Identifying and characterizing functional noncoding mutations in multiple myeloma

识别和表征多发性骨髓瘤的功能性非编码突变

• 批准号: 10586759
• 负责人: Benjamin Tycko
• 金额: $ 77.39万
• 依托单位: HACKENSACK UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-13 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586759
• 关键词: 3-Dimensional; Address; Alleles; Apoptosis; Area; B-Lymphocytes; Binding Sites; Bioinformatics; Biological; Biological Assay; Bone Marrow; CRISPR/Cas technology; Cell Line; Cell Proliferation; Cells; Chromatin; Chromatin Structure; Chromosome Mapping; Clinical; Code; DNA Methylation; DNA methylation profiling; Data; Disease Progression; Elements; Enhancers; Epigenetic Process; Gene Expression; Genes; Genetic; Genetic Enhancer Element; Goals; Hematologic Neoplasms; Human; Insulator Elements; Malignant Neoplasms; Maps; Methods; Methylation; Multiple Myeloma; Mutagenesis; Mutation; Oncogenes; Oncogenic; Pathway interactions; Patients; Pharmaceutical Preparations; Point Mutation; Procedures; Prognosis; Recurrence; Regulatory Element; Research; Resistance; Sampling; Series; Signal Transduction; Site; Somatic Mutation; System; Testing; Triage; Tumor Bank; Tumor Suppressor Genes; Untranslated RNA; Xenograft procedure; bioinformatics pipeline; bisulfite sequencing; cancer subtypes; candidate identification; clinically relevant; genome sequencing; genome-wide; in vivo; lymphoblastoid cell line; mRNA Expression; methylation pattern; mimetics; molecular subtypes; mutant; neoplastic cell; peripheral blood; promoter; targeted sequencing; three dimensional cell culture; transcription factor; treatment response; whole genome

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.

除了对癌基因编码序列突变的了解日益深入之外， 肿瘤抑制基因，人类癌症的全基因组测序揭示了肿瘤抑制基因的丰富突变 非编码序列。其中一些非编码突变存在于调控序列元件中 决定基因表达的（启动子、增强子、绝缘子）。然而，即使发现这样的 序列中，大多数突变预计是中性的，即乘客，只有一小部分 具有功能性影响。在这里，我们寻求解决识别致癌非编码的挑战 突变，重点关注一种常见且难以治疗的人类血液恶性肿瘤——多发性骨髓瘤 （毫米）。我们的方法需要结合遗传-表观遗传作图，重点关注等位基因特异性 DNA 甲基化 （ASM），然后进行功能测定。我们将进行全基因组测序（WGS）和全基因组 30 个 MM 病例中的亚硫酸氢盐测序（WGBS；甲基测序）代表了两种最常见的分子 这种癌症的亚型，每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 程序将识别在 ASM 中从头出现的差异甲基化区域（ASM DMR） 体细胞突变破坏或产生转录因子结合位点 (TFBS) 导致肿瘤细胞 启动子/增强子元件或绝缘体元件中的 CTCF 位点。这些调查结果将提名候选人 功能性非编码突变，通过赋予局部物理特性来声明其功能活性 突变型和野生型等位基因之间的不对称性，即 ASM。我们将审查这些突变的功能 通过使用 CRISPR-Cas9 诱变在 MM 细胞系中创建它们，然后进行表观遗传和 对野生型和突变细胞系的同基因对进行生物学测定。使用我们的映射方法 来自 MM 患者的配对样本的小型试点系列，我们已经确定了候选监管 突变，包括 TEAD1 基因的假定增强子元件的点突变，我们有 经功能验证，其侧翼 CpG 甲基化缺失。最后，我们将测试是否复发 我们的肿瘤库中数百个 MM 病例中相同调控序列的突变，适用 生物信息学方法来确定是否从头 ASM 相关突变，即使不是高度 复发性，优先参与驱动 MM 进展的生物途径，并进行序贯研究 临床样本以确定这些相同途径中是否存在额外的非编码调节突变 MM 疾病进展期间出现。我们的总体目标是开发和验证高度实用且 识别人类癌症中功能性非编码突变的通用方法，并使用 该项目的具体数据可确定 MM 治疗的新目标。