Functions of the KDM5C and KDM5D tumour suppressor genes in clear cell renal cell carcinoma

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Epigenetic control of chromatin function governs gene transcription patterns that are maintained and transmitted to daughter cells to preserve human health. Breakdown of this control in cancer is evidenced by the predominance of mutations in genes regulating chromatin and the post-translational modification of histones. This project investigates the functional consequences of combined mutations in the VHL tumour suppressor gene and the sex chromosome-linked KDM5C and KDM5D tumour suppressor genes in the context of clear cell renal cell carcinoma (ccRCC), the most frequent form of kidney cancer. Genomic analyses have revealed that ccRCC has a unique genetic make-up, with almost all tumours exhibiting mutations in VHL, as well as one or more mutations in a series of genes that regulate different epigenetic processes, including the X-chromosome gene KDM5C, which is mutated in approximately 20% of cases of metastatic ccRCC, and KDM5D which is frequently lost in male ccRCC cases due to somatic loss of the Y-chromosome. The VHL protein indirectly regulates methylation of histone H3 lysine 27 via HIF-α-dependent mechanisms and the highly homologous KDM5C/D proteins encode histone H3 lysine 4 di-/tri-methyl demethylases. It is however currently not understood if and how cooperative mutations in these tumour suppressor genes affect transcriptional outputs or cellular and organismal phenotypes. We will undertake a reductive experimental approach to simplify the complexity of ccRCC tumours using clean genetics to address these issues. This will involve the generation of several new mouse models to delete Kdm5c and/or Kdm5d in renal epithelial cells alone or together with Vhl to analyse the effects on renal epithelial cell homeostasis and tumour formation. To complement these mouse-based experiments we will use CRISPR-Cas9 mutagenesis to introduce KDM5C mutation into a series of female human ccRCC cell lines and KDM5C/KDM5D single and double mutations into a series of male ccRCC cell lines. The availability of primary cells and tumour cell lines from our various mouse lines, as well as engineered ccRCC cells, will provide relevant material to analyse the effects of these genetic alterations on cancer-relevant cellular phenotypes using cell culture and xenograft assays and will also allow detailed molecular analyses involving RNA-Seq, ChIP-Seq and Exome-Seq to characterise the effects of these mutations on histone modifications and on transcriptional and mutational profiles. Using publically available gene expression and mutation databases from human ccRCCs and archival ccRCC tissue resources, we aim correlate the findings from our experimental systems to human ccRCC tumours. These studies are expected to lay the foundation for a better understanding of the pathogenesis of a molecular subset of ccRCC and to provide cellular and mouse models that will be highly useful for the identification and testing of genotype-specific therapies in future studies.