Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DnA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1′′-2′) ribose-ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodaninecontaining mammalian PARG inhibitor RBPI-3. our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors.

Summary PARP inhibitors (PARPi) are a promising class of targeted cancer drugs, but their individual target profiles beyond the PARP family, which could result in differential clinical utility or toxicity, are unknown. Using an unbiased, mass spectrometry-based chemical proteomics approach, we generated a comparative proteome-wide target map of the four clinical PARPi olaparib, veliparib, niraparib, and rucaparib. PARPi as a class displayed high target selectivity. However, in addition to the canonical targets PARP1, PARP2 and several of their binding partners, we also identified hexose-6-phosphate dehydrogenase (H6PD) and deoxycytidine kinase (DCK) as previously unrecognized targets of rucaparib and niraparib, respectively. Subsequent functional validation suggested that inhibition of DCK by niraparib could have detrimental effects when combined with nucleoside analog pro-drugs. H6PD silencing can cause apoptosis and further sensitize cells to PARPi, suggesting that H6PD may be, in addition to its established role in metabolic disorders, a new anticancer target.

Several selective CDK4/6 inhibitors are in clinical trials for non-small cell lung cancer (NSCLC). Palbociclib (PD0332991) is included in the phase II/III Lung-MAP trial for squamous cell lung carcinoma (LUSQ). We noted differential cellular activity between palbociclib and the structurally related ribociclib (LEE011) in LUSQ cells. Applying an unbiased mass spectrometry-based chemoproteomics approach in H157 cells and primary tumor samples, we here report distinct proteome-wide target profiles of these two drug candidates in LUSQ, which encompass novel protein and, for palbociclib only, lipid kinases. In addition to CDK4 and 6, we observed CDK9 as a potent target of both drugs. Palbociclib interacted with several kinases not targeted by ribociclib, such as casein kinase 2 and PIK3R4, which regulate autophagy. Furthermore, palbociclib engaged several lipid kinases, most notably PIK3CD and PIP4K2A/B/C. Accordingly, we observed modulation of autophagy and inhibition of AKT signaling by palbociclib, but not ribociclib.

The poly(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG in vitro and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1, and thus will be useful tools to study the biochemistry of PAR signaling.

The selection of an appropriate therapy and dosing regimen is a significant challenge in the treatment of cancer. Although there are recommended standardized chemotherapy protocols for some types of cancer, protocol changes that usually only occur after large clinical trials demonstrate improvements and individual patients often require dose modifications (amount or interval) or delays in dose administration as toxicities arise. In other areas of medicine, therapeutic drug monitoring is commonly and successfully used to ensure appropriate drug exposure and to limit dose-related toxicities. Currently, the wide pharmacokinetic variability of cytotoxic chemotherapies is addressed clinically by the use of body surface area to determine drug doses; however, this is outdated and demonstrably ineffective for this purpose. This review discusses the challenges of dosing cytotoxic chemotherapies, dose determination strategies for cytotoxic, targeted, and antibody-based biological anticancer drugs, and provides an overview of the recent literature regarding the use of therapeutic drug monitoring in cancer.