Abstract A71: Differential proteome-wide target profiles of clinical poly(ADP-ribose) polymerase inhibitors in breast cancer
Authors: Knezevic Claire , Wright Gabriela , Kuenzi Brent , Remsing Rix Lily , Luo Yunting , Lawrence Harshani , Rix Uwe 
Published: May 2016

Due to the clinical promise of PARP inhibitors (PARPi) in both breast and ovarian cancers, an understanding of the full protein target profile of each clinical PARPi is important for understanding the differential activity within this drug class.

Modified versions of clinical PARPi niraparib, olaparib, rucaparib, and veliparib containing propylamine linkers were synthesized to allow for coupling to solid supports. The modified PARPi were immobilized onto NHS-ester sepharose beads which were then incubated with CAL51 breast cancer cell lysate. Free unmodified drug was added to the bead/lysate mixtures as a competition control to aid in the identification of specific drug-protein interactions. After washing, elution, and trypsin digest, bound proteins were identified by LC-MS/MS and analyzed by Significance Analysis of Interactome (SAINT) Express. Immunoprecipitation was performed by pre-binding Protein A/G beads to a polyclonal PARP-1 antibody before incubation with lysate. H6PD activity was assessed by monitoring consumption of NADP in H6PD-overexpressing HEK293 cell lysate. Cell viability was measured by CellTiter Glo after incubation with drug for three days.

Target profiles of PARPi were generated by unbiased chemical proteomics. Target candidates were identified using both SAINT and a score-based analysis method. Within the PARP protein family, most PARPi interacted with PARP 1 and 2 as well as tankyrase 1 and 2. SAINT Express analysis of the chemical proteomics data revealed deoxycytidine kinase (DCK) as a potential target of niraparib and hexose-6-phosphate dehydrogenase (H6PD) as a potential target of rucaparib. Analysis of H6PD activity in cellular lysate of H6PD-overexpressing cells revealed inhibition of H6PD by rucaparib. In whole cell culture, niraparib reduces the cytotoxicity of cytarabine, an anticancer agent that is activated by DCK-mediated phosphorylation. Inosine monophosphate dehydrogenase 2 (IMPDH2), which was initially identified as a candidate target of all PARPi, was co-immunoprecipitated from CAL51 lysate with PARP-1, confirming its PARP-1 binding abilities.

Each clinical PARPi's molecular target profile was characterized with chemical proteomics. The clinical implications of inhibition of H6PD are currently unclear, but H6PD is of interest for its role in glucocorticoid activation and is a possible therapeutic target for diabetes. Rucaparib is the only small molecule known to inhibit H6PD and as such rucaparib may provide a starting point for the development of chemical tool compounds and therapeutics. Inhibition of DCK by niraparib suggests that combination strategies with niraparib and either cytarabine or gemcitabine should not be pursued. Understanding the full protein target profiles of these promising cancer drugs is important for their clinical usage and these results will enable a more complete understanding of their clinical activity.

Citation Format: Claire Knezevic, Gabriela Wright, Brent Kuenzi, Lily Remsing Rix, Yunting Luo, Harshani Lawrence, Uwe Rix. Differential proteome-wide target profiles of clinical poly(ADP-ribose) polymerase inhibitors in breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A71.