Using CRISPR screening to uncover aneuploidy-specific genetic dependencies

使用 CRISPR 筛选揭示非整倍体特异性遗传依赖性

• 批准号: 10661533
• 负责人: Klaske Marijke Schukken
• 金额: $ 7.18万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10661533
• 关键词: Affect; Aneuploid Cells; Aneuploidy; Apoptosis; Binding Proteins; Bioinformatics; Biological; CRISPR library; CRISPR screen; Cancer cell line; Cell Aging; Cell Line; Cell division; Cell physiology; Cells; Cellular Stress; Chemoresistance; Chromosome 7; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Colon Carcinoma; Communities; Complementary DNA; Data; Defect; Dependence; Diploidy; Drug Screening; Drug Targeting; Drug resistance; Embryo; Environment; Epigenetic Process; Evolution; Exhibits; Family; Fibroblasts; Frequencies; Funding; Gene Dosage; Genes; Genetic; Genetic Screening; Glioblastoma; Goals; Growth; Human; Human Cell Line; Karyotype; Knock-out; Knowledge; Libraries; Malignant Neoplasms; Malignant neoplasm of ovary; Mass Spectrum Analysis; Metabolic; Metabolic stress; Methodology; Molecular; Mus; Neoplasm Metastasis; Nocodazole; Pathway interactions; Patient-Focused Outcomes; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Pituitary Gland Adenoma; Proteins; Proteomics; Research Personnel; Role; Solid Neoplasm; Specificity; Stress; Survival Rate; Techniques; Tertiary Protein Structure; Toxic effect; Training; Universities; Work; cancer cell; cancer type; carcinogenesis; career; career development; cell growth; chromosome missegregation; colon cancer cell line; congenic; design; experimental study; follow-up; gene function; inhibitor; insight; knockout gene; malignant breast neoplasm; medical schools; screening; secondary analysis; segregation; senescence; small molecule; small molecule inhibitor; stressor; targeted treatment; therapeutic development; transcription factor; transcriptomics; tumor; ubiquitin ligase

Project Summary Aneuploidy is a cellular state in which cells contain extra or missing chromosomes. Over 90% of solid tumors are aneuploid. Aneuploidy has been shown to contribute to drug resistance and metastasis, and aneuploid cancers have a worse patient survival rate than euploid cancers. Despite aneuploidy’s role in cancer, aneuploidy itself causes growth defects and induces several ongoing stressors within the cell. Gaining or losing chromosomes leads to transcriptomic and proteomic stress, metabolic deregulation, an altered secretome, and induces further chromosome mis-segregation. We hypothesize that aneuploidy-induced cellular stresses can be targeted to specifically eliminate aneuploid cells, and we aim to discover genetic dependencies that are specific to aneuploid cells. We will use CRISPR to screen multiple near-euploid human cell lines and aneuploid clones that we derive from these near euploid cell lines. We will then compare the effect of aneuploidy on gene dependency, independent of cell line-specific effects. Toward this goal, we have generated 54 aneuploid clones from nine near euploid human cancer cell lines, and we have access to an additional ten aneuploid clones from two human cell lines. We will screen these aneuploid clones, and their near-euploid controls, with a domain- specific CRISPR library. This library targets multiple druggable protein domains, including all kinase, ubiquitinase, transcription factor, epigenetic modulator, “royal family” epigenetic factor, protease, and ubiquitin ligase genes. In preliminary work, we have screened multiple euploid and aneuploid cell lines with a smaller kinase domain-focused CRISPR library, and we identified several potential aneuploid-specific dependencies. More aneuploid cancer cell lines and their corresponding controls will be screened to confirm these potential hits. Cancer aneuploidy is not entirely random, as specific chromosome gains and losses are selected for in certain cancer types. In addition to uncovering general aneuploid dependencies, we aim to uncover chromosome specific dependencies. Once we have uncovered potential aneuploidy dependencies, we will validate their aneuploid-specificity, and then we will use cDNA to rescue gene knockout and rule out off-target effects. We will use IP mass spectrometry to uncover any differences in hit protein binding between euploid and aneuploid conditions, or to identify the protein binding partners of poorly characterized genes. Next, we will perform RT- qPCR and IF to screen for aneuploidy-associated phenotypes including chromosome missegregation, proteomic stress, senescence, and apoptosis. Additional follow up experiments will be performed to uncover their aneuploidy-targeting mechanisms and better understand the targetable stressors induced by aneuploidy. During this period I will be trained in CRISPR screening, bioinformatic analysis, and mass spectrometry techniques. Aneuploid dependencies and chromosome-specific aneuploid dependencies could serve as promising targets for aneuploid-cancer therapeutic development.

项目概要 非整倍体是一种细胞状态，其中超过 90% 的实体瘤细胞含有额外或缺失的染色体。 非整倍体已被证明有助于耐药性和转移，并且非整倍体。 尽管非整倍性在癌症中发挥着重要作用，但癌症的患者生存率比整倍体癌症要低。 它本身会导致生长缺陷并诱发细胞内的一些持续的压力源的获得或损失。 染色体导致转录组和蛋白质组应激、代谢失调、分泌组和 诱导进一步的染色体错误分离。我们认为非整倍体诱导的细胞应激可能是由非整倍体引起的。 旨在特异性消除非整倍体细胞，我们的目标是发现特定的遗传依赖性 我们将使用 CRISPR 筛选多个近整倍体人类细胞系和非整倍体克隆。 然后我们将比较非整倍体对基因的影响。 为了实现这一目标，我们已经产生了 54 个非整倍体克隆。 来自九个近整倍体人类癌细胞系，我们还可以获得另外十个非整倍体克隆 我们将筛选这些非整倍体克隆及其近整倍体对照，其结构域为 特定的 CRISPR 文库。该文库针对多个可药物蛋白结构域，包括所有激酶， 泛素酶、转录因子、表观遗传调节剂、“皇室”表观遗传因子、蛋白酶、泛素 在前期工作中，我们筛选了多个具有较小的整倍体和非整倍体细胞系。 我们利用以激酶结构域为中心的 CRISPR 文库，发现了几种潜在的非整倍体特异性依赖性。 将筛选更多的非整倍体癌细胞系及其相应的对照，以确认这些潜力 癌症非整倍体不是随机的，因为特定的染色体增益和丢失完全是选择的。 除了揭示一般的非整倍体依赖性之外，我们的目标还在于揭示某些癌症类型。 一旦我们发现了潜在的非整倍体依赖性，我们将验证它们。 非整倍体特异性，然后我们将使用 cDNA 来挽救基因敲除并排除脱靶效应。 使用 IP 质谱法揭示整倍体和非整倍体之间命中蛋白结合的任何差异 接下来，我们将进行 RT- 分析。 qPCR 和 IF 筛选非整倍体相关表型，包括染色体错误分离、蛋白质组学 我们将进行额外的后续实验来揭示它们的作用。 非整倍性靶向机制并更好地了解非整倍性引起的靶向应激源。 这段时间我将接受CRISPR筛选、生物信息分析和质谱技术方面的培训。 非整倍体依赖性和染色体特异性非整倍体依赖性可以作为有希望的目标 用于非整倍体癌症治疗的开发。