Discovery of kinase targets in chromosome 10 deleted glioma cells

发现 10 号染色体缺失的神经胶质瘤细胞中的激酶靶标

• 批准号: 9343972
• 负责人: Jayne Stommel
• 金额: $ 3.7万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9343972
• 关键词: 10q; Agar; Anoikis; Apoptosis; Astrocytes; Back; Biological Assay; CCR; Cell Death; Cell Fraction; Cell Line; Cell Proliferation; Cell Survival; Cells; Chromosomal Loss; Chromosome Deletion; Chromosomes; Chromosomes, Human, Pair 10; Collaborations; Data; Data Set; Drug Targeting; Family; Gene Expression; Genes; Genetic Screening; Genomic DNA; Glioma; Growth; Harvest; Histologic; Human; Immunocompromised Host; Implant; In Vitro; Infection; Laboratories; Libraries; Malignant Neoplasms; Measures; Medical Oncology; Memorial Sloan-Kettering Cancer Center; Methods; Mus; Outcome; Phosphotransferases; Preparation; Process; Prostate; Protein Kinase; Puromycin; Sampling; Signal Pathway; Signal Transduction; Stem cells; Testing; Time; Toxic effect; Tumor Suppressor Proteins; United States National Institutes of Health; Validation; Viral; Virus; cancer genetics; candidate validation; cell growth; chromosome 10 loss; comparative genomic hybridization; design; experience; in vivo; knock-down; melanoma; migration; neoplastic cell; novel therapeutics; research study; restoration; small hairpin RNA; small molecule inhibitor; stem; targeted agent; therapeutic target; transduction efficiency; tumor; tumor xenograft

Thus far, we have been in the process of performing experiments in preparation for the screen. We have performed array CGH on a panel of glioma stem-like cell lines that were obtained from Dr. Cameron Brennan (TS600, TS603, TS616, MSKCC) or Dr. Phil Tofilon (GBAM1, NCI ROB), and an immortalized normal human astrocyte line which will be used as a control. We determined that two glioma stem cell lines have a single copy of chromosome 10 (TS600 and TS616), one has a single copy deletion of 10q (TS543), and three cell lines have primarily intact chromosome 10 (TS603, GBAM1). There are 15 kinases on chromosome 10 representing 15 kinase families. We have also performed extensive optimization of conditions under which the stem-like cells will be infected with the murine stem cell virus (MSCV) hairpin library. We have established a viral purification and infection method that reduces stem-like cell toxicity and differentiation, while optimizing transduction efficiency. Validating chromosome 10 kinase status in GBM stem-like cell lines. We will first measure the gene expression of the 16 protein kinases on chromosome 10 in the deleted and intact cells in Table 2 by real time quantitative PCR. We will then compare these results to the array CGH data in Figure 2 to validate that single copy loss of chromosome 10 coincides with reduced expression levels of the deleted kinases. Synthetic lethal kinase screen in chromosome 10-deleted glioma stem-like cells. We will perform a pooled shRNA screen with a validated library of hairpins directed against the entire kinase family in collaboration with Dr. Ji Luo of the NCI Medical Oncology Branch. Dr. Luo has extensive experience with large-scale genetic screens and will provide the library and assistance with the design of the experiment. The glioma stem-like cells will each be infected with the kinase hairpin pool in triplicate at an MOI of 1-2 and an average representation of 1,000 infected cells per shRNA. Virus will be removed 24 hrs later, and a fraction of the cells will be collected at 48 post-infection and saved for later sequencing as the initial samples (PD=0). Puromycin-selected cells will then be continuously passaged until final harvesting at PD=12. Genomic DNA will be extracted from the PD=0 and PD=12 cells, and shRNAs will be PCR-amplified and sequenced with Solexa sequencing by the NIH CCR Cancer Genetics Branch sequencing facility. A false-discovery rate of 20% as well as a 2-fold shRNA decrease cutoff will be applied to the dataset for each cell line to identify shRNAs which reduce cell viability in the screen. Validation of candidate kinases for on-target effects. Kinase shRNAs absent from the chromosome 10 deleted cell lines will be further analyzed to determine whether their absence is due to synthetic lethality or cell death due to off-target effects of the hairpin. Candidate kinases will be inhibited individually with commercially-available siRNAs or pharmacological agents and cell viability will be compared between chromosome 10 intact and deleted cells. Identification of the critical redundant kinases lost on chromosome 10. To determine the identity of the lost kinases that are redundant with the positive hits in the shRNA screen, we will add back each kinase in Table 2 to the chromosome 10 deleted cells and assess viability upon knockdown of the candidate kinase. Validating the synthetically lethal kinase as a therapeutic target in chromosome 10-deleted tumors. We will enlist the candidate kinases in multiple in vitro and in vivo assays to determine its relevance as a potential therapeutic target. We will perform validation assays for cell proliferation, apoptosis, invasion, anoikis, and soft agar growth that are routinely performed in our laboratory. We will also determine the feasibility of therapeutically targeting the candidate kinases in cancers other than GBM by performing these assays in additional tumor lines with frequent deletions of chromosome 10, such as prostate and melanoma. An advantage of using the glioma stem-like cells in this screen is that they generate orthotopic xenograft tumors in immunocompromised mice that are indistinguishable histologically from human gliomas. Therefore, to determine the relevance of the synthetically lethal kinases as drug targets, we will measure tumor formation and overall survival in mice implanted with glioma stem-like cells in which the kinases are inhibited with shRNAs or small molecule inhibitors, and compare these outcomes between the chromosome 10-deleted and intact cells.

到目前为止，我们一直在进行实验以准备屏幕。我们已经在胶质瘤干细胞系上进行了阵列CGH，该细胞系从Cameron Brennan博士（TS600，TS603，TS603，TS616，MSKCC）或Phil Tofilon博士（GBAM1，NCI ROB）获得，以及不代之一的正常人类星形细胞线，该线将作为对照使用。我们确定两种神经胶质瘤干细胞系具有单个染色体10（TS600和TS616）的副本，一个副本具有10Q（TS543）的单副本删除（TS543），并且三个细胞系主要具有完整的染色体10（TS603，GBAM1）。 10染色体上有15种激酶，代表15个激酶家族。我们还对条件进行了广泛的优化，在这些条件下，干细胞将被鼠类干细胞病毒（MSCV）发夹库感染。我们已经建立了一种病毒纯化和感染方法，可降低茎样细胞的毒性和分化，同时优化转导效率。在GBM茎样细胞系中验证染色体10激酶状态。通过实时定量PCR，我们将首先测量表2中已删除和完整细胞中染色体10的16种蛋白激酶的基因表达。然后，我们将将这些结果与图2中的阵列CGH数据进行比较，以验证染色体10的单个拷贝丢失与已删除激酶的表达水平降低相吻合。染色体10删除神经胶质瘤干细胞中的合成致命激酶筛选。我们将与NCI医学肿瘤学分支的Ji Luo博士合作，使用针对整个激酶家族的经过验证的发夹库进行合并的shrna屏幕。 Luo博士在大规模的遗传筛选方面拥有丰富的经验，并将为实验设计提供图书馆和协助。胶质瘤茎样细胞将分别以1-2的MOI三份被激酶发夹池感染，每个shRNA的平均表现为1,000个感染细胞。 24小时后将去除病毒，并在感染后48点收集一部分细胞，并保存以作为初始样品（PD = 0）进行测序。然后，纯霉素选择的细胞将连续传递，直到最终收获为Pd = 12。基因组DNA将从PD = 0和PD = 12个细胞中提取，并将通过NIH CCR癌症遗传学分支测序设施对SHRNA进行PCR扩增并通过Solexa测序进行测序。对于每个细胞系，将应用于数据集的虚假发现率为20％以及2倍SHRNA降低的截止率，以识别降低屏幕中细胞活力的shRNA。验证候选激酶对靶向效应的验证。将进一步分析10个缺失的细胞系中缺乏的激酶SHRNA，以确定它们的缺失是由于发夹的非靶向作用而导致的合成致死性或细胞死亡。候选激酶将被市售的siRNA或药理学剂单独抑制，并且将比较10个完整细胞和已删除细胞之间的细胞活力。鉴定染色体10上丢失的临界冗余激酶。为了确定丢失的激酶的身份，这些激酶与ShRNA筛网中的正命中率相关，我们将在表2中的每个激酶在染色体10已删除的细胞中添加回来，并评估候选激酶敲低时的短暂性。验证合成性致命激酶作为染色体10删除肿瘤的治疗靶标。我们将在多个体外和体内测定中征收候选激酶，以确定其作为潜在治疗靶点的相关性。我们将对我们的实验室通常执行的细胞增殖，凋亡，入侵，Anoikis和软琼脂生长进行验证测定。我们还将通过在频繁缺失10号染色体10的其他肿瘤系中（例如前列腺和黑色素瘤）中进行这些测定法，从而确定除GBM以外的其他癌症中候选激酶的可行性。在此屏幕中使用神经胶质瘤干细胞的一个优点是它们在免疫功能低下的小鼠中产生原位异种移植肿瘤，这些小鼠与人神经胶质瘤在组织学上是无法区分的。因此，为了确定合成性致命激酶作为药物靶标的相关性，我们将测量植入胶质瘤干细胞的小鼠中的肿瘤形成和总生存率，其中激酶被shrnas或小分子抑制剂抑制，并比较10止卵母细胞和完整细胞之间的这些脱位。