Analysis of PARP inhibitor toxicity by multidimensional fluorescence microscopy reveals mechanisms of sensitivity and resistance

Abstract

Exploiting the full potential of anti-cancer drugs necessitates a detailed understanding of their cytotoxic effects. While standard omics approaches are limited to cell population averages, emerging single cell techniques currently lack throughput and are not applicable for compound screens. Here, we employed a versatile and sensitive high-content microscopy-based approach to overcome these limitations and quantify multiple parameters of cytotoxicity at the single cell level and in a cell cycle resolved manner. Applied to PARP inhibitors (PARPi) this approach revealed an S-phase-specific DNA damage response after only 15 min, quantitatively differentiated responses to several clinically important PARPi, allowed for cell cycle resolved analyses of PARP trapping, and predicted conditions of PARPi hypersensitivity and resistance. The approach illuminates cellular mechanisms of drug synergism and, through a targeted multivariate screen, could identify a functional interaction between PARPi olaparib and NEDD8/SCF inhibition, which we show is dependent on PARP1 and linked to PARP1 trapping.

Phase separation of 53 BP 1 determines liquid‐like behavior of DNA repair compartments

“…would react to changes in osmolarity, we monitored the 53BP1 response to ionizing radiation (IR) by quantitative image-based cytometry (QIBC), a high-content microscopy approach that allows for cell cycle resolved profiling of DNA damage responses Toledo et al, 2013;Ochs et al, 2016;Pellegrino et al, 2017;Michelena et al, 2018). As observed previously, we measured a strong IR-induced increase in nuclear 53BP1 foci in G1, which gradually declined in S-phase when increasing amounts of replicated chromatin promote DSB repair by homologous recombination (Chapman et al, 2012;Saredi et al, 2016;Pellegrino et al, 2017), and which rose again in late G2 (Fig 1A).…”

“…Automated multichannel wide-field microscopy for quantitative image-based cytometry (QIBC) was performed on the Olympus ScanR Screening System described above as done previously (Pellegrino et al, 2017;Michelena et al, 2018). Images were analyzed with the Olympus ScanR Image Analysis Software version 3.0.0, a dynamic background correction was applied, nuclei segmentation was performed using an integrated intensity-based object detection module using the DAPI signal, and foci segmentation was performed using an integrated spot-detection module.…”

PARP and PARG inhibitors in cancer treatment

“…6). This leads to an accumulation of DNA damage in S-phase cells, S-phase stalling, and G2 delay (Sugimura et al 2008;Bryant et al 2009;Ray Chaudhuri et al 2012;Berti et al 2013;Dale Rein et al 2015;Farrés et al 2015;Maya-Mendoza et al 2018;Michelena et al 2018;Ronson et al 2018). In PARP1-depleted or inhibited cells, DSBs arise due to deprotection of stalled replication forks and their degradation by the MRE11 nuclease, due to impaired fork reversal, or from unligated Okazaki fragments encountered by replication forks (Lonn and Lonn 1985;Ray Chaudhuri et al 2012;Ying et al 2012;Hanzlikova et al 2018).…”

A RAD 51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation

“…or in the RNASEH2 complex(Zimmermann et al, 2018); secondly, when PARPi sensitivity occurs via mechanisms that preserve RAD51 foci formation, e.g., alterations in the MRN complex, RAD51AP1, polymerase eta, or ERCC1(Kawamoto et al, 2005;Wiese et al, 2007;Oplustilova et al, 2012;Postel-Vinay et al, 2013); thirdly, when HRR-deficient tumors have acquired PARPi resistance via RAD51independent mechanisms such as loss of PARG, mutations in PARP1, or those that involve replication fork stabilization(Guillemette et al, 2015;Chaudhuri et al, 2016;Kais et al, 2016; Yazinski et al, 2017;Gogola et al, 2018;Michelena et al, 2018;Pettitt et al, 2018);…”