Date of Award

12-2022

Document Type

Dissertation

Degree Name

Ph.D.

Department

Basic Medical Sciences

Committee Chair

Robert W. Sobol, Ph.D.

Abstract

High-grade gliomas (HGGs) are malignant, highly metabolically active brain tumors. HGGs are associated with poor patient outcome, attributed to resistance to current therapies, with a survival rate between 12 to 15 months. Gliomas are highly complex tumors, making targeted therapy difficult, highlighting the need for novel approaches and new treatment options. In addition, a large percentage of HGGs are comprised of glioma stem cells (GSCs) that further contribute to therapeutic resistance. Notable characteristics of GSCs are a heightened DNA damage response (DDR) and elevated replication stress that could provide opportunities for therapeutic targeting. A notable feature of many glioma tumors that harbor mutations in isocitrate dehydrogenase isoforms 1 or 2 (IDH 1/2) mutations is reduced levels of the cellular metabolite nicotinamide adenine dinucleotide (NAD+). NAD+ is essential for cellular energy homeostasis and is responsible for the regulation of cellular processes such as fatty acid oxidation, glycolysis and the tricarboxylic acid cycle. IDH1/2 mutations are more sensitive to NAD+ depletion than wild-type cells and, therefore, may be a rational target for chemotherapeutics. In addition to a role in cellular metabolism, NAD+ serves as an important cofactor to poly-(ADP-ribose) polymerases (PARPs) and NAD-dependent deacetylases (Sirtuins) in chromatin remodeling. In our lab, we have found that GSCs have increased levels of the DDR protein Poly-(ADP-Ribose) polymerase 1 (PARP1) at both the mRNA and protein expression levels. However, we find that GSCs lack sufficient cellular NAD+ levels for xx robust PARP1 activation. PARP1 is involved in several DNA repair pathways that have evolved to repair specific types of DNA damage. Importantly, the NAD+ dependent enzymes PARP1 and PARP2, along with the sirtuin isoforms SIRT1 and SIRT6 (NAD+-dependent deacetylases), comprise a PARP-NAD+-SIRT axis that plays an essential role in the regulation and coordination of the base excision repair (BER) and single-stranded break repair (SSBR) pathways. The BER/SSBR pathway is responsible for repairing base damage and DNA single-strand breaks that result from both endogenous and exogenous sources, which are essential for genome maintenance. Defects in these pathways have been associated with the onset of cancer and other diseases. The activation of PARP1 is crucial to the cellular response to both base and SSB damage across the genome (referred to as canonical BER/SSBR) and the response to such lesions that impact replication associated BER/SSBR. The recruitment of and activation of PARP1 is essential for the relaxation of chromatin and recruitment of important BER/SSBR proteins to sites of DNA damage. However, central to that role is the hydrolysis of NAD+ by PARP1 to form poly-(ADP-ribose) (PAR) polymers. PARP1 covalently modifies itself and other proteins with PAR to facilitate and regulate DNA repair processes. We find that NAD+ is an important regulator of BER/SSBR as an essential substrate for PARP1. My hypothesis is that biological variation in cellular NAD+ levels modulate PARP1 activity, PAR metabolism and the PARP1-interactome to alter both canonical and replication-associated base excision and single-strand break repair. This variation impacts the efficacy of PARG inhibitors as a targeted cancer therapeutic option.

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