A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage.

The Comet or single-cell gel electrophoresis assay is a highly sensitive method to measure cellular, nuclear genome damage. However, low throughput can limit its application for large-scale studies. To overcome these limitations, a 96-well CometChip platform was recently developed that increases throughput and reduces variation due to simultaneous processing and automated analysis of 96 samples. To advance throughput further, we developed a 384-well CometChip platform that allows analysis of ∼100 cells per well. The 384-well CometChip extends the capacity by 4-fold as compared to the 96-well system, enhancing application for larger DNA damage analysis studies. The overall sensitivity of the 384-well CometChip is consistent with that of the 96-well system, sensitive to genotoxin exposure and to loss of DNA repair capacity. We then applied the 384-well platform to screen a library of protein kinase inhibitors to probe each as enhancers of etoposide induced DNA damage. Here, we found that 3-methyladenine significantly increased levels of etoposide-induced DNA damage. Our results suggest that a 384-well CometChip is useful for large-scale DNA damage analyses, which may have increased potential in the evaluation of chemotherapy efficacy, compound library screens, population-based analyses of genome damage and evaluating the impact of environmental genotoxins on genome integrity.

Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens.

Poly(ADP-ribose) (PAR) is a molecular scaffold that aids in the formation of DNA repair protein complexes. Tools to sensitively quantify PAR in live cells have been lacking. We recently described the LivePAR probe (EGFP fused to the RNF146-encoded WWE PAR binding domain) to measure PAR formation at sites of laser micro-irradiation in live cells. Here, we present two methods that expand on the use of LivePAR and its WWE domain. First, LivePAR enriches in the nucleus of cells following genotoxic challenge. Image quantitation can identify single-cell PAR formation following genotoxic stress at concentrations lower than PAR ELISA or PAR immunoblot, with greater sensitivity to genotoxic stress than CometChip. In a second approach, we used the RNF146-encoded WWE domain to develop a split luciferase probe for analysis in a 96-well plate assay. We then applied these PAR analysis tools to demonstrate their broad applicability. First, we show that both approaches can identify genetic modifications that alter PARylation levels, such as hyper-PARylation in BRCA2-deficient cancer cells. Second, we demonstrate the utility of the WWE split luciferase assay to characterize the cellular response of genotoxins, PARP inhibitors, and PARG inhibitors, thereby providing a screening method to identify PAR modulating compounds.

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

Glioblastoma multiforme (GBM) is an incurable brain cancer with an average survival of approximately 15 months. Temozolomide (TMZ) is a DNA alkylating agent for the treatment of GBM. However, at least 50% of the patients treated with TMZ show poor response, primarily due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or due to defects in the mismatch repair (MMR) pathway. These resistance mechanisms are either somatic or arise in response to treatment, highlighting the need to uncover treatments to overcome resistance. We found that administration of the NAD+ precursor dihydronicotinamide riboside (NRH) to raise cellular NAD+ levels combined with PARG inhibition (PARGi) triggers hyperaccumulation of poly(ADP-ribose) (PAR), resulting from both DNA damage-induced and replication-stress-induced PARP1 activation. Here, we show that the NRH/PARGi combination enhances the cytotoxicity of TMZ. Specifically, NRH rapidly increases NAD+ levels in both TMZ-sensitive and TMZ-resistant GBM-derived cells and enhances the accumulation of PAR following TMZ treatment. Furthermore, NRH promotes hyperaccumulation of PAR in the presence of TMZ and PARGi. This combination strongly suppresses the cell growth of GBM cells depleted of MSH6 or cells expressing MGMT, suggesting that this regimen may improve the efficacy of TMZ to overcome treatment resistance in GBM.

A specific inhibitor of ALDH1A3 regulates retinoic acid biosynthesis in glioma stem cells.

Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.

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

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.