NAD+ bioavailability mediates PARG inhibition-induced replication arrest, intra S-phase checkpoint and apoptosis in glioma stem cells

Abstract

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.

Overcoming Temozolomide Resistance in Glioblastoma via Enhanced NAD+ Bioavailability and Inhibition of Poly-ADP-Ribose Glycohydrolase

“…In contrast, MSH6-proficient control cells were highly sensitive to TMZ ( Figure 1 B,D). We previously reported that NRH treatment strongly potentiated the cytotoxicity of PARGi against glioma stem cells (GSCs) [ 25 ]. Therefore, we reasoned that the addition of both NRH (N) and PARGi (P) to the TMZ (T)-treated cells may overcome the resistance seen in the LN428/MSH6-KD and LN428/MSH6-KO cells [ 25 ].…”

“…We previously reported that NRH treatment strongly potentiated the cytotoxicity of PARGi against glioma stem cells (GSCs) [ 25 ]. Therefore, we reasoned that the addition of both NRH (N) and PARGi (P) to the TMZ (T)-treated cells may overcome the resistance seen in the LN428/MSH6-KD and LN428/MSH6-KO cells [ 25 ]. As predicted, pre-treatment of the cells with NRH and PARGi, followed by TMZ treatment (T + N + P), restored the sensitivity to TMZ in both the LN428/MSH6-KD and LN428/MSH6-KO cells to that seen in the MSH6-proficient control cells ( Figure 1 B,D).…”

“…The shuttle vector pLenti-U6-sgRNA(MSH6)-SFFV-Cas9-2A-Puro was used to generate lentivirus for the expression of Cas9 and gRNA specific to the human MSH6 gene, pLenti-CRISPR-cas9-Scramble for the expression of the Cas9/gRNA control (SCR-gRNA), pLV-CMV-XRCC1-EGFP-Hygro for the expression of XRCC1-EGFP, pLKO.1-puro-shSCR for the expression of the scrambled shRNA, or pLKO.1-puro-shMSH6.1 for the expression of MSH6-specific shRNA (see plasmid details in Supplementary Table S1 ). Viral particles were then isolated 48 h after transfection by filtering the supernatant through 0.45-μM filters, as described previously [ 25 , 32 ]. A Lenti-X Concentrator (Takara Bio, Cat# 631231, Kusatsu, Japan) was then used to concentrate the lentivirus particles, according to the manufacturer’s instructions.…”

“…One promising target is poly(ADP-ribose) glycohydrolase (PARG), an important component of the BER pathway as the primary hydrolase responsible for the degradation of poly(ADP-ribose) (PAR) after the recruitment of DNA repair factors [ 24 ]. Genetic depletion of PARG or inhibition of PARG strongly increased the level of DNA damage induced by TMZ, methyl methanesulfonate (MMS), ionizing radiation (IR), or cisplatin and significantly reduced cell survival in different types of cancer cells [ 6 , 25 , 26 , 27 , 28 , 29 ]. We recently reported that supplementation with the NAD + precursor dihydronicotinamide riboside (NRH) rapidly increased NAD + levels in glioma stem cells (GSCs) and GBM cells, enhancing PARP1 activation.…”

“…We recently reported that supplementation with the NAD + precursor dihydronicotinamide riboside (NRH) rapidly increased NAD + levels in glioma stem cells (GSCs) and GBM cells, enhancing PARP1 activation. Further, we found that co-administration of NRH and a PARG inhibitor (PARGi) triggers hyperaccumulation of PAR, intra S-phase arrest, and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes [ 25 ]. Therefore, in this study, we hypothesized that co-treatment of T MZ with N RH + P ARGi (T + N + P) would enhance the cytotoxicity of TMZ in TMZ-resistant GBM cells.…”