PO-019 Phosphoproteomics to characterise DNA damage response in mouse mammary tumours of different PARP inhibitor susceptibility

Abstract

IntroductionTriple negative breast cancers show very poor prognosis, with only limited treatment options. A subgroup of these tumours is deficient in homologous recombination (HR) and therefore sensitive to DNA damaging drugs or PARP inhibitors (PARPi). Unfortunately, resistance to PARPi is often observed. Therefore, the identification of biomarkers for PARPi response or status of HR are of importance for personalised therapy. The DNA damage response is regulated via post-translational modifications such as phosphorylation. We thus study differential phosphorylation events in the context of different PARPi susceptibility.Material and methodsWe used a collection of biopsies from either untreated or irradiated (15 Gy, 2 hour) HR-deficient mouse mammary tumours and matched PARPi resistant tumours which restored HR. Changes in phosphorylation were examined via titanium dioxide based phosphopeptide enrichment or protein expression profiling using single-shot LC-MS/MS (label-free).Results and discussionsIn total, we identified 14 695 phosphopeptides and 11 138 high-confidence phosphosites (PS) from 48 individual tumours samples that were measured in duplicates. As proof of concept, we analysed changes in PS in PARPi naive tumours upon induction of the DNA damage response via ionising irradiation. We detect known events of the DNA damage response such as increased phosphorylation of proteins involved in DNA damage checkpoint or double strand break repair. Further evaluation of these PS, like sequence motif analysis, prediction of upstream kinases and kinase substrate enrichment analysis revealed activation of ATM/ATR in irradiated tumours as expected. To further proof the validity of our approach, we analysed samples with known PARPi resistance mechanism – loss of 53 BP1 expression – and observed downregulation of PS specific to 53 BP1. We are now extending our analysis to additional samples sets with known and unknown PARPi resistance mechanisms.ConclusionOur data point to the feasibility of using phosphoproteomics as a tool to study DNA damage response in BRCA1 deficient tumours with different status of HR and thus contrasting susceptibility to PARPi.