MYC, MITOSIS AND SUMOYLATION
Can we define what causes polyploidy when MYC is over expressed in the absence of SAE2?
EXPLOITING THE SUMO PATHWAY TO TARGET MYC-DRIVEN OVARIAN CANCERS
As MYC is a potent oncogene, is amplified in many cancers, and is a super-controller of biogenesis and proliferation, it has attracted a huge amount of attention as an anti-cancer target. Ovarian cancer exhibits possibly the highest frequency of MYC amplification of all cancers; in 579 samples in the TCGA data set with copy number analysis, MYC is amplified in 42%. Therefore, an alternative avenue to target HR-proficient ovarian cancers is to exploit MYC overexpression.
While analysing MYC and mitotic cell fate, we noted that MYC influences an unperturbed mitosis, and while MYC has been implicated in mitotic control, this had not been fully explored. Therefore, we wanted to dissect MYC’s ability to modulate mitosis. To generate a model system whereby MYC is tightly regulated, we first generated MYC-null clones using CRISPR/Cas9, then “flipped in” a tetracycline-responsive MYC transgene. However, following MYC mutation, these cells adapted such that cell cycle control was no longer MYC-dependent. Therefore, we reversed the strategy and first “flipped in” the transgene, then mutated endogenous MYC in the presence of tetracycline to minimise adaptation. Then tetracycline induction modulated doubling times, indicating that MYC-dependent cell cycle controls are intact.
One way by which we have used this model to dissect MYC’s ability to modulate mitosis is isobaric tag for relative and absolute quantification (iTRAQ) proteomics, under MYC-High and MYC-Low conditions (Figure). In samples from cells in G2, we identified 54 proteins involved in various mitotic functions demonstrating that MYC has a pervasive impact on the mitotic proteome. This proteomic screen also identified differentially modulated SUMO signalling components, the SUMO E2 (UBC9) and a SUMO E3 (RANBP2), which are key nodal regulators of multiple mitotic processes. Importantly, it has previously been shown that inhibition of the SUMO E1 enzyme SAE1/2 is synthetic lethal with MYC overexpression.
Targeting MYC directly is challenging because it lacks features typically amenable to small molecule docking, however an alternative strategy is to identify druggable factors that are synthetic lethal with MYC overexpression. In light of this, and our iTRAQ proteomics analysis, we set out to revisit the SAE2-MYC interaction with a view to exploiting SUMO signalling therapeutically in ovarian cancer. Initially, we suppressed SAE2 by RNAi in our tuneable MYC cell line, with striking results revealing polyploidy and apoptosis, specifically in MYC-High cells. We have now evaluated several SAE2 inhibitors including ML-792, a pyrazolecarbonylpyrimidine that inhibits SAE2 in the low nanomolar range, via a SUMO adduct mechanism. Importantly, ML-792 phenocopies siSAE2, potently inducing polyploidy and apoptosis specifically in cells overexpressing MYC.
Moving forwards we are using our tuneable MYC cell line to determine what causes polyploidy when MYC is overexpressed in the absence of SAE2. For example, is it a direct effect on the cell division machinery, or an indirect effect via gene expression? In addition, we will use our patient-derived ex vivo cultures to evaluate this therapeutic strategy in the context of ovarian cancer, using drug-sensitivity profiling combined with multi-omics to confirm sensitivity of ovarian cancers overexpressing MYC. Furthermore, we aim to mine multi-omics datasets from the ex vivo cultures to identify potential molecular signatures predicting which MYC-High tumours are more likely to respond to SUMOylation inhibitors.