DNA damage is implicated as playing a causal role in numerous disease processes. Hence, it is suggested that DNA repair proteins, which maintain the integrity of the nuclear and mitochondrial genomes, play a critical role in reducing the onset of multiple disease phenotypes. Conversely, the requirement for DNA repair and genome maintenance in response to radiation and genotoxic chemotherapeutics implicates DNA repair proteins as prime targets for improving response to currently available anti-cancer regimens. Further, cancer-specific DNA repair defects offer novel approaches for tumor-selective therapy. It is now expected that all cancer cells will be found to be defective in some aspect of DNA repair encoded by one of the 150 different proteins that catalyze DNA repair. To help in our understanding and treatment of cancer, geneticists and molecular biologists must explore the detailed consequences of an alteration in each of these repair pathways. It is our expectation that a detailed genetic and mechanistic understanding of the cellular phenotypes associated with specific DNA repair deficiencies will offer the opportunity to identify novel drug targets, optimize and validate new small molecule inhibitors of DNA repair and provide a mechanistic underpinning for the development of tumor selective therapeutic strategies.
In my lab, we have identified two compensatory pathways that respond to defects in DNA repair mediated by the base excision repair pathway, including proteins involved in the synthesis and degradation of poly-ADP-ribose (PAR) and those involved in the biosynthesis of NAD+. In particular, we study the convergent roles of DNA Repair, PAR metabolism and NAD+ biosynthesis in response to chemotherapy.
Model for methylpurine DNA glycosylase-initiated BER
This model depicts the proteins and chemical structures of a TMZ-induced base lesion (N3-MeA) and the corresponding BER intermediates following BER initiated by the methylpurine DNA glycosylase, (MPG). The chemistry of the lesion and the repair intermediates throughout the repair process are shown on the right, highlighting the three major steps for BER: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation. The structures on the left depict the protein complexes required for completion of each step in BER initiated by MPG.
1) Novel approaches to enhance tumor cell cytotoxicity of alkylating agents: Glioblastoma is the most commonly diagnosed brain malignancy and a major cause of cancer related death in the United States. Limited success in the treatment of glioblastoma has been demonstrated with the alkylating agent Temozolomide (TMZ). The overall goals of this project are to discover strategies to circumvent resistance to TMZ and enhance the cytotoxicity and efficacy of this alkylating agent. The base excision repair (BER) pathway provides significant resistance to TMZ by repairing damaged bases but some of the activity of TMZ is due to the accumulation of cytotoxic BER intermediates that result from incomplete or failed repair (termed BER Failure). As the rate-limiting enzyme in BER, DNA polymerase ß (Polß) is important to facilitate repair and to maintain cell survival following DNA damage. Therefore, inhibition of Polß will enhance TMZ response. We posit that BER mediated by Polß is a regulated process that signals BER failure through poly(ADP)ribose (PAR) synthesis and NAD+/ATP depletion by a process that requires activation of PARP1 & PARP2 and is regulated by the enzyme PARG. Specifically, we hypothesize that TMZ efficacy can be increased in glioma cells by increasing post-translational modification and inhibition of Polß. BER failure-induced cell death results from energy (NAD+ & ATP) depletion due to elevated PAR synthesis mediated by the PARP1/PARP2 BER sensor complex, suggesting that the response to TMZ can be enhanced via increased PAR synthesis or further depletion of cellular NAD+ synthesis and/or deregulation of the BER enzyme PARG. Overall, we are testing the hypothesis that the BER pathway is a determinant of resistance to TMZ and therefore selective targeting of the BER pathway will significantly enhance TMZ efficacy.
2) Development and characterization of isogenic DNA Repair deficient human cell lines: To extend our analysis beyond base excision repair, we have embarked on a large-scale project for the development, characterization and transcriptome analysis of isogenic human cell lines deficient in all known DNA repair genes (>150) in three unique cell backgrounds (glioma, breast cancer and neuronal). These include genes involved in Base Excision Repair, Direct Reversal of Damage, Mismatch Excision Repair, Nucleotide Excision Repair, Homologous Recombination, Non-homologous End-Joining, the modulation of nucleotide pools, DNA polymerases, editing and processing nucleases, the Rad6 pathway, Chromatin Structure, DNA Repair genes defective in diseases and conserved DNA Damage Response genes and Fanconi Anemia/DNA crosslink repair. Further, each will be analyzed for alterations in PAR metabolism and NAD+ biosynthesis. With the expectation that DNA repair capacity impacts basic cellular functions both spontaneously and in response to genotoxic stress, alters the transcriptional and epigenetic landscape and dictates the cellular response to stress, the development of a complete panel of isogenic DNA repair deficient cell lines across multiple backgrounds will be a valuable platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations.
Ongoing and future projects also include the identification of novel DNA Repair targets for improved chemotherapy response, testing and evaluation of novel DNA repair inhibitors, tumor tissue analysis for defects in expression of enzymes critical for chemotherapy response and evaluating the potential of NAD+ biosynthesis inhibitors in the development and testing of tumor specific, synthetically lethal, chemotherapy combinations.
Proposed Rotation Projects
Probing synthetic lethality in glioblastoma - This project will use small molecule inhibitors of DNA repair and lentiviral-based shRNA to exploit a defect in the de novo pathway of NAD+ biosynthesis in glioblastoma.
Viral-based insertional mutagenesis as a tool to discover chemotherapy-resistance genes - This project will use a lentiviral-based selection tool to identify genes that provide resistance to chemotherapies such as the alkylating agent temozolomide and inhibitors of the DNA repair protein PARP1. Transduced cells are selected and genes that confer resistance will be identified by deep sequencing and validated using select shRNA and cellular response analyses.
Investigating the kinome for involvement in chemotherapeutic response - This project will use pools of lentivirus expressing shRNA specific to the human kinome to identify protein kinases that provide resistance or sensitivity to chemotherapies such as the alkylating agent temozolomide. Transduced cells are selected and shRNAs that confer resistance or sensitivity will be identified by deep sequencing and validated using select shRNA and cellular response analyses.
Transcriptional regulation by DNA repair proteins - This project will utilize a newly developed library of isogenic DNA repair deficient human cells to evaluate the transcriptional reprogramming that is induced following loss of a specific DNA repair gene (e.g., BRCA1).
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