Using CRISPR screening to uncover aneuploidy-specific genetic dependencies

项目摘要 非整倍性是一种细胞状态，其中细胞含有额外或缺失的染色体。超过90%的实体瘤 是非整倍体非整倍体已被证明有助于耐药性和转移， 癌症的患者存活率比整倍体癌症差。尽管非整倍体在癌症中的作用， 其本身引起生长缺陷并在细胞内诱导几种持续的应激源。获得或失去 染色体导致转录组和蛋白质组应激、代谢失调、分泌组改变， 诱导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以 有针对性地消除非整倍体细胞，我们的目标是发现特定的遗传依赖性， 非整倍体细胞。我们将利用CRISPR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获得。然后我们将比较非整倍体对基因的影响， 依赖性，独立于细胞系特异性效应。为了实现这一目标，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，我们可以获得另外10个非整倍体克隆， 两种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，用一个结构域- CRISPR文库。该文库靶向多个可药物化的蛋白质结构域，包括所有激酶， 泛素酶、转录因子、表观遗传调节因子、“皇家家族”表观遗传因子、蛋白酶和泛素 连接酶基因在前期工作中，我们筛选了多个整倍体和非整倍体细胞系， 我们在激酶结构域聚焦的CRISPR文库中，鉴定了几种潜在的非整倍体特异性依赖性。 更多的非整倍体癌细胞系及其相应的对照将被筛选以确认这些潜力 点击率了癌症非整倍体并不完全是随机的，因为特定的染色体获得和丢失是有选择的， 某些癌症类型。除了揭示一般的非整倍体依赖性，我们的目标是揭示染色体 具体的依赖性。一旦我们发现了潜在的非整倍体依赖性，我们将验证它们的 然后我们将使用cDNA来拯救基因敲除并排除脱靶效应。我们将 使用IP质谱来揭示整倍体和非整倍体之间命中蛋白结合的任何差异 条件下，或确定蛋白质结合伴侣的不良特征的基因。接下来，我们将进行RT- qPCR和IF用于筛选非整倍体相关表型，包括染色体错误分离、蛋白质组学 应激、衰老和凋亡。将进行额外的后续实验，以揭示其 非整倍体靶向机制，并更好地了解非整倍体诱导的靶向应激源。期间 在此期间，我将接受CRISPR筛选，生物信息学分析和质谱技术的培训。 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靶点 用于非整倍体癌症的治疗开发。

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.