In order to circumvent evolutionary selection and resistance to environmental context and anti-cancer agents, novel approaches that result in synthetic (synergistic) cell lethality (dependencies) that are specific for the cancer cell are most likely to have translational impact. The aim of this project is to discover and evaluate mechanisms of synthetic lethality in cancers derived from mesenchyme (sarcoma). Translation of NGS sequencing observations in cancer has resulted in the discovery of numerous gain-of-function driver variants, only some of which have predicted functional impact when inhibited. Targeting of loss-of-function variants has proved to be more challenging, resulting in forward genetic approaches with large-scale pan-cancer single gene CRISPR/Cas9 screens. Importantly, genomic copy number context, paralogs and the epigenome are all factors that influence the context of synthetic lethality and have identified functional targets for translational validation. Variants and their epistatic context that exist in all cell clones are preferred targets because of tumour evolutionary conservation, and so their identification offer important potential for mechanism based synthetic lethality.
Cancers derived from mesenchyme are rare, poorly studied and have unmet need compared to common cancers. In humans, there are approximately 80 different genotype-phenotype ‘sarcoma’ diagnostic categories. Importantly, these tumours have unique genotype-phenotype correlation, with specific independent genomic driver mechanisms based on mutation, genomic amplification, structural variants, deletions, chromosomal loss, loss of heterozygosity associated with tumour suppressor genes, chimeric functional proteins through chromosomal translocations and associated differentiation (e.g. bone, muscle, nerve, fat, fibrous tissue, blood vessels). Here, the experimental focus is modified CRISPR/Cas12a screens in mesenchymal cell lines with specific genomic contexts, such as with and without addition of selective modifiers of chromatin, DNA repair and signalling. Unbiased and novel designs of essentialome screens in defined contexts, ideally using a combination of inducible CRISPRi and Cas12a with single and dual (multiplex) guide RNA libraries, will be applied to representative specific WGS sequenced cell lines. Single cell CRISPR approaches that combine forward and reverse genetic approaches may be considered in order to enable discovery of potential variant type-specific synthetic lethality mechanisms. For example, mechanisms under current evaluation in the laboratory include: Ewing sarcoma (ES), driven by a transcriptional gain-of-function translocation (EWSR1-FLI1) where context dependencies through chromatin and transcriptional repression also establish signalling lethality dependencies that can be co-targeted, and lethality dependencies in malignant peripheral nerve sheath tumours with PRC2 complex loss of function in the genomic context of loss-of-function of the NF1 tumour suppressor. The student will join a multi-disciplinary laboratory with day-to-day post-doctoral supervision, gain experience in cancer biology, genome engineering, genomics, bioinformatics and translational research.