Intra and Extra-Chromosomal Probes for Mutagenesis by Carcinogens

用于致癌物诱变的染色体内和染色体外探针

基本信息

批准号：
10440503
负责人：
JOHN M ESSIGMANN
金额：
$ 38万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10440503
关键词：
8-hydroxyguanosine Address Adjuvant Therapy Advanced Malignant Neoplasm Aflatoxin B1 Alkylating Agents Analytical Chemistry Antigens Area Base Pairing Binding Biological Biological Models Bypass Cancer Biology Carcinogens Cells Characteristics Chemicals Cisplatin Collection Complex Consensus Custom Cytotoxic Chemotherapy DNA DNA Adduction DNA Adducts DNA Binding DNA Damage DNA Repair DNA lesion DNA sequencing Data Data Set Databases Deoxycytidine Dependence Development Embryo Environmental Carcinogens Epoxy Compounds Etiology Evaluation Event Fibroblasts Frequencies Genetic Genetic Engineering Genome Genomics Goals Grant Guanine Human Individual Inflammation Informatics Investigation Isotopes Kinetics Label Laboratories Lesion Malignant Neoplasms Mammalian Cell Maps Mass Spectrum Analysis Mathematics Measures Methylnitrosourea Modeling Modernization Molecular Mus Mutagenesis Mutagens Mutation Mutation Spectra N-nitrosodimethylamine Normal Cell Nucleosides Nucleotides Oligonucleotides Oxidants Oxidative Stress Oxides PD-1/PD-L1 Pattern Plasmid Cloning Vector Polymerase Primary carcinoma of the liver cells Process Property Protocols documentation Reaction Reagent Refractory Replication Error Resolution Role Series Site Somatic Mutation Source Specificity Spottings Sterigmatocystin Streptozocin Synthesis Chemistry Techniques Testing Time Viral Genome Viral Vector Work adduct anti-PD-L1 therapy base cancer cell cancer genome cancer immunotherapy cancer therapy carcinogenesis chemical carcinogen chemotherapy chloroacetaldehyde complex data cytotoxic design experimental study genetic analysis genome sequencing immune checkpoint in vivo knock-down neoantigens novel novel strategies oxidation pre-clinical repaired sugar temozolomide tool tumor

项目摘要

Project Summary/Abstract Human cancer genomes show a complex pattern of mutations that, upon computational deconvolution, resolves into a systematic series of over 80 “Mutational Signatures” (the pattern of mutations across all possible trinucleotide sequence contexts). Some signatures are common to many cancers (e.g., CGàTA in CpG sites) whereas others appear in single tumor types (e.g., signatures involving GCàTA mutations that are presumably associated with aflatoxin B1 (AFB1) exposure in hepatocellular carcinoma (HCC)). Current sequencing efforts sometimes provide hints, via examination of the details of mutational signatures, as to cancer mechanistic etiology. The first two Aims of the present proposal are a bottom up approach to determine which specific chemical insults to DNA cause mutational patterns that match mutational signatures in tumors. We have formulated a model that describes three variables that contribute to the complexity of mutational spectra: The likelihood that a DNA lesion will form in a particular context, the likelihood that it will evade repair, and the likelihood that it will miscode when traversed by a polymerase. Aim 1 uses a combination of synthetic and analytical chemistry to generate the adduct-formation spectrum of a host of DNA damaging agents (AFB1, sterigmatocystin, N-methyl-N-nitrosourea, streptozotocin, temozolomide, chloroacetaldehyde and the oxidant SIN-1). A strategy involving the use of heavy isotope containing defined-sequence oligonucleotides will be used along with mass spectrometry to map the binding specificities of electrophiles derived from these agents. Aim 2 takes a two-pronged approach to define the biological effects of DNA damage from the studied agents. Since the DNA adducts of the compounds evaluated are known, we shall insert those adducts one at a time into defined-sequence oligonucleotides in all 16 possible 3-base contexts (i.e., 5’-NXN-3’, where X is the adduct and N is A, G, C or T). The oligonucleotides will be inserted into viral genomes, replicated in cells of defined repair or replication status, and the areas of the genome that contained the lesion will be characterized to define the type, amount, genetic requirements for - and sequence context dependency of - mutagenesis. The second part of Aim 2 is to use a newly developed mouse embryo fibroblast (MEF) line to define, using Duplex Consensus Sequencing (DCS), the high-resolution mutational spectra (HRMS) of the agents under study. These data are compared via informatics techniques (e.g., cosine similarity) to human cancer mutational signatures and to the DNA binding (Aim 1) and mutagenic properties of individual adducts. Lastly, a damaged nucleotide pool is an often-overlooked source of mutations. Aim 3 uses the MEF line of Aim 2 to probe the opportunity of a pool damaged with products of oxidative stress (e.g., 8-oxoG and 5-chlorocytosine) to impact the HRMS of the MEF line. Further, we have custom-designed a pool mutagen (fKP1212), which we believe might be useful to diversify the neoantigen arrays of cancer cells in advance of cancer immunotherapy. We plan to apply our HRMS tools to investigate the pre-clinical potential of a novel adjuvant therapy for cancer. 1

项目摘要/摘要人类癌症基因组显示出复杂的突变模式，在计算反卷积后，解决了突变。进入一个系统的80多个“突变特征”（所有可能的突变模式）三核苷酸序列上下文）。一些签名对于许多癌症（例如，CPG站点中的CGàta）很常见）而其他则出现在单个肿瘤类型中（例如，涉及GCàta突变的特征与肝细胞癌（HCC）中的黄曲霉毒素B1（AFB1）暴露有关。当前的测序工作有时通过检查突变特征的细节来提供有关癌症机理的提示病因。本提案的前两个目标是确定哪种特定的自下而上的方法对DNA的化学侮辱会导致突变模式与肿瘤中的突变特征匹配。我们有伪造了一个描述三个变量的模型，这些变量有助于突变光谱的复杂性： DNA病变会在特定情况下形成的可能性，逃避修复的可能性以及当聚合酶穿越时，它可能会错过。 AIM 1结合了合成和分析化学以生成许多DNA损伤剂的加合形成谱（AFB1， stigmatocystin，N-甲基-N-硝酸盐，链霉菌素，替莫唑胺，氯乙醛醛和氧化物 sin-1）。将使用涉及使用含有定义序列寡核苷酸的重量同位素的策略目标2 采用两管齐的方法来定义研究毒剂剂的DNA损伤的生物学作用。自从评估化合物的DNA加合物是已知的，我们将一次将这些加合物插入在所有16个可能的3碱基上下文（即5'-nxn-3'，其中x是加合物， n是A，G，C或T）。寡核苷酸将插入病毒基因组中，在定义的修复细胞中复制或复制状态，并且包含病变的基因组区域将被特征在于定义诱变的类型，数量，遗传要求和序列上下文依赖性。第二部分 AIM 2的使用是使用新开发的鼠标成纤维细胞（MEF）线来定义，使用双工共识测序（DCS），研究药物的高分辨率突变光谱（HRM）。这些数据是通过信息信息（例如余弦相似性）与人类癌症突变特征进行比较 DNA结合（AIM 1）和单个加合物的诱变特性。最后，受损的核苷酸池是通常被忽视的突变来源。 AIM 3使用AIM 2的MEF线来探测池的机会用氧化应激的产物（例如8-氧化物和5-氯环肽）损坏，以影响MEF的HRM 线。此外，我们已经定制设计了一个泳池诱变者（FKP1212），我们认为这对于多元化可能是有用的癌细胞的新抗原阵列在癌症免疫疗法之前。我们计划将我们的HRMS工具应用于研究新型调整癌症的临床前潜力。 1