The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin

Dragomir B Krastev,Yves Pommier,Christopher J Lord,Kristijan Ramadan,Jiri Bartek,Yilun Sun,Lu Yu,Gwendoline Hoslett,Tanaji T Talele,Shudong Li,Rebecca Marlow,Daniel Weekes,Luned M Badder,Eleanor G Knight,Mercedes Calvo Pardo,Jyoti S Choudhary,Andrew N J Tutt,Stephen J Pettitt,Andrew J Wicks

doi:10.1038/s41556-021-00807-6

Abstract

Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors.

Highlights

(ADP-ribose)-polymerase (PARP) inhibitors (PARPi) selectively kill tumour cells with impaired homologous recombination and are approved for use in homologous recombination-defective breast, ovarian, pancreatic and prostate cancers[1]
We used a PAR-binding PBZ–mRuby[2] probe and an ultraviolet light (UV) micro-irradiation assay to demonstrate that PARP1WT– Apex2–enhanced green fluorescent protein (eGFP) localized to DNA-damage sites where it generated PAR
Consistent with the idea that RNF4 is an upstream factor involved in the processing of trapped poly(ADP-ribose) polymerase 1 (PARP1), we found that gene silencing of RNF4 led to the persistence of PARP1–γH2AX proximity ligation assays (PLAs) foci (Fig. 5f)

Summary

Introduction

(ADP-ribose)-polymerase (PARP) inhibitors (PARPi) selectively kill tumour cells with impaired homologous recombination and are approved for use in homologous recombination-defective breast, ovarian, pancreatic and prostate cancers[1]. PARP1 ( known as ARTD1), the key target of PARPi, is a ubiquitously expressed nuclear enzyme that uses NAD+ to synthesise poly(ADP-ribose) (PAR) chains on substrate proteins (heteromodification) and itself (automodification). This catalytic activity (PARylation), which is enhanced by PARP1 binding to damaged DNA, initiates DNA repair by driving the recruitment/concentration of DNA-repair effectors and modulating chromatin structure. Most clinical PARPi bind the NAD+-binding site (catalytic domain) and inhibit catalytic activity, and induce chromatin retention of PARP1 (PARP trapping); this latter characteristic is a key driver of PARPi-mediated cytotoxicity[2]. Our findings suggest that the PARP1–p97 axis is essential for the removal of trapped PARP1 and the cellular response to PARPi

Methods

Results

Conclusion