Mechanisms of generation and maintenance of extrachromosomal DNA in lung cancer

DNA损伤和DNA修复效率影响ecDNA生物合成，并在肿瘤演变过程中调节癌基因激活。通过敲除DNA修复基因或用化疗药物治疗来损害DNA修复将使得能够研究它们对ecDNA形成、维持和肿瘤演变的贡献。我们将使用荧光原位杂交与癌基因特异性探针的组合来研究候选DNA修复基因的缺失对ecDNA形成和维持的影响，在含有ecDNA的NSCLC细胞系中，使用癌基因特异性探针和基于荧光的ecDNA特异性报告基因的空间转录组学（4，5）。目标二：我们将研究化疗过程中ecDNA的形成和稳定性，以拓宽我们对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。工作计划第一年：学生将培养一组已知在ecDNA上携带EGFR的正常肺和肺癌细胞系，如HCC 827和H460。将优化转染条件，以使用CRISPR在这些细胞系中实现基因敲除。还将优化DNA损伤修复抑制药物（DDRi）的最佳浓度，以建立具有最小毒性的合适工作浓度。还将建立几种方法来跟踪ecDNA的形成和稳定性。为了使ecDNA扩增的基本特征，学生将使用细胞遗传学方法测量ecDNA的水平，如荧光原位杂交探针到癌基因，如EGFR和Myc。与此同时，根据最近的出版物，将对量化ecDNA形成的更高通量方法进行研究（4，5）。死Cas9系统将用于用荧光蛋白特异性靶向含有致癌基因的ecDNA，以允许通过免疫荧光或FACS进行检测（5）。或者，可以在ecDNA上插入特异性荧光报告基因（4）。如果我们用这些方法不成功，可以建立ecDNA探针和RNA探针，用于通过免疫荧光在细胞核中空间检测ecDNA/ ecDNA转录物（4;可在AZ获得的RNAView技术）。这种高通量策略将允许在第2/3年筛选ecDNA生物发生和维持的决定因素。第2年：阿斯利康提供了一个包含约100个参与DNA损伤修复（DDR）的基因的CRISPR文库，并已在PC 9肺细胞上成功测试了主要DDR基因的敲除。我们将包括39个潜在的新的DDR基因中确定TRACERx到这个库。利用我们的高通量ecDNA检测分析，我们将筛选这个组合库，以评估DDR对ecDNA形成和维持的贡献。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形成和维持的贡献和调节。第3年：在第3年的CRISPR和药物筛选中鉴定的DDR基因将使用额外的含有ecDNA的细胞系和不含有ecDNA的细胞系进行验证，同时使用下调和敲除策略。第四年：探索ecDNA形成对耐药性的功能性后果，特别是肺癌对受体酪氨酸激酶抑制剂（TKI）如吉非替尼、厄洛替尼和奥希替尼的耐药性。学生将追踪在TKI存在下细胞增殖过程中ecDNA结构的分离和定位，以及它们通过调节含EGFR的ecDNA水平而产生抗性的能力。将评估与DDRi或前几年鉴定的基因的组合策略对ecDNA依赖性耐药性的影响。

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.