Abstract S5-06: Large-scale identification of cell-specific PARP substrates

Abstract

Abstract PARPs (poly-ADP-ribose polymerases) catalyze a protein posttranslational modification termed Poly-ADP-ribosylation (PARylation). PARylation is composed of linear and/or branched repeats of ADP-ribose, whose lengths can reach up to 200 units. The first gene encoding a poly-ADP-ribose polymerase, PARP1, was cloned in 1987. Numerous efforts have now led to the identification of 16 additional PARP enzymes. PARP1 is a nuclear protein that is activated as a result of sensing DNA strand breaks. PARylation levels in a quiescent cell are usually very low. In response to genotoxic stress, PARP1 is recruited to nicked DNA and is rapidly activated. This triggers the synthesis of a large number of PARylated proteins and the initiation of DNA damage repair mechanisms. Cancer cells with defects in double-strand break (DSB) repair, such as BRCA1/2-mutated cells, are reliant on PARP1 activity for genome integrity. These cells undergo unsustainable genetic damage, and eventually, apoptosis upon PARP1 inhibition. Indeed, recent late-stage clinical studies revealed that PARP1 inhibitor treatment significantly prolonged progression-free survival of BRCA-deficient breast cancer patients. Contrary to the fruitful efforts in characterizing the upstream inputs regulating PARP1, its genuine downstream targets, however, remain poorly defined. We recently developed the first mass spectrometry-based approach to a global mapping of the human aspartic acid- and glutamic acid-ADP-ribosylated proteome (Zhang et al., Nature Methods 2013). This method allowed us to identify 1,048 in vivo PARylation sites on 340 proteins. These modified proteins are involved in a wide array of nuclear functions including DNA damage repair, transcription regulation, epigenetic modulation, and mRNA processing. Using a quantitative mass spectrometry experiment, we also identified hundreds of novel PARP1 downstream effectors. The central hypothesis of our current study is that ADP-ribosylation levels of PARP1 substrates can reflect the activity of PARP1 in a cell. We predict that a signature composed of multiple PARylated proteins can then be used to identify cells that are "addicted" to PARP1 activity for genome integrity. To this end, we performed a large scale profiling of the Asp- and Glu-ADP-ribosylated proteome of a benign breast epithelial cell line (MCF10A), and compared it to that of a panel of eight breast carcinoma cell lines, including the ER+ (MCF7, T47D and ZR-75-1), HER2+ (SKBR3), and triple negative (MDA-MB-231, MDA-MB-468, SUM159 and HCC1937) subtypes. Among these lines, MDA-MB-468 (PTEN null), SKBR3 (HER2+) and HCC1937 (BRCA1 null) are known to be sensitive to PARP inhibitors. We correlated the pattern of protein ADP-ribosylation to their IC50, and found events of predictive value. In addition, we also observed that the differential PARylation pattern among these cells can be divided into a "public" (proteins that are commonly modified across all cell lines) class, and a "private" one (proteins that specifically modified in certain cell lines). We envision that this dataset will also serve as a valuable resource for the PARP1/DNA damage research community to investigate cell line-specific Asp- and Glu-ADP-ribosylation events. Citation Format: Yonghao Yu, Yajie Zhang. Large-scale identification of cell-specific PARP substrates [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S5-06.