Controlling survival of aneuploid cancer cells through UBUK, an emerging ubiquitin ligase module

BACKGROUND A balanced (euploid) chromosome number is a key feature of normal cells in our body.

However, aneuploidy (an unbalanced chromosome number) is highly prevalent in tumours and drives cancer progression and drug resistance. When a euploid cell becomes aneuploid, the cell dies due to genotoxic and proteotoxic stress. Cancer aneuploid cells must thus employ mechanisms to ensure their survival.

Recent evidence shows four ubiquitin ligases UBA6, BIRC6, UBR4, KCMF1 (UBUK) are required for survival of aneuploidy cancer cells through uncharacterised processes. We have uncovered how BIRC6 ubiquitinates and inhibits caspases in vitro. UBR4 and KCMF1 form an N-degron E3 ligase pair recognising exposed features on proteins targeting them for degradation.

We will take integrated structural, biochemical and cellular approaches to delineate how UBUK is exploited to enable survival of highly aneuploidy cells.

Our findings will provide critical molecular and cellular understanding of fundamental mechanisms of UBUK regulation and function and will facilitate and inform future small molecule screening efforts to inhibit this promising therapeutic target.

AIMS We will delineate how UBUK is exploited in aneuploidy cancer cells through four interrelated aims dissecting module components individually and as whole.

First we will screen a panel of dependent and non-dependent cell lines to determine the relationship with UBUK and cell death pathways.

Second, we will couple detailed biochemical and structural understanding of BIRC6 function with characterisation studies in cellulo. Third, we will determine the first UBR4/KCMF1 interactome and substrate profile. Lastly, we will establish molecular and functional cross-talk between BIRC6 and UBR4.

METHOD We will use complementary structural biology techniques (single particle cryoEM, NMR) to provide molecular understanding of BIRC6 and UBR4 substrate recognition and mechanisms of action.

We will employ CRISPR/Cas9 genome editing to generate cell lines with endogenously tagged UBUK module components for characterisation in cells.

Finally, we will use advanced proteomics to uncover in depth comparative interactome and substrateomes of BIRC6 and UBR4.

HOW THE RESEARCH WILL BE USED Our research will define the mechanisms by which UBUK functions to alleviate proteotoxic stress and concurrently inhibit cell death.

Our findings will be particularly focussed on these mechanisms in aneuploidy cancer; however, our research will similarly impact on the fundamental understanding of cell survival mechanisms more broadly.

Our studies will catapult future work into design of small molecule inhibitors targeting this ubiquitin ligase module, ultimately offering novel avenues of therapeutic intervention.