Transcriptional mechanisms and melanoma

转录机制和黑色素瘤

• 批准号: 10227093
• 负责人: LEONARD Ira ZON
• 金额: $ 36.45万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-12 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227093
• 关键词: ATAC-seq; Automobile Driving; BRAF gene; Behavior; Binding; CD8B1 gene; Cell physiology; Cells; Cessation of life; Chemicals; Chromatin; Chromatin Structure; Clinical; Collaborations; Confocal Microscopy; DNA Packaging; Data; Development; Disease; Drug resistance; Drug resistance pathway; ETV1 gene; Enhancers; Epigenetic Process; Fishes; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Genotype; Goals; Grant; Human; Image; Immune response; Immune system; Immunologics; Immunotherapy; Lead; MAP Kinase Gene; MEKs; Maintenance; Malignant Neoplasms; Melanoma Cell; Metastatic Melanoma; Methods; Mitogen-Activated Protein Kinase Inhibitor; Modeling; Molecular; Mus; Mutate; Mutation; Neoplasm Metastasis; Oncogenes; Oncogenic; Outcome; Paper; Pathway interactions; Patients; Pharmaceutical Preparations; Primary Neoplasm; Proteomics; Reporter; Research Personnel; Resistance; Role; Shapes; Signal Transduction; Skin; Survival Rate; System; T-Lymphocyte; Techniques; Therapeutic; Tissues; Transgenic Organisms; Tumor Suppressor Proteins; Work; Zebrafish; anti-CTLA4; anti-PD1 antibodies; base; cancer imaging; cell motility; chromatin immunoprecipitation; clinical efficacy; confocal imaging; conventional therapy; effective therapy; exhaust; immune checkpoint blockade; immune resistance; ineffective therapies; inhibitor/antagonist; interest; melanoma; neoplastic cell; new therapeutic target; novel therapeutics; pre-clinical; prevent; promoter; recruit; resistance mechanism; screening; small molecule libraries; targeted treatment; trafficking; transcription factor; tumor; tumor initiation; tumor microenvironment; two photon microscopy; vector

Abstract Melanoma is a tumor of the skin that frequently metastasizes and causes many deaths annually. Although there are effective therapies for melanoma such as checkpoint blockade or BRAF inhibitors, resistance mechanisms are frequently activated. Immunotherapy has become a frontline treatment for metastatic melanoma, but only 20-30% of patients have long term benefit from the treatment. Resistance correlates to the number of CD8 positive T cells that have infiltrated the tumor. Epigenetic regulators that are mutated in melanoma are associated with drug resistance to checkpoint blockade. Understanding the pathways regulated by these chromatin factors will allow for better therapies. We have developed a rapid melanoma model using zebrafish and can model many of the mutations associated with human melanoma. We have created stable transgenic fish with the CD8a promoter driving fluorescent markers and generated primary melanomas in this line to live image the T cells as they enter the tumor. Using this approach, we have found distinct behaviors of T cells that interact directly with melanoma cells. Slow migrating T cells appear exhausted at the borders of tumors, whereas active faster moving T cells interact with the melanoma cells. Based on human melanoma genetics defined by our clinical colleagues, we plan to quantify this behavior in melanomas with mutations in epigenetic regulators such as ARID2 and G9a. Preliminary data shows that G9a inhibition suppresses the enhanced tumor initiation by ARID2 deficiency. Other regulators will be investigated based on human tumor genetics. The effects of these epigenetic regulators on T cell migration into the melanoma will be assessed by live imaging using two photon microscopy. We will study chromatin accessibility of these tumors using ATAC-seq and correlate the results with human tumor accessibility as defined by our group. The correlation of live imaging with chromatin accessibility will help define the mechanisms of resistance of these transcription factors and will provide preclinical information about G9a inhibitors on ARID2 deficient tumors. We will create models with mutations in a variety of transcription factors and epigenetic regulators, particularly those of interest to the other investigators on this grant. Enhancer reporters will also be developed to examine target genes in the tumors and T cells. We also will study the chromatin effects driven by BRAF treatment, focusing on the mechanism of resistance. ETV1, a gene that is amplified in 8% of melanoma, is reorganized on chromatin to activate a network of genes that drive resistance. ETV1 is known to be phosphorylated by MAPK and we plan to interrogate this mechanism using proteomics. By screening an epigenetic chemical library, we hope to reverse the resistance network for therapeutic purposes. We will also investigate the role of ETV1 in the recruitment of T cells to tumors. Our studies, greatly strengthened by our collaborations with Dr. Fisher (G9a and mouse melanoma models), Dr. Liu (epigenetic analyses), and Drs. Wucherpfennig and Rodig (immunological mechanisms and cell profiling) have great impact on the basic mechanism of resistance and will lead to new therapies for the treatment of drug resistant melanoma.

摘要 黑色素瘤是一种皮肤肿瘤，经常转移，每年造成许多人死亡。虽然 是黑色素瘤的有效疗法，如检查点阻断剂或BRAF抑制剂，耐药机制 经常被激活。免疫疗法已成为转移性黑色素瘤的一线治疗方法， 20-30%的患者从治疗中获得长期受益。耐药性与CD 8数量相关 已经渗入肿瘤的阳性T细胞黑色素瘤中突变的表观遗传调节因子与 对检查站封锁有抗药性。了解这些染色质因子调控的途径 会带来更好的治疗方法我们已经开发了一个快速的黑色素瘤模型使用斑马鱼，可以模拟许多 与人类黑色素瘤相关的突变。我们已经创造了稳定的转基因鱼与CD 8a 启动子驱动荧光标记物，并在该细胞系中产生原发性黑素瘤，以将T细胞实时成像为 它们进入肿瘤。使用这种方法，我们发现了T细胞的不同行为，它们直接与 黑素瘤细胞。缓慢迁移的T细胞在肿瘤边缘似乎已经耗尽，而活跃的T细胞则移动得更快。 T细胞与黑素瘤细胞相互作用。基于我们临床同事定义的人类黑色素瘤遗传学， 我们计划在具有表观遗传调节因子如ARID 2和G9 a突变的黑色素瘤中量化这种行为。 初步数据显示，G9 a抑制抑制了由ARID 2缺陷引起的增强的肿瘤起始。其他 将基于人类肿瘤遗传学研究调节因子。这些表观遗传调节因子对T 通过使用双光子显微镜的活体成像来评估细胞迁移到黑素瘤中。我们将研究 使用ATAC-seq分析这些肿瘤的染色质可及性，并将结果与人类肿瘤可及性相关联 根据我们的定义。实时成像与染色质可及性的相关性将有助于定义 这些转录因子的耐药机制，并将提供有关G9 a的临床前信息 ARID 2缺陷型肿瘤的抑制剂。我们将创建各种转录因子突变的模型 和表观遗传调节因子，特别是那些对这项资助的其他研究人员感兴趣的。增强子 报告基因也将被开发来检测肿瘤和T细胞中的靶基因。我们还将研究 BRAF处理驱动的染色质效应，重点关注抗性机制。ETV 1是一种基因， 在8%的黑色素瘤中扩增，在染色质上重组以激活驱动抗性的基因网络。 ETV 1已知被MAPK磷酸化，我们计划使用蛋白质组学来询问这种机制。通过 通过筛选表观遗传化学文库，我们希望逆转耐药网络以达到治疗目的。 我们还将研究ETV 1在T细胞向肿瘤募集中的作用。我们的研究，大大加强 通过我们与Fisher博士（G9 a和小鼠黑色素瘤模型），Liu博士（表观遗传学分析）和 Drs. Wucherpfennig和Rodig（免疫机制和细胞分析）对基础免疫学有很大影响。 耐药机制，并将导致新的治疗耐药黑色素瘤的疗法。