Replication associated base excision repair in cancer

This project focuses on base excision repair (BER) activity and BER complex regulation of PARP1-mediated and PARP2-mediated poly-ADP-ribosylation (PARylation). Further, we are exploring the coordinated role of substrate availability (NAD+) and BER in the regulation of poly-ADP-ribose (PAR) induced activation of the intra-S-phase checkpoint and the onset of cell death in cell (2D), organoid (3D) and tumor (xenograft) model systems.

This project employs systematic approaches, together with the Ideker group at University of California, San Diego (https://idekerlab.ucsd.edu/ddram/), to map and model DNA damage response pathways (DDRAM) that maintain integrity of our genetic material following exposure to genotoxins. Here, we are investigating the molecular response mechanisms of cells exposed to genotoxic agents and are measuring DNA damage & repair, DNA damage response signaling (PARP activation, etc), double strand break repair and base excision repair/single strand break repair capacity.

These studies use novel expression systems, CRISPR/cas9-mediated gene editing and mouse models to mimic genetic ancestry informatic markers (AIMS) that modulate expression of POLB and other DNA repair genes (working with the Ragin group at Fox Chase Cancer Center). The goal is to understand the functional mechanisms related to altered POLB expression and radiation/cisplatin resistance in oral cavity and laryngeal cancer. We are evaluating how the interaction of POLB with key base excision repair (BER) factors (e.g., XRCC1) impacts both canonical and replication associated BER in response to genotoxins and radiation/cisplatin.  Further, the goal is to define treatment responses that overcome the resistance observed in POLB-over-expressing tumor cells. Finally, we have developed a novel mouse model to explore this altered POLB phenotype.

The Paternal Age Effect – Enhanced Germ Cell Mutagenesis modulated by the TRP53/APE1/MDM2 Tumor Suppressor Axis

Mutations increase in male gametes as men get older, leading to older fathers being more likely to have children with a genetic disease and creating a reproductive concern for older men. Our studies, together with the Walter group at the University of Texas Health Science Center San Antonio, elucidate mechanisms with the goal of reducing risk of genetic disease in children born to older fathers, with specificity on discovery and characterization of the interactome of mouse Apex1 and human APEX1, in germ cells. In addition to its role in base excision repair (BER), Apex1/APEX1 may have additional biological roles mediated through protein-protein interactions including mRNA repair, microRNA/ncRNA processing, protein stability, and p53 signaling. 

DNA and RNA are master instructional and regulatory molecules that control human cellular and organismal health. Base modifications of both DNA and RNA add a disease-driven or developmental level of gene regulation. The primary enzymatic pathway for the repair of base modifications is the Base Excision Repair (BER) pathway. Our goal will be to identify the complete protein interactome among these critical BER factors in organs (brain, liver), in cancer cells, upon differentiation and upon disease relevant genotoxic conditions. The goal here is to uncover protein-protein, protein-DNA, and protein-RNA complexes, along with functional outcome analyses, that will yield novel analytical tools important for the development of biomarkers (protein, gene expression) for patient diagnosis, monitoring, treatment, and stratification.

This project is designed to create a panel of barcoded, human cells with genetic diversity in genotoxin-response gene families: DNA damage response/repair, cell death and stress response. This system will provide a rapid and high-throughput, barcode-based analysis of toxicodynamic variability coupled with mechanistic insight that contributes to the variability in genotoxin response. 

These studies, together with the Pursell group at Tulane University, will use novel mouse models and human cell systems, combined with structural and enzymological approaches, to define the mechanism linking APOBEC3 mediated deamination at the replication fork to base excision repair. Together these studies are designed to better understand the role of the human APOBEC3 enzymes and their deamination in mutagenesis, cancer, and disease.

Poly(ADP-ribose) (PAR) is a molecular scaffold that aids the formation of DNA repair protein complexes. We recently described a split luciferase probe for the quantitative analysis of PAR levels in cells (Split-Luciferase-LivePAR). This split luciferase assay is useful in the characterization of PARP and poly(ADP-ribose) glycohydrolase (PARG) inhibitors, thereby providing a method to identify PAR modulating compounds. Our goals in this new project are to advance our Split-Luciferase-LivePAR assay based on improved PAR binding domain (PBD) candidates. Once optimized, our Split-Luciferase-LivePAR assay will be used, in collaboration with the Legorreta Cancer Center Drug Discovery core facility, to identify novel PAR-modulating compounds, PARG inhibitors, that can be considered lead compounds in drug development to affect PARG activity in cancer cells.

Our goal in this project is to use novel proteomic technologies to identify protein factors that are involved in  base excision repair (BER) mediated regulation of the replication stress response, focused on ovarian cancer cells as a model system (those that have or do not have mutations in the BRCA1 or BRCA2 genes). We will then evaluate the requirement of these protein factors in the survival of these cancer cell types and in response to clinical and pre-clinical treatment options.

The splicing factor SON regulates the expression of stress response genes, DNA repair (DR) genes and DNA damage response (DDR) genes. Here, we are collaborating with Erin Ahn (University of Alabama Birmingham) to define the DNA repair factors regulated by SON in glioma and other cancers, working to clarify the mechanism of SON-mediated regulation of these DR and DDR genes.

These studies use novel expression systems, CRISPR/cas9-mediated gene editing and mouse models to mimic genetic ancestry informatic markers (AIMS) that modulate expression of POLB and other DNA repair genes (working with the Ragin group at Fox Chase Cancer Center). The goal is to understand the functional mechanisms related to altered POLB expression and radiation/cisplatin resistance in oral cavity and laryngeal cancer. We are evaluating how the interaction of POLB with key base excision repair (BER) factors (e.g., XRCC1) impacts both canonical and replication associated BER in response to genotoxins and radiation/cisplatin.  Further, the goal is to define treatment responses that overcome the resistance observed in POLB-over-expressing tumor cells. Finally, we have developed a novel mouse model to explore this altered POLB phenotype.

The Paternal Age Effect – Enhanced Germ Cell Mutagenesis modulated by the TRP53/APE1/MDM2 Tumor Suppressor Axis

Measuring genomic DNA damage and DNA repair capacity in longitudinal population samples

DNA and RNA are master instructional and regulatory molecules that control human cellular and organismal health. Base modifications of both DNA and RNA add a disease-driven or developmental level of gene regulation. The primary enzymatic pathway for the repair of base modifications is the Base Excision Repair (BER) pathway. Our goal will be to identify the complete protein interactome among these critical BER factors in organs (brain, liver), in cancer cells, upon differentiation and upon disease relevant genotoxic conditions. The goal here is to uncover protein-protein, protein-DNA, and protein-RNA complexes, along with functional outcome analyses, that will yield novel analytical tools important for the development of biomarkers (protein, gene expression) for patient diagnosis, monitoring, treatment, and stratification.

This project is designed to create a panel of barcoded, human cells with genetic diversity in genotoxin-response gene families: DNA damage response/repair, cell death and stress response. This system will provide a rapid and high-throughput, barcode-based analysis of toxicodynamic variability coupled with mechanistic insight that contributes to the variability in genotoxin response. 

These studies, together with the Pursell group at Tulane University, will use novel mouse models and human cell systems, combined with structural and enzymological approaches, to define the mechanism linking APOBEC3 mediated deamination at the replication fork to base excision repair. Together these studies are designed to better understand the role of the human APOBEC3 enzymes and their deamination in mutagenesis, cancer, and disease.

Poly(ADP-ribose) (PAR) is a molecular scaffold that aids the formation of DNA repair protein complexes. We recently described a split luciferase probe for the quantitative analysis of PAR levels in cells (Split-Luciferase-LivePAR). This split luciferase assay is useful in the characterization of PARP and poly(ADP-ribose) glycohydrolase (PARG) inhibitors, thereby providing a method to identify PAR modulating compounds. Our goals in this new project are to advance our Split-Luciferase-LivePAR assay based on improved PAR binding domain (PBD) candidates. Once optimized, our Split-Luciferase-LivePAR assay will be used, in collaboration with the Legorreta Cancer Center Drug Discovery core facility, to identify novel PAR-modulating compounds, PARG inhibitors, that can be considered lead compounds in drug development to affect PARG activity in cancer cells.

Our goal in this project is to use novel proteomic technologies to identify protein factors that are involved in  base excision repair (BER) mediated regulation of the replication stress response, focused on ovarian cancer cells as a model system (those that have or do not have mutations in the BRCA1 or BRCA2 genes). We will then evaluate the requirement of these protein factors in the survival of these cancer cell types and in response to clinical and pre-clinical treatment options.

The splicing factor SON regulates the expression of stress response genes, DNA repair (DR) genes and DNA damage response (DDR) genes. Here, we are collaborating with Erin Ahn (University of Alabama Birmingham) to define the DNA repair factors regulated by SON in glioma and other cancers, working to clarify the mechanism of SON-mediated regulation of these DR and DDR genes.

THE SOBOL LAB

THE SOBOL LAB