Activated NAD+ biosynthesis pathway induces olaparib resistance in BRCA1 knockout pancreatic cancer cells.

PARP inhibitors have been developed as anti-cancer agents based on synthetic lethality in homologous recombination deficient cancer cells. However, resistance to PARP inhibitors such as olaparib remains a problem in clinical use, and the mechanisms of resistance are not fully understood. To investigate mechanisms of PARP inhibitor resistance, we established a BRCA1 knockout clone derived from the pancreatic cancer MIA PaCa-2 cells, which we termed C1 cells, and subsequently isolated an olaparib-resistant C1/OLA cells. We then performed RNA-sequencing and pathway analysis on olaparib-treated C1 and C1/OLA cells. Our results revealed activation of cell signaling pathway related to NAD+ metabolism in the olaparib-resistant C1/OLA cells, with increased expression of genes encoding the NAD+ biosynthetic enzymes NAMPT and NMNAT2. Moreover, intracellular NAD+ levels were significantly higher in C1/OLA cells than in the non-olaparib-resistant C1 cells. Upregulation of intracellular NAD+ levels by the addition of nicotinamide also induced resistance to olaparib and talazoparib in C1 cells. Taken together, our findings suggest that upregulation of intracellular NAD+ is one of the factors underlying the acquisition of PARP inhibitor resistance.

Dysfunction of poly (ADP-ribose) glycohydrolase suppresses osteoclast differentiation in RANKL-stimulated RAW264 cells.

Poly (ADP-ribose) glycohydrolase (PARG) is an enzyme that mainly degrades poly (ADP-ribose) (PAR) synthesized by poly (ADP-ribose) polymerase (PARP) family proteins. Although PARG is involved in many biological phenomena, including DNA repair, cell differentiation, and cell death, little is known about the relationship between osteoclast differentiation and PARG. It has also not been clarified whether PARG is a valuable target for therapeutic agents in the excessive activity of osteoclast-related bone diseases such as osteoporosis. In the present study, we examined the effects of PARG inhibitor PDD00017273 on osteoclast differentiation in RANKL-induced RAW264 cells. PDD00017273 induced the accumulation of intracellular PAR and suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. PDD00017273 also downregulated osteoclast differentiation marker genes such as Trap, cathepsin K (Ctsk), and dendrocyte expressed seven transmembrane protein (Dcstamp) and protein expression of nuclear factor of activated T cells 1 (NFATc1), a master regulator of osteoclast differentiation. Taken together, our findings suggest that dysfunction of PARG suppresses osteoclast differentiation via the PAR accumulation and partial inactivation of the NFATc1.

Inhibition of Poly (ADP-Ribose) Glycohydrolase Accelerates Osteoblast Differentiation in Preosteoblastic MC3T3-E1 Cells.

Poly ADP-ribosylation (PARylation) is a post-translational modification catalyzed by poly (ADP-ribose) polymerase (PARP) family proteins such as PARP1. Although PARylation regulates important biological phenomena such as DNA repair, chromatin regulation, and cell death, little is known about the relationship between osteoblast differentiation and the PARylation cycle involving PARP1 and the poly (ADP-ribose)-degrading enzyme poly (ADP-ribose) glycohydrolase (PARG). Here, we examined the effects of PARP inhibitor olaparib, an approved anti-cancer agent, and PARG inhibitor PDD00017273 on osteoblast differentiation. Olaparib decreased alkaline phosphatase (ALP) activity and suppressed mineralized nodule formation evaluated by Alizarin Red S staining in preosteoblastic MC3T3-E1 cells, while PDD00017273 promoted ALP activity and mineralization. Furthermore, PDD00017273 up-regulated the mRNA expression levels of osteocalcin and bone sialoprotein, as osteoblast differentiation markers, and osterix as transcription inducers for osteoblast differentiation, whereas olaparib down-regulated the expression of these genes. These findings suggest that PARG inhibition by PDD00017273 accelerates osteoblast differentiation in MC3T3-E1 cells. Thus, PARG inhibitor administration could provide therapeutic benefits for metabolic bone diseases such as osteoporosis.