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: 10464002
• 负责人: Klaske Marijke Schukken
• 金额: $ 6.76万
• 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/10464002
• 关键词: Affect; Aneuploid Cells; Aneuploidy; Apoptosis; Binding Proteins; Bioinformatics; Biological; CRISPR library; CRISPR screen; Cancer cell line; Cell Line; Cell division; Cell physiology; Cells; Cellular Stress; Chromosome 7; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Colon Carcinoma; Colorectal Cancer; Communities; Complementary DNA; Data; Defect; Dependence; Diploidy; Drug Screening; Drug Targeting; Drug resistance; Embryo; Environment; Epigenetic Process; Exhibits; Family; Fibroblasts; Frequencies; Funding; Gene Dosage; Genes; Genetic; Genetic Screening; Goals; Growth; Human; Human Cell Line; Knock-out; Knowledge; Libraries; Light; Malignant Neoplasms; Malignant neoplasm of ovary; Mass Spectrum Analysis; Metabolic; 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; career; career development; chemotherapy; chromosome missegregation; congenic; design; experimental study; follow-up; gene function; inhibitor; insight; knockout gene; malignant breast neoplasm; medical schools; screening; secondary analysis; 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.

项目摘要 非整倍体是一种细胞状态，在这种状态下，细胞含有额外或缺失的染色体。90%以上的实体瘤 都是非整倍体。非整倍体已被证明与耐药和转移有关，而非整倍体 癌症的患者存活率比整倍体癌症更差。尽管非整倍体在癌症中的作用，但非整倍体 它本身会导致生长缺陷，并在细胞内诱导几个持续的应激源。得或失 染色体导致转录和蛋白质组应激，代谢失调，分泌组改变，以及 导致进一步的染色体错误分离。我们假设非整倍体诱导的细胞应激可能是 我们的目标是专门消除非整倍体细胞，我们的目标是发现特定的遗传依赖性 到非整倍体细胞。我们将使用CRISPR来筛选多个近整倍体人类细胞系和非整倍体克隆 我们从这些接近整倍体的细胞系中衍生出来的。然后我们将比较非整倍体对基因的影响 依赖性，独立于细胞系特定的效应。为了实现这个目标，我们已经产生了54个非整倍体克隆 从9个接近整倍体的人类癌细胞系中，我们获得了另外10个来自 两个人类细胞系。我们将筛选这些非整倍体克隆，以及它们的近整倍体对照，带有一个结构域- 特定的CRISPR文库。该文库针对多个可药物的蛋白结构域，包括所有的激酶， 泛素酶、转录因子、表观遗传调节剂、“皇室”表观遗传因子、蛋白酶和泛素 连接酶基因。在初步工作中，我们筛选了多个整倍体和非整倍体细胞系，并用较小的 我们鉴定了几个潜在的非整倍体特异性依赖关系。 将对更多的非整倍体癌细胞株及其相应的对照进行筛选，以确认这些潜力 点击率。癌症的非整倍体不是完全随机的，因为特定的染色体获得和丢失是在 某些癌症类型。除了发现一般的非整倍体依赖关系外，我们的目标是发现染色体 特定的依赖项。一旦我们发现了潜在的非整倍体依赖关系，我们将验证它们的 非整倍体的特异性，然后我们将用cDNA来挽救基因敲除，排除非靶点效应。我们会 使用电感耦合等离子体质谱揭示整倍体和非整倍体之间HIT蛋白结合的任何差异 条件，或确定特征不佳的基因的蛋白质结合伙伴。接下来，我们将执行RT- QPCR和IF筛查非整倍体相关表型，包括染色体错误分离、蛋白质组学 应激、衰老和细胞凋亡。将进行更多的后续实验，以发现他们的 非整倍体靶向机制和更好地理解非整倍体引起的靶向应激源。在.期间 在此期间，我将接受CRISPR筛查、生物信息学分析和质谱学技术方面的培训。 非整倍体依赖和特定于染色体的非整倍体依赖可以作为有希望的目标 用于非整倍体癌症治疗的发展。