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.

抽象的 黑色素瘤是皮肤的肿瘤，每年经常转移并导致许多死亡。虽然那里 是黑色素瘤的有效疗法，例如检查点阻滞或BRAF抑制剂，抗性机制 经常被激活。免疫疗法已成为转移性黑色素瘤的一线治疗，但仅 20-30％的患者长期从治疗中受益。电阻与CD8的数量有关 浸润肿瘤的阳性T细胞。黑色素瘤突变的表观遗传调节剂与 具有对检查点阻滞的耐药性。了解这些染色质因子调节的途径 将允许更好的疗法。我们已经使用斑马鱼开发了快速黑色素瘤模型，可以建模许多 与人黑色素瘤相关的突变。我们用CD8A创建了稳定的转基因鱼 启动子驱动荧光标记物并在此系列中生成原发性黑色素瘤，以将T细胞成像为T细胞 他们进入肿瘤。使用这种方法，我们发现了直接与T细胞相互作用的T细胞的不同行为 黑色素瘤细胞。缓慢迁移的T细胞在肿瘤的边界上显得疲惫，而活动速度更快地移动 T细胞与黑色素瘤细胞相互作用。基于我们的临床同事定义的人类黑色素瘤遗传学 我们计划在黑色素瘤中使用表观遗传调节剂（例如ARID2和G9A）中的突变进行量化这种行为。 初步数据表明，G9A抑制作用抑制了ARID2缺乏症的增强肿瘤起始。其他 将根据人类肿瘤遗传学对调节剂进行调查。这些表观遗传调节剂对T的影响 细胞迁移到黑色素瘤中将通过使用两个光子显微镜实时成像来评估。我们将学习 这些肿瘤使用ATAC-SEQ的染色质可及性，并将结果与​​人类肿瘤可及性相关联 由我们的小组定义。实时成像与染色质可访问性的相关性将有助于定义 这些转录因子的抗性机制，并将提供有关G9A的临床前信息 ARID2缺乏肿瘤的抑制剂。我们将创建具有多种转录因子突变的模型 以及表观遗传调节器，特别是其他研究人员对此赠款感兴趣的调节者。增强剂 还将开发记者来检查肿瘤和T细胞中的靶基因。我们还将研究 由BRAF处理驱动的染色质作用，重点是抗药机制。 ETV1，一个基因 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。 已知ETV1被MAPK磷酸化，我们计划使用蛋白质组学询问这种机制。经过 筛选表观遗传化学文库，我们希望以治疗目的扭转阻力网络。 我们还将研究ETV1在T细胞募集到肿瘤中的作用。我们的研究大大加强了 通过我们与Fisher博士（G9A和小鼠黑色素瘤模型），Liu博士（表观遗传学分析）和 博士。 Wucherpfennig和Rodig（免疫机制和细胞分析）对基本的影响很大 耐药机制，并将导致新的抗药性黑色素瘤治疗疗法。