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.

项目摘要 非整倍性是细胞含有额外或缺失染色体的细胞状态。超过90％的实体瘤 是非整倍体。已证明非整倍性有助于耐药性和转移，而非整倍性 癌症患者的存活率比整倍体癌症差。尽管非整倍性在癌症中的作用，但非整倍性 本身会导致生长缺陷，并引起细胞内的几个持续应力源。获得或输 染色体会导致转录组和蛋白质组学胁迫，代谢失调，分泌剂改变，并且 诱导进一步的染色体错误分离。我们假设非整倍性诱导的细胞应力可以是 针对特异性消除非整倍性细胞的针对性，我们旨在发现特定的遗传依赖性 到非整倍体细胞。我们将使用CRISPR筛选多个近欧上型人类细胞系和非整倍型克隆 我们源自这些近乎整倍的细胞系。然后，我们将比较非整倍性对基因的影响 依赖，与细胞系特异性效应无关。为了实现这一目标，我们产生了54个非整倍性克隆 从九个近乎整倍体的人类癌细胞系中，我们可以从其他十个非整倍型克隆中 两个人类细胞系。我们将使用域 - 特定的CRISPR库。该库针对多个可药物蛋白质结构域，包括所有激酶， 泛素酶，转录因子，表观遗传调节剂，“王室”表观遗传因子，蛋白酶和泛素 连接酶基因。在初步工作中，我们已经筛选了多个多倍体和非整倍性细胞系，较小 激酶结构域的CRISPR库，我们确定了几个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 命中。癌症非整倍性不是完全随机的，因为选择了特定的染色体增长和损失 某些癌症类型。除了发现一般的非整倍体依赖性外，我们还旨在发现染色体 特定依赖性。一旦我们发现了潜在的非整倍依赖性，我们将验证它们 非整倍体特异性，然后我们将使用cDNA挽救基因敲除并排除脱靶效应。我们将 使用IP质谱法揭示了Euploid和Anuploid之间的HIT蛋白结合的任何差异 条件，或确定特征较差的基因的蛋白结合伴侣。接下来，我们将执行RT- QPCR，如果要筛选针对非整倍性相关的表型，包括染色体错误分类，蛋白质组学 压力，感应和凋亡。将进行其他后续实验，以发现他们的 非整倍性靶向机制，并更好地了解非整倍性引起的靶向应力源。期间 在此期间，我将接受CRISPR筛查，生物信息学分析和质谱技术的培训。 非整倍性依赖性和染色体特异性的非整倍体依赖性可以作为承诺的目标使用 用于非整倍体癌热发育。