120 名胸部恶性肿瘤（主要是肺癌）患者按照胸部肿瘤科启动的方案接受了 DAC、DP 或连续 DAC/DP 输注。临床毒性和治疗反应已分别按照 CTCAE 和 RECIST 标准进行评估。血浆 DAC 和 DP 水平已通过 LC-MS 和 HPLC 技术进行评估。定量 RT-PCR、甲基化特异性 PCR、免疫组织化学和 ELISA 技术已用于评估治疗前和治疗后肿瘤活检和血清中的各种分子终点。微阵列技术已用于全面检查激光捕获的肿瘤细胞的基因表达谱，这些肿瘤细胞来自接受 DAC、DP 或连续 DAC/DP 输注的 21 名个体治疗前和治疗后的活检。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。尽管尚未观察到客观的临床反应，但一些患者在 DAC、DP 或序贯 DAC/DP 输注后表现出疾病的长期稳定。血浆 DAC 和 DP 浓度接近培养肺癌细胞中基因诱导和凋亡的阈值水平。大约 30% 接受 DAC 或 DP 输注的患者在肿瘤活检中表现出 NY-ESO-1、p16、p21 或乙酰化组蛋白 H3 和 H4 表达增强，表明对治疗的分子反应。微阵列分析揭示了激光捕获的肺癌细胞对 DAC、DP 和 DAC/DP 的复杂、异质反应，基因表达谱向组织学正常支气管上皮细胞中观察到的转变。这些发现表明，可能需要更长时间的暴露才能介导癌症消退。在其他研究中，我们证明 cdk 抑制剂 Flavopiridol (FLA) 可以部分通过消除 HDAC 抑制剂介导的 p21 上调来显着增强 DP 介导的细胞凋亡。这些发现为评估胸部恶性肿瘤患者连续 DP/FLA 输注的毒性和潜在疗效的方案提供了临床前基本原理。迄今为止，已有 21 名患者接受了这些输注，且毒性可接受。尽管在这项剂量递增研究中尚未发现肿瘤消退，但 7 名患者的疾病已表现出持续 4-12 个月的稳定。这些发现表明对治疗的分子反应。该研究将在另外 4-6 名患者接受治疗后结束。总的来说，这一经验值得进一步分析用于治疗胸部恶性肿瘤的染色质重塑剂，并使用能够实现长期药物暴露的时间表。尽管 CT-X 基因在肺癌中协调去抑制，但在患有这些恶性肿瘤的患者中，对 CT-X 抗原的免疫反应极其罕见，部分原因是抗原表达水平低于免疫识别阈值，以及这些个体的肿瘤和体循环内存在免疫抑制性 T 调节细胞。因此，我们寻求开发组合疗法，以增强肺癌患者对 CT-X 抗原的免疫反应。一种潜在的策略涉及使用表观遗传修饰的自体肿瘤细胞来使肺癌患者针对多种 CT-X 抗原进行免疫，这些抗原可能通过全身基因诱导方案在各自的原发性癌症中上调。迄今为止，还没有此类努力的报道。为了检验这种方法的潜在可行性，对来自不同组织学的胸部肿瘤患者的组织/液体进行了原代培养。肿瘤细胞的来源包括腹膜液、内窥镜胸膜或纵隔活检以及 CT 引导的 FNA。迄今为止，已经建立了 26 个细胞系，其中一些细胞系在超过临床环境中可达到的暴露条件下暴露于 DAC+/-DP。这些实验揭示了强大的、异质的 CT-X 基因诱导；药物治疗后，原发性 HLA-A*0201 癌细胞系被表达 NY-ESO-1 和 MAGE-A3 的 HLA-A*0201 T 细胞受体的同种异体 PBL 识别。这些研究为两项 IRB 批准的方案提供了临床前基本原理，这两项方案评估了自体表观遗传修饰肿瘤细胞疫苗的使用，作为胸部肿瘤患者在恶性肿瘤完全切除后针对多种 CT-X 抗原进行广泛免疫的一种手段。与这些试验相关的临床前研究的各个方面已在美国胸外科协会年会以及国际癌症表观遗传学会议上以口头或海报形式展示。描述这一经历的手稿正在准备出版。另一种可能更便宜的策略是利用同种异体癌细胞，这些细胞无需药物操作即可表达高水平的多种临床相关 CT-X 抗原。在正在进行的实验中，我们观察到 K562 红白血病细胞广泛表达 CT-X 抗原。此外，H1299肺癌细胞表达的CT-X抗原水平比K562更高。两种细胞系的 CT-X 抗原表达水平均显着高于睾丸对照。已启动两项方案，以确定使用组成型表达 GM-CSF (K562-GM) 的 K562 细胞在原发性胸部恶性肿瘤完全切除或胸外肿瘤转移至胸部的患者中诱导对 CT-X 抗原的免疫的可行性。这些疫苗每月与节拍性口服环磷酰胺和塞来昔布联合注射，以抑制/消除免疫抑制性 T 调节细胞。迄今为止，已有 6 名患者按照最近启动的方案接受了疫苗接种。一旦主细胞库建立并获得 IND，将立即开始利用 H1299 细胞进行额外的疫苗工作，以全细胞或 Iscomatrix（一种强效佐剂）中的裂解物形式施用。