Cellular Determinants Of AURKA Degradation By Small Molecule Proteolysis-Targeting Chimeras

In recent years the development of proteolysis-targeting chimeras (PROTACs) has enhanced the field of ubiquitin signalling through advancing therapeutic targeted protein degradation (TPD) strategies and generating tools to explore the ubiquitin landscape. However, the cellular factors that modulate PROTAC activity, such as substrate characteristics and their interaction with components of the ubiquitin-proteasome system (UPS), remain poorly understood. This thesis addresses fundamental questions about how cellular factors, such as substrate localisation and the activity of deubiquitinating enzymes (DUBs), influence PROTAC efficacy, using the oncogenic kinase Aurora kinase A (AURKA) as a model substrate. Many small molecule inhibitors of AURKA have failed during clinical development due to dose-limiting toxicities and the emergence of resistance mechanisms. MLN8237 (alisertib) is an example of a high-affinity, selective kinase inhibitor of AURKA which has mostly failed in clinical trials. More recently, kinase-independent functions of AURKA (e.g., oncogenic signalling, transcriptional activation) have been identified. These functions contribute to its oncogenic activity but are not targeted by conventional ATP-competitive inhibitors. TPD using PROTACs offers a promising alternative approach, with the potential to overcome resistance associated with small-molecule inhibitors and to eliminate non-catalytic functions of AURKA that contribute to tumour progression. Previous work characterised small molecule PROTACs targeting AURKA that combine MLN8237 and the CRBN-recruiting ligand pomalidomide via a PEG linker. While the PROTACs caused degradation of AURKA in a proteasome-dependent manner, different pools of AURKA were not equally sensitive to PROTAC-mediated degradation, with centrosomal AURKA showing resistance to degradation. This thesis further investigates how AURKA localisation impacts PROTAC sensitivity. I use fluorescence live-cell imaging to show that forced nuclear-localisation leads to improved degradation of AURKA by either AURKA-targeting or orthogonal PROTACs from the dTAG toolkit that recruit AURKA via an FKBP12F36V tag. Orthogonal tagging experiments reveal this effect to be independent of the recruited E3 but specific to AURKA. Through an siRNA screen to identify deubiquitinating enzymes (DUBs) influencing PROTAC sensitivity of AURKA, I identify several candidate DUBs. One of these, OTUD6A, is able to specifically protect the cytoplasmic pool of AURKA from PROTAC-mediated degradation and its activity is sufficient to explain the enhanced degradation of nuclear-localised AURKA. I also identify a PROTAC-opposing activity of the proteasomal DUB UCHL5 against both AURKA and dTAG PROTACs that is independent of the substrate tested but specific to CRBN-recruiting dTAG PROTACs. I also explore the PROTAC-independent role of OTUD6A in regulating AURKA stability and function in the cell cycle, by examining cellular consequences of modulating OTUD6A expression. I show that OTUD6A maintains steady state AURKA levels and is required for proper mitotic spindle formation. Knockdown of OTUD6A impairs proliferation in breast cancer cell lines, highlighting its potential as a therapeutic target, both alone and in combination with AURKA-targeting PROTACs. Together, this thesis provides new insights into the mechanistic underpinnings of TPD and demonstrates how PROTACs can reveal previously unrecognised regulators of protein stability, such as OTUD6A. The identification of cellular determinants that govern PROTAC sensitivity will be broadly relevant to many other therapeutic targets and disease pathways, and elucidating such determinants will allow for the design of more effective TPD strategies in the future.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Cardno, Annabel

Advisors dc:contributor.advisor

• Lindon, Catherine
• Roberts, Karen

• AURKA
• PROTAC
• Targeted Protein Degradation
• Deubiquitinase

Licence

• https://www.repository.cam.ac.uk/bitstreams/ec4f9716-933f-4b47-ad80-a05edc9b7fe4/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.122339

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/391068