Identifying and characterizing functional noncoding mutations in multiple myeloma

除了人们越来越了解的癌基因和癌基因编码序列的突变谱系 肿瘤抑制基因，人类癌症的全基因组测序发现了大量的突变 非编码序列。这些非编码突变中的一些存在于调控序列元件中 (启动子、增强子、绝缘子)决定基因表达。然而，即使在这样的环境中发现 序列，这些突变中的大多数预计是中性的，即乘客，只有一小部分 有功能上的影响。在这里，我们试图解决识别致癌非编码的挑战 突变，专注于一种常见且难以治疗的人类血液系统恶性肿瘤--多发性骨髓瘤 (Mm)。我们的方法需要结合遗传-表观遗传作图，重点是等位基因特定的DNA甲基化 (ASM)，然后进行功能分析。我们将进行全基因组测序和全基因组测序 30例MM患者的亚硫酸氢盐测序(WGBS；甲基序列)，代表两种最常见的分子 这种癌症的每一种亚型都与来自同一患者的非肿瘤性外周血B细胞配对。这 程序将识别差异甲基化区域与ASM(ASM DMRS)，这些区域已经出现在 破坏或产生转录因子结合位点(TFBS)的体细胞突变所致的肿瘤细胞 绝缘体元件中的启动子/增强子元件或CTCF位。这些调查结果将提名候选人 功能性非编码突变，它们通过赋予局部物理 突变型和野生型等位基因之间的不对称性，即ASM。我们将对这些突变进行功能性检查 通过使用CRISPR-Cas9突变在MM细胞系中创建它们，然后进行表观遗传和 野生型和突变型细胞系等基因对的生物学检测。使用我们的映射方法在 来自多发性骨髓瘤患者的配对样本的小试点系列，我们已经确定了候选调控 突变，包括TEAD1基因的一个假定的增强子元件的点突变，我们有 功能验证为产生其侧翼CPGS的甲基化缺失。最后，我们将测试 在我们肿瘤库的数百例MM病例中，相同调控序列的突变适用于 生物信息学方法确定从头开始的ASM相关突变，即使不是很高 反复发作，优先参与推动多发性骨髓瘤进展的生物途径，并进行连续研究 临床样本以确定在这些相同的通路中是否存在额外的非编码调控突变 在MM疾病进展过程中出现。我们的总体目标是开发和验证一个高度实用和 用于识别人类癌症中功能性非编码突变的通用方法，并使用 来自该项目的具体数据，以确定MM治疗的新靶点。

In addition to the increasingly well understood repertoire of mutations in coding sequences of oncogenes and tumor suppressor genes, genome-wide sequencing of human cancers has revealed abundant mutations in non-coding sequences. Some of these non-coding mutations are found in regulatory sequence elements (promoters, enhancers, insulators) that determine gene expression. However, even when found in such sequences, most of these mutations are expected to be neutral, i.e., passengers, with only a small percentage having a functional impact. Here we seek to address the challenge of identifying oncogenic non-coding mutations, focusing on a common and difficult to treat human hematologic malignancy, multiple myeloma (MM). Our approach entails combined genetic-epigenetic mapping, focusing on allele-specific DNA methylation (ASM), followed by functional assays. We will perform whole-genome sequencing (WGS) and whole-genome bisulfite sequencing (WGBS; methyl-seq) in 30 MM cases, representing two of the most common molecular subtypes of this cancer, each paired with non-neoplastic peripheral blood B cells from the same patients. This procedure will identify differentially methylated regions with ASM (ASM DMRs) that have arisen de novo in the tumor cells due to somatic mutations that destroy or create transcription factor binding sites (TFBS) in promoter/enhancer elements or CTCF sites in insulator elements. These findings will nominate candidate functional non-coding mutations, which have declared their functional activity by conferring the local physical asymmetry, namely ASM, between mutant and wild-type alleles. We will vet these mutations for functional effects by creating them in MM cell lines using CRISPR-Cas9 mutagenesis, followed by epigenetic and biological assays on the isogenic pairs of wild-type and mutant cell lines. Using our mapping approach in a small pilot series of paired samples from MM patients, we have already identified candidate regulatory mutations, including a point mutation in a putative enhancer element of the TEAD1 gene, which we have functionally validated as producing loss of methylation of its flanking CpGs. Lastly, we will test for recurrence of mutations in the same regulatory sequences in several hundred MM cases in our tumor banks, apply bioinformatic approaches to determine whether the de novo ASM-associated mutations, even if not highly recurrent, preferentially participate in biological pathways that drive MM progression, and study sequential clinical samples to determine whether additional non-coding regulatory mutations in these same pathways arise during MM disease progression. Our overall goals are to develop and validate a highly practical and generalizable approach for identifying functional non-coding mutations in human cancers, and to use the specific data from this project to identify new targets for treatment of MM.