Identification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1

Thesis

Our lab has recently identified a conserved synthetic lethality between loss of SETD2-dependent H3K36 trimethylation and inhibition of WEE1 kinase (Pfister et al., 2015). In order to explore the possible mechanisms of resistance to WEE1 inhibitor-treatment in H3K36me3-deficient cells, a yeast genetic screen was performed to isolate mutants that suppressed the set2Δ wee1-50 temperature sensitivity. Whole-genome sequencing of 23 spontaneous suppressor mutants revealed mutations in eight genes involved in cell cycle regulation (bub1, cdc2, cut2, cut1, gtb1, myo52, slp1 and wee1), five genes involved in cell communication (gpa1, shk1, ssp2, ste20 and wis1), and seven genes involved in transcription (bdf1, brf1, bye1, epl1, lsm4, paf1 and taf10).

Further analysis in mammalian cells revealed that knockdown of RNA Polymerase II-associated factor 1 (PAF1) partially rescued cell viability at higher concentrations (300 and 600 nM) of the WEE1 inhibitor AZD1775 in both SETD2 wild-type and SETD2 CRISPR knockout U2OS cells. Data were verified in fission yeast as well as in the renal cell carcinoma cell lines 786-O (SETD2 wild-type) and A498 (bearing a truncating mutation in SETD2), indicating functional conservation of Paf1/PAF1-dependent resistance to Wee1/WEE1 inhibition from fission yeast to mammalian cells.

Investigations into the molecular mechanisms responsible for PAF1-mediated suppression in human cells suggested involvement of the CDK inhibitor p21^Cip1/Waf1. Knockdown of PAF1 induced relocalisation of p21^Cip1/Waf1 to the cytoplasm where it is thought to suppress caspase-3 dependent apoptosis as well as the degradation of RRM2 via CDK2 inhibition in the presence of AZD1775. These findings suggest that targeting cytoplasmic p21^Cip1/Waf1 may offer new strategies for the treatment of resistance to WEE1-inhibition and knowledge of this pathway should be taken into account in the design of trials of targeted therapies for H3K36me3-deficient cancers.

Authors: van Bijsterveldt, L

• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford