Transcriptional mechanisms and melanoma

摘要 黑色素瘤是一种皮肤肿瘤，经常转移，每年导致许多人死亡。尽管在那里 对黑色素瘤是否有有效的治疗方法，如检查点阻断或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，但仅限于 20%-30%的患者从治疗中长期受益。耐药性与CD8的数量相关 肿瘤内的阳性T细胞。黑色素瘤中突变的表观遗传调节因子相关 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。我们已经开发了一种使用斑马鱼的快速黑色素瘤模型，并可以模拟许多 与人类黑色素瘤相关的突变。我们已经用CD8a创造了稳定的转基因鱼 启动子驱动荧光标记，并在该品系中产生原发黑色素瘤，将T细胞活成像为 它们进入肿瘤。使用这种方法，我们已经发现了T细胞直接与 黑色素瘤细胞。缓慢迁移的T细胞在肿瘤边缘看起来疲惫不堪，而活跃的T细胞移动得更快 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等表观遗传调控因子的突变来量化黑色素瘤的这种行为。 初步数据显示，G9a抑制可抑制ARID2缺乏所增强的肿瘤起始。其他 监管机构将根据人类肿瘤遗传学进行调查。这些表观遗传调控因子对T细胞的影响 细胞向黑色素瘤的迁移将通过使用双光子显微镜的实时成像进行评估。我们会研究 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 正如我们团队所定义的那样。活体成像与染色质可及性的相关性将有助于确定 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缺陷性肿瘤的抑制剂。我们将创建具有多种转录因子突变的模型 和表观遗传调节器，特别是那些对这项拨款的其他调查人员感兴趣的。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 染色质效应受BRAF处理驱动，侧重于耐药机制的研究。ETV1，一种基因 在8%的黑色素瘤中被放大，在染色质上重组以激活驱动耐药性的基因网络。 已知ETV1被MAPK磷酸化，我们计划用蛋白质组学来研究这一机制。通过 筛选表观化学文库，我们希望逆转耐药网络用于治疗目的。 我们还将研究ETV1在T细胞向肿瘤募集中的作用。我们的学习，大大加强了 由我们与Fisher博士(G9a和小鼠黑色素瘤模型)、刘博士(表观遗传学分析)以及 Wucherpfennig博士和Roig博士(免疫机制和细胞图谱)对基础 耐药机制，并将导致新的治疗方法来治疗耐药黑色素瘤。

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.