Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool

Richard Wang,Ahmed Abdelbaki,Iacovos Michaelides,Alex Fung,Catherine Lindon,Karen Roberts,Tim Rasmusson,Camilla Ascanelli

doi:10.1038/s42003-021-02158-2

Abstract

Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (PROteolysis Targeting Chimeras, PROTACs) that recruit ubiquitin ligases to targets of interest, leading to ubiquitination and destruction of the targets. Several PROTACs against targets of clinical interest have been described, but detailed descriptions of the cell biology modulated by PROTACs are missing from the literature. Here we describe the functional characterization of a PROTAC derived from AURKA inhibitor MLN8237 (alisertib). We demonstrate efficient and specific destruction of both endogenous and overexpressed AURKA by Cereblon-directed PROTACs. At the subcellular level, we find differential targeting of AURKA on the mitotic spindle compared to centrosomes. The phenotypic consequences of PROTAC treatment are therefore distinct from those mediated by alisertib, and in mitotic cells differentially regulate centrosome- and chromatin- based microtubule spindle assembly pathways. In interphase cells PROTAC-mediated clearance of non-centrosomal AURKA modulates the cytoplasmic role played by AURKA in mitochondrial dynamics, whilst the centrosomal pool is refractory to PROTAC-mediated clearance. Our results point to differential sensitivity of subcellular pools of substrate, governed by substrate conformation or localization-dependent accessibility to PROTAC action, a phenomenon not previously described for this new class of degrader compounds.

Highlights

Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (PROteolysis Targeting Chimeras, PROTACs) that recruit ubiquitin ligases to targets of interest, leading to ubiquitination and destruction of the targets
We set out to investigate the action of AURKA-directed targeted protein degradation tools (PROTACs) against AURKA in single cell time-lapse assays using cell lines that we have previously described[25]: an AURKA-Venus knock-in line in RPE1 cells (AURKA-VenusKI), where AURKA-Venus recapitulates expression of the endogenous protein, and a line expressing exogenous AURKA-Venus under tetracycline control (RPE1FRT/ TO-AURKA-Venus, AURKA-VenusTO) where higher levels of expression occur throughout the cell cycle
We used AURKAVenusKI and AURKA-VenusTO cells arrested in mitosis by an agonist of the Spindle Assembly Checkpoint (SAC), S-trityl L-cysteine (STLC), to test the activity of PROTAC compounds (Fig. 1)

Summary

Introduction

Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (PROteolysis Targeting Chimeras, PROTACs) that recruit ubiquitin ligases to targets of interest, leading to ubiquitination and destruction of the targets. One novel way to target proteins for degradation is through PROteolysis Targeting Chimeras (PROTACs), consisting of a chimeric molecule that binds at one end to a protein target, and at the other to a ubiquitin ligase (E3), most commonly the Cereblon (CRBN) substrate recognition protein together with the CUL4A E3 ligase complex, or to the von Hippel Lindau (VHL) protein in association with the CUL2 complex[2,3] This PROTAC-mediated ternary complex formation between functional E3 and target protein facilitates ubiquitin transfer, leading to ubiquitination of the target and its proteolysis at the 26S proteasome. In vitro tests show that binding by TPX2 and T-loop phosphorylation independently activate AURKA approximately 100fold[15,16] These separable intracellular AURKA activities (defined by pT288 at the centrosomes and TPX2 binding around chromatin) contribute to distinct pathways of MT nucleation that act together to achieve mitotic spindle assembly. Recent work from our lab has shown that un-degraded AURKA retains activity after mitosis[24]

Methods

Results

Conclusion