The Genetics of Ocular Melanoma

项目摘要/摘要 葡萄膜黑色素瘤(UM)是第二种最常见的黑色素瘤形式，也是最常见的原发癌症 这不仅会导致视力丧失，还会导致多达一半患者的转移性死亡。没有有效的方法 一旦肿瘤转移，患者通常在确诊后几个月内死亡，尽管 系统治疗。UMS存在早期发生的GNAQ、GNA11或CySLTR2的致癌基因突变 在肿瘤形成中的作用，并且与转移风险无关。我们显示，超过80%的2级UM是在港口 BAP1的功能突变丢失和SF3B1的R625的反复突变被发现在 转移率较低。EIF1AX基因突变与转移几率最小的肿瘤相关。 BAP1、SF3B1和EIF1AX突变的存在几乎总是相互排斥的。但是，覆盖了 这是基因组重排，包括8q和6p染色体的获得，以及1p染色体的丢失， 6Q和8P。我们对UM中这些核型/拷贝数改变的研究发现了一种关键的染色质 染色体6q(PHF10)的修饰定位和重复染色体其他区域的候选基因 更改。人们对UM发展中的这些变化的后果知之甚少。要扩展和 完善我们的拷贝数研究，我们现在将对DNA和RNA进行长时间阅读的单分子测序 从具有不同驱动程序突变和CNA的原发UM中分离。在目标1和目标2中，我们将进行长期阅读 单分子测序(SMRT)，以开发基因组结构变异(SVS)的详细图谱 在这些肿瘤中，识别和表征关键断裂点、基因融合和肿瘤特异性异构体。 除了对染色体改变的产生有深入的了解外，我们还将验证和完善焦点 删除并确定这些区域中的关键异构体。细胞系中的功能分析将检查 基因融合和异构体的后果与拷贝数的变化高度相关。并行并瞄准 3我们将利用苍蝇遗传学的力量来研究D。 黑猩猩。我们将首先产生并鉴定BAP1同源基因功能突变缺失的果蝇 Calypso，并产生激活突变的GNAQ同源基因GαQ，使其综合作用丧失 Calypso和G-α-Q的激活可以被研究。我们将重点关注幼虫眼盘和翼盘 代表有序的上皮单分子层并检查表型、信号和表观基因组 由这些突变引起的变化。Fly模型能够解决 通过执行快速的遗传相互作用，来自所有三个目标的基因组洞察。未来，UM候选司机 从AIMS 1-2中识别出来的人将接受测试，以确定是否有能力修改我们在两个热门节目中看到的癌症样表型 GαQ和Calypso苍蝇模型。过度生长、组织转化和转移的基因抑制因子 表型将是未来研究中令人兴奋的治疗候选对象。

PROJECT SUMMARY/ABSTRACT Uveal melanoma (UM) is the second most common form of melanoma and the most common primary cancer of the eye, resulting not only in vision loss, but in metastatic death in up to half of patients. There are no effective treatment options once the tumor metastasizes and patients usually die within a few months of diagnosis despite systemic therapy. UMs harbor activating oncogenic mutations in GNAQ, GNA11 or CYSLTR2 that occur early in tumor formation and that are unrelated to metastatic risk. We showed that over 80% of class 2 UMs harbor loss of function mutations of BAP1 and that recurrent mutations altering R625 of SF3B1 are found in tumors with slower rates of metastasis. Mutations in EIF1AX are associated with tumors with minimal chance of metastasis. The presence of BAP1, SF3B1 and EIF1AX mutations, are nearly always mutually exclusive. However, overlaid on this are genomic rearrangements that include gain of chromosomes 8q and 6p, and loss of chromosomes 1p, 6q and 8p. Our studies of these karyotypic/copy number alterations in UM have identified a critical chromatin modifier mapping to chromosome 6q (PHF10) and candidate genes in other regions of recurrent chromosomal alteration. The consequences of these changes in the development of UM are poorly understood. To extend and refine our copy number studies we will now will perform long-read single molecule sequencing of DNA and RNA isolated from primary UMs with different driver mutations and CNAs. In Aims 1 and 2 we will perform long-read single molecule sequencing (SMRT) to develop detailed maps of the structural variations (SVs) of the genomes of these tumors, identifying and characterizing critical breakpoints, gene fusions and tumor specific isoforms. Besides obtaining insights into the generation of chromosomal alterations we will validate and refine focal deletions and identify critical isoforms in such regions. Functional analyses in cell lines will examine the consequences of gene fusions and isoforms highly correlated with copy number changes. In parallel and in Aim 3 we will use the power of fly genetics to investigate the consequence of major alterations in UM in D. Melanogaster. We will first generate and characterize flies with loss of function mutations in the BAP1 ortholog calypso, and generate activating mutations of the GNAQ ortholog GαQ so that the combined effect of loss of calypso and activation of GαQ can be investigated. We will focus on larval eye discs and wing discs that represented well-ordered epithelial monolayers and examine the the phenotypic, signaling and epigenomic changes that arise from these mutations. The fly model is capable of addressing the functional relevance of the genomic insights from all three Aims by performing rapid genetic interactions. In the future, candidate UM drivers identified from Aims 1-2 will be tested for the ability to modify the cancer-like phenotypes we see in our two hit GαQ and calypso fly model. Genetic suppressors of overgrowth, tissue transformation, and metastasis phenotypes would represent exciting therapeutic candidates to pursue in future studies.