Abstract A02: PARP inhibition selectively kills RNF20-depleted cells
Authors: Guppy Brent J. , McManus Kirk J. 
Published: August 2014

Chromosomal instability (CIN) is an early event that drives the development of tumors and it is associated with virtually all tumor types. CIN is defined as an increase in the rate at which whole chromosomes or large parts thereof, are gained or lost. Consequently, CIN drives large-scale genomic changes, which has adverse consequences for oncogenes and tumor suppressor genes that localize to those altered regions. Genes that normally function to maintain chromosome stability are often found altered in human cancers. As a result, synthetic lethal strategies designed to target cancer cells harboring defects in chromosome stability genes has potential broad-spectrum applicability. RNF20, a histone H2B mono-ubiquitin ligase, is one such chromosome stability gene that is frequently found mutated in various cancers. The prevalence of cancer-associated RNF20 mutations across multiple tumor types and its role in homologous recombination repair makes RNF20 an attractive target for therapeutic intervention. Previous groups have shown that cells harboring defects in homologous recombination repair proteins (BRCA1/2) can be specifically killed through PARP inhibition. As a result, we hypothesized that RNF20-depleted cancer cells may be targeted in a similar manner.

Consistent with the hallmarks of CIN, siRNF20 and flow cytometry revealed increases in DNA content compared to controls. In addition, indirect immunofluorescent imaging demonstrated the appearance of micronuclei and lagging chromosomes in RNF20-silenced populations. We subsequently showed that RNF20-depleted cells exhibit defects in homologous recombination repair through delayed repair kinetics as indicated by the persistence of γH2AX and 53BP1 foci. Using real-time cell analyses and RNAi, we show that simultaneous silencing of RNF20 and PARP can specifically kill these populations compared to RNF20-silenced plus GAPDH-silenced controls. In addition, specific killing was recapitulated using Olaparib (a chemical PARP inhibitor) in combination with RNF20 knockdown. Consistent with real-time cell analysis data, we show a reduced number of viable cells in RNF20 and PARP-silenced populations and RNF20-silenced and PARP inhibited populations. To evaluate the underlying mechanism accounting for the cell cytotoxicity detailed above, Western blot analyses and immunofluorescent imaging revealed activated Caspase-3 and suggests an apoptotic response.

Our data suggests that RNF20 is a CIN gene that when downregulated, can be selectively targeted and killed by either silencing or inhibiting PARP. Using a synthetic lethality strategy we have identified a candidate drug target with potential broad spectrum applicability across multiple tumor types.

Citation Format: Brent J. Guppy, Kirk J. McManus. PARP inhibition selectively kills RNF20-depleted cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A02.