Mechanisms of generation and maintenance of extrachromosomal DNA in lung cancer

Project Hypothesis

DNA 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.

Objectives

Aim 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 plan

Year 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.