Drug target development and analysis of genome stability in cancer cells lacking the BAF180 subunit of the PBAF remodelling complex

In eukaryotes, DNA is packaged into a highly condensed structure, known as chromatin. Several complexes facilitate the remodelling of chromatin, for example, INO80, NURD and SWI/SNF, which attach to tightly bound chromatin, allowing its relaxation by nucleosome sliding, unwrapping, histone eviction and exchange of histone variants. The activities carried out by these chromatin remodelling complexes are thought to be integral in the prevention of cancer cell formation. Recently, whole exome sequencing has identified frequent mutations in subunits of the SWI/SNF chromatin remodelling complex, at a frequencythat rivals p53. Strikingly, the BAF180 (PBRM1) subunit of the PBAF variant of SWI/SNF remodelers is mutated in over 40% of clear cell renal cell carcinoma (ccRCC), a cancer with typically poor prognosis and limited treatment options to date.This work embodiesfour main results chapters that aim to identify novel synthetic lethal gene candidates with BAF180, with a view to targeting these gene candidates with chemotherapeutic drugs. In the first chapter we work through a short list of hypothesis driven potential synthetic lethal candidates and identify the genes KAT2A, RNF4, EZH2 and BAP1 as potential synthetic lethal partners for BAF180. Chapter twodescribes the development of both stableshRNAand CRISPR/Cas9-derived BAF180-deficient cell lines that were used both inthis studyas well as for other ongoing projects. The third chapter outlines the set-up of a high-throughput synthetic lethal siRNA (HTS) screen and determinespotential synthetic lethal interactions identified here. The final chapter examinesvarious PARP genes, identified ashits in HTS screening, to further explore the interaction between PARP and BAF180. We find that PARP1 and PARP3are synthetic lethal with BAF180 and treatment with various siRNA’s and PARP inhibitors in BAF180 deficient mammalian cells results in specific cell death. A phenotype that could be clinically exploited for treatment of ccRCC.