Transcriptional mechanisms and melanoma

转录机制和黑色素瘤

基本信息

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
项目类别：
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

项目摘要

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.

抽象的黑色素瘤是一种经常转移的皮肤肿瘤，每年导致许多人死亡。虽然有是治疗黑色素瘤的有效疗法，例如检查点阻断或 BRAF 抑制剂、耐药机制被频繁激活。免疫疗法已成为转移性黑色素瘤的一线治疗方法，但仅 20-30% 的患者从治疗中获得长期获益。耐药性与 CD8 的数量相关已浸润肿瘤的阳性 T 细胞。黑色素瘤中突变的表观遗传调节因子与对检查点封锁具有耐药性。了解这些染色质因子调节的途径将有助于更好的治疗。我们使用斑马鱼开发了一种快速黑色素瘤模型，可以模拟许多与人类黑色素瘤相关的突变。我们用 CD8a 培育出了稳定的转基因鱼启动子驱动荧光标记并在该细胞系中产生原发性黑色素瘤，以对 T 细胞进行实时成像它们进入肿瘤。使用这种方法，我们发现了与 T 细胞直接相互作用的独特行为。黑色素瘤细胞。迁移缓慢的 T 细胞在肿瘤边界处显得疲惫不堪，而移动速度较快的 T 细胞则活跃 T 细胞与黑色素瘤细胞相互作用。根据我们的临床同事定义的人类黑色素瘤遗传学，我们计划通过 ARID2 和 G9a 等表观遗传调节因子突变来量化黑色素瘤中的这种行为。初步数据表明，G9a 抑制可抑制 ARID2 缺陷导致的肿瘤发生增强。其他监管机构将根据人类肿瘤遗传学进行研究。这些表观遗传调节因子对 T 的影响将使用双光子显微镜进行实时成像来评估细胞向黑色素瘤的迁移。我们将学习使用 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正如我们小组所定义的。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 G9a 的临床前信息 ARID2 缺陷肿瘤的抑制剂。我们将创建具有多种转录因子突变的模型和表观遗传调节因子，特别是其他研究人员对此资助感兴趣的调节因子。增强剂还将开发报告基因来检查肿瘤和 T 细胞中的靶基因。我们还将研究 BRAF 治疗驱动的染色质效应，重点关注耐药机制。 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耐药性的基因网络。已知 ETV1 会被 MAPK 磷酸化，我们计划使用蛋白质组学来探究这一机制。经过筛选表观遗传化学库，我们希望扭转耐药网络以达到治疗目的。我们还将研究 ETV1 在向肿瘤招募 T 细胞中的作用。我们的学习，大大加强通过我们与 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（免疫学机制和细胞分析）对基础研究有很大影响耐药机制，并将带来治疗耐药黑色素瘤的新疗法。