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.

除了癌基因编码序列中的突变越来越好理解之外， 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中的丰富突变 非编码序列。其中一些非编码突变存在于调控序列元件中 （启动子，增强子，绝缘子）决定基因表达。然而，即使在这种情况下， 序列，这些突变中的大多数预期是中性的，即，乘客，只有一小部分 具有功能性影响。在这里，我们试图解决识别致癌非编码基因的挑战， 突变，重点是一种常见的和难以治疗的人类血液恶性肿瘤，多发性骨髓瘤 （MM）。我们的方法需要结合遗传-表观遗传作图，重点是等位基因特异性DNA甲基化 (ASM)，随后进行功能测定。我们将进行全基因组测序（WGS）和全基因组 亚硫酸氢盐测序（WGBS; methyl-seq）在30例MM病例中，代表了两种最常见的分子 这种癌症的亚型，每一种都与来自相同患者的非肿瘤性外周血B细胞配对。这 该程序将鉴定ASM（ASM DMR）的差异甲基化区域，这些区域在细胞中从头出现， 肿瘤细胞由于体细胞突变，破坏或创建转录因子结合位点（TFBS）， 启动子/增强子元件或绝缘子元件中的CTCF位点。这些调查结果将提名候选人 功能性非编码突变，其通过赋予局部物理活性来宣布其功能活性。 不对称性，即ASM，突变体和野生型等位基因之间。我们会检查这些突变的功能 通过使用CRISPR-Cas9诱变在MM细胞系中产生它们，然后进行表观遗传和 对野生型和突变细胞系的等基因对的生物学测定。使用我们的映射方法， 从MM患者的配对样本的小试验系列，我们已经确定了候选的监管 突变，包括TEAD 1基因的推定增强子元件中的点突变， 在功能上被验证为产生其侧翼CpG的甲基化缺失。最后，我们将测试 我们的肿瘤库中数百例MM病例中相同调控序列的突变， 生物信息学方法，以确定是否从头ASM相关的突变，即使不是高度 复发，优先参与驱动MM进展的生物学途径，并研究序贯 临床样本，以确定是否有额外的非编码调控突变，在这些相同的途径 在MM疾病进展期间出现。我们的总体目标是开发和验证一个高度实用和 本发明涉及用于鉴定人类癌症中的功能性非编码突变的可推广的方法，并使用所述方法， 从这个项目的具体数据，以确定治疗MM的新目标。