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.

除了越来越充分了解癌基因的编码序列中突变的曲目和 肿瘤抑制基因，人类癌的全基因组测序揭示了大量突变 非编码序列。这些非编码突变中的一些是在调节序列元件中发现的 （启动子，增强子，绝缘子）确定基因表达。但是，即使在这样发现 序列，这些突变中的大多数被预期为中性，即乘客，只有一百分比 具有功能影响。在这里，我们试图解决识别致癌非编码的挑战 突变，重点是常见且难以治疗人血液学恶性肿瘤，多发性骨髓瘤 （毫米）。我们的方法需要结合遗传性景观图，重点是等位基因特异性DNA甲基化 （ASM），然后进行功能测定。我们将执行全基因组测序（WGS）和全基因组 在30毫米的情况下，亚硫酸氢盐测序（WGB；甲基seq），代表两个最常见的分子 该癌症的亚型，每种与同一患者的非塑性外周血液B细胞配对。这 程序将使用从头出现的ASM（ASM DMR）鉴定差异甲基化区域 肿瘤细胞是由于在销毁或产生转录因子结合位点（TFB）的体细胞引起的 绝缘子元件中的启动子/增强子元素或CTCF位点。这些发现将提名候选人 功能性非编码突变，通过授予局部物理来宣布其功能活动 突变体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以进行功能 通过使用CRISPR-CAS9诱变在MM细胞系中创建效果，然后表观遗传和 在野生型和突变细胞系的等源性对上进行生物测定。使用我们的映射方法 来自MM患者的小型试验系列配对样品，我们已经确定了候选调节性 突变，包括Tead1基因的假定增强子元件中的点突变，我们已经具有 在功能上证实了其侧翼CPG的甲基化丧失。最后，我们将测试 在我们的肿瘤库中几百个MM病例中相同的调节序列中的突变，应用 生物信息学的方法来确定从头ASM相关的突变，即使不是很高的突变 经常性，优先参与驱动MM进展的生物学途径，并研究顺序 临床样品确定是否在这些相同途径中的其他非编码调节突变 在MM疾病进展过程中出现。我们的总体目标是开发和验证高度实用的和 可识别人类癌症功能非编码突变的可概括方法，并使用 来自该项目的具体数据以确定用于治疗MM的新目标。