Mechanisms of generation and maintenance of extrachromosomal DNA in lung cancer

项目假设DNA损伤和DNA修复效率影响ecDNA的生物发生，并在肿瘤进化过程中调节癌基因的激活。通过敲除DNA修复基因或用化疗药物治疗来损害DNA修复，将有助于研究它们在ecDNA形成、维持和肿瘤演变中的作用。目的：我们将结合荧光原位杂交与癌基因特异性探针、空间转录与癌基因特异性探针和基于荧光的报告基因在含有ecDNA(4，5)的非小细胞肺癌细胞系中，研究候选DNA修复基因缺失对ecDNA形成和维持的影响。目的2：我们将研究ecDNA在化疗方案中的形成和稳定性，以加深我们对ecDNA的形成及其对肿瘤异质性、进化和靶向药物耐药性的贡献的理解。工作计划1：学生将培养一组已知含有ecDNA上EGFR的正常肺癌和肺癌细胞系，如HCC827和H460。将优化转染条件，以使用CRISPR在这些品系中进行基因敲除。DNA损伤修复抑制药物(DDRI)的最佳浓度也将得到优化，以建立毒性最小的适当工作浓度。还将建立几种方法，以跟踪ecDNA的形成和稳定性。为了能够对ecDNA扩增进行基本表征，学生将使用细胞遗传学方法测量ecDNA水平，例如针对癌基因的荧光原位杂交探针，如EGFR和Myc。同时，在最近的出版物(4，5)之后，将确定用于量化ecDNA形成的更高吞吐量的方法的范围。一个失效的Cas9系统将被用来特异性地靶向含有带有荧光蛋白的癌基因的ecDNA，以允许通过免疫荧光或FACS进行检测(5)。或者，可以在ecDNA(4)上插入特定的荧光报告。如果这些方法不成功，可以建立ecDNA探针和RNA探针，通过免疫荧光对细胞核中的ecDNA/ecDNA转录本进行空间检测(4；可在AZ获得的RNAView技术)。这一高通量策略将允许在第二年/3年筛选ecDNA生物发生和维护的决定因素。第二年：阿斯利康公司提供了一个包含约100个参与DNA损伤修复(DDR)的基因的CRISPR文库，并已在PC9肺细胞上成功测试了主要DDR基因的敲除。我们将把TRACERx中发现的39个潜在的新DDR基因纳入这个文库。利用我们的高通量ecDNA检测方法，我们将筛选这个组合文库，以评估DDR对ecDNA形成和维护的贡献。同时，学生还将测试一些化疗药物，并有针对性地评估它们对ecDNA形成和维护的贡献和调控。这将确定涉及ecDNA生物学的DDR基因。第3年：在CRISPR和药物筛选中确定的DDR基因将使用额外的含有ecDNA的细胞系和含有非ecDNA的细胞系进行验证，同时采用下调和敲除策略。还将进行救援实验，以验证与表型相关的基因和特定基因活性。第4年：探索ecDNA形成对耐药性的功能后果，特别是肺癌对受体酪氨酸激酶抑制剂(TKIs)的耐药性，如吉非替尼、厄洛替尼和奥西美替尼。学生将在TKI存在的情况下追踪细胞增殖过程中ecDNA结构的分离和定位，以及它们通过调节含有EGFR的ecDNA水平而产生抵抗力的能力。将评估与DDRI或前几年确定的基因联合使用策略对ecDNA依赖的耐药性的影响。

Project HypothesisDNA damage and DNA repair efficiency impacts ecDNA biogenesis and regulate oncogene activation during tumour evolution. Impairing DNA repair by knockout of DNA repair genes or treatment with chemotherapeutic drugs will enable to study their contribution of ecDNA formation, maintenance and tumour evolution.ObjectivesAim 1: We will study the impact of depletion of candidate DNA repair genes on ecDNA formation and maintenance using a combination of fluorescence in-situ hybridisation with oncogene specific probes, spatial transcriptomics with oncogene specific probes and fluorescence based reporters specific for ecDNA in NSCLC lines containing ecDNA (4,5). Aim 2: We will study the formation and stability of ecDNA during chemotherapeutic regimens to broaden our understanding of the formation of ecDNA and its contribution to tumour heterogeneity, evolution and resistance to targeted drugs.Work planYear 1: Student will culture a panel of normal lung and lung cancer cell lines known to harbour EGFR on ecDNA such as HCC827 and H460. Transfection conditions will be optimised to enable gene knockout in those lines using CRISPR. Optimal concentration of DNA damage repair inhibitory drugs (DDRi) will also be optimised to establish suitable working concentrations with minimal toxicity. Several approaches will also be established to enable the tracking of ecDNA formation and stability. To enable basic characterisation of ecDNA amplification, the student will measure levels of ecDNA using cytogenetic approaches such a fluorescent in situ hybridisation probes to oncogenes such as EGFR and Myc. In parallel, a higher throughput approach to quantify ecDNA formation will be scoped, following recent publications (4,5). A dead Cas9 system will be used to specifically target ecDNA containing oncogenes with fluorescent proteins to allow detection by immunofluorescence or FACS (5). Alternatively, a specific fluorescence reporter can be inserted on ecDNA (4). If we are not successful with those approaches, ecDNA probes and RNA probes can be established for spatial detection of ecDNA/ ecDNA transcripts in cell nuclei by immunofluorescence (4; RNAView technology available at AZ). This high-throughput strategies will allow to screen for determinants of ecDNA biogenesis and maintenance in year 2/3.Year 2: A CRISPR library of about 100 genes involved in DNA damage repair (DDR) is available at AstraZeneca and it has been successfully tested on PC9 lung cells for the knockout of major DDR genes. We will include the 39 potentially novel DDR genes identified within TRACERx to this library. Using our high throughput ecDNA detection assays, we will screen this combined library to assess the contribution of DDR to formation and maintenance of ecDNA.In parallel, the student will also test a number of chemotherapeutic drug and targeted to assess their contribution and modulation of ecDNA formation and maintenance. This should identify DDR genes involved in ecDNA biology.Year 3: DDR genes identified in the CRISPR and drug screen in year 3 will be validated using additional ecDNA containing cell lines and non ecDNA containing lines with both downregulation and knock out strategies. Rescue experiments will also be performed to validate genes and specific gene activity relevant for the phenotype.Year 4:Explore the functional consequence of ecDNA formation on drug resistance, specifically lung cancer resistance to receptor tyrosine kinase inhibitors (TKIs) such as gefitinib, erlotinib and osimertinib. The student will trace the segregation and localisation of the ecDNA structures during cell proliferation in the presence of TKIs and their ability to develop resistance with modulation of EGFR-containing ecDNA levels. The effect of combination strategies with DDRi or genes identified in the previous years on ecDNA-dependent drug resistance will be evaluated.