Aims

The main aims of the EpiDRIVERS project are:

Work Package 1: Identify epidriver candidates associated with tumorigenesis and cancer phenotypes. EGM will expand its preliminary CRISPR/Cas9 screen to additional cell types and phenotypic readouts, by taking advantage of its custom lentiviral CRISPR library (consisting of 1649 gRNAs targeting all 426 ERGs; Halaburkova et al., 2020), and perform its screens in additional non-tumorigenic and tumorigenic cell lines, with a focus on cancer sites studied in other EGM priority projects (including breast, colon, and oesophageal cancer and B-cell and Burkitt lymphoma). Because EGM’s recent in silico data mining shows a significant number of epidrivers that may be overexpressed, through gene amplification or other non-mutational mechanisms, the CRISPR/Cas9 (loss-of-function) screens (Halaburkova et al., 2020) will be complemented with recently developed CRISPRa approaches (Josipović et al., 2019). Immortalized and cancer cells will be targeted through CRISPR/Cas9 or CRISPR/dCas9 using a sgRNA library and specific sgRNAs, respectively, and assessed for the acquisition of cancer hallmarks characteristic of transformed cells. The top epidriver candidates resulting from these screens will be taken further for in-depth functional characterization (see Work Package 2).

Work Package 2: Perform in-depth functional characterization of individual epidriver candidates by multiparametric phenotyping and organoid models. The top epidriver candidates resulting from the screens in Work Package 1 that also show a high rate of deregulation in data mining of clinical samples and a high driver score (based on EGM’s driver prediction algorithm) will be subjected to a detailed functional characterization, using the tools for high-resolution morphological and functional phenotyping (Figure 4). First, changes in the cell phenotype of cell clones harbouring individual epidrivers (disrupted by CRISPR or CRISPRa) will be examined using an Operetta High-Content Imaging System (PerkinElmer), which allows fully automated image acquisition of multiple phenotypic features or markers (collaboration with the Cancer Research Centre of Lyon [CRCL]). In addition, EGM will perform different functional assays that enable detection of the acquisition of new oncogenic features and phenotypes, such as self-sufficiency in growth signals, insensitivity to anti-growth signals, invasion, evasion of apoptosis, deregulated metabolism, anchorage-independent growth, and the ability to induce tumours (and metastasis) in mice xenograft experiments. For example, EGM has identified several epidriver genes related to the epithelial-to-mesenchymal transition (EMT) in a preliminary study (Halaburkova et al., 2020). EGM will also consider validating and expanding these findings by testing whether disruption of specific epidrivers will confer to the cells stemness properties by applying single-cell (epi)genomics analysis (see Work Package 3) and evaluating their capacity to reconstitute tumour heterogeneity in organoid cultures.

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Figure 4. Experimental study design. Schematic representation of different experimental steps aimed at identifying and characterizing epidriver genes involved in tumorigenesis and cancer cell phenotypes.

EGM will also implement the organoid culture, microscopic self-organizing three-dimensional structures starting from a single stem cell (or stem-like cells) that remain genetically stable over a long time period and recapitulate more closely many structural and functional aspects of their in vivo counterparts. This work will be done in collaboration with Dr Matteo Boretto (Hubrecht Institute, Utrecht, Netherlands), Dr Alain Puisieux (Institut Curie, Paris, France), and Dr Maria Ouzounova and Dr Julie Caramel (CRCL, Lyon, France), who are involved in the LYriCAN project (one work package of which is aimed at investigating epidrivers in tumour cell plasticity).

Work Package 3: Investigate the effect of epidriver deregulation on the epigenome reconfiguration and transcriptional reprogramming of cells. The products of ERGs are involved in the tight control of the gene expression programmes required for the establishment and maintenance of cell identity; therefore, EGM will investigate the (epi)genome remodelling resulting from deregulation of epidrivers. To this end, epidrivers selected according to those main readouts will be taken further for downstream assessment of their mechanisms based on their known or putative functional role (e.g. DNA methylation, histone modifications). Cells with an individual targeted epidriver (or combinations thereof) will be subjected to several analyses: (i) DNA methylome patterns will be studied with BeadChip arrays (Illumina 850K, established by EGM); (ii) open chromatin regions will be mapped using ATAC-seq (collaboration with Dr Emmanuel Bachy, INSERM/CNRS Lyon-Sud) to define changes in accessibility to chromatin regions after disrupting specific epidrivers (motif analysis will be performed on these regions to identify consensus binding signatures for ERG-specific transcription factor binding); (iii) changes in histone marks associated with chromatin accessibility states (open vs repressed chromatin) and regulatory elements (enhancers, promoters, and gene bodies) will be investigated using ChIP-seq; (iv) RNA-seq will be performed, and differentially expressed genes (DEGs) between targeted and untargeted cells will be identified as well as differentially enriched pathways and biological processes (GO and KEGG enrichment analyses); and (v) based on the genome-wide data (DNA methylation, histone modifications, chromatin accessibility, and transcriptome), EGM will perform an integrative bioinformatics analysis of changes that are associated with specific epidriver events and phenotypic features. This will be complemented by genome (whole-genome sequencing), epigenome (DNA methylome), chromatin accessibility (ATAC-seq), and transcriptome (RNA-seq) profiling of carcinogen-immortalized and corresponding primary cells (described under the MUTSPEC project).

Work Package 4: Test the role of epidrivers in potentiating genotoxic and non-genotoxic agents in tumorigenesis. To test whether changes in chromatin features associated with epidriver deregulation may determine the impact of specific carcinogens, EGM will use human and mouse cell-based models of exposure that monitor early steps of cell transformation (such as biological barrier bypass for immortalization, anchorage-independent growth, and EMT). This is based on the premise that the presence of an epidriver deregulation could further aggravate the effects of environmental mutagens or other non-mutagenic carcinogens. This notion is supported by the finding that mutation density in tumours is negatively correlated with open chromatin regions, consistent with the hypothesis that epigenetic modifications are involved in DNA repair (Murr et al., 2006). EGM will first assess whether immortalized non-tumorigenic cell lines and established organoids harbouring inactivation of single epidriver genes are prone to transformation after controlled exposure to specific carcinogens (Table 1). EGM will further investigate how the cells exhibiting individual deregulated epidrivers (or specific combinations thereof) and exposed to specific carcinogens differ from the control cells in terms of chromatin organization (chromatin accessibility and structure, by ATAC-seq and ChIP-seq), gene expression (RNA-seq), and mutation profile (whole-genome sequencing), and how this affects subsequent cellular transformation and cell phenotypes.

Table 1. Established carcinogens and other agents included in testing the potentiating role of epidrivers in carcinogenesis and representing compounds selected by EGM (among which are included several priority exposures that relate to the EpiEARLY, EpiMARKS, and EpiESCC projects)