Defective Base Excision Repair Research Articles

Pan-Cancer Interrogation of MUTYH Variants Reveals Biallelic Inactivation and Defective Base Excision Repair Across a Spectrum of Solid Tumors.

Biallelic germline pathogenic variants of the base excision repair (BER) pathway gene MUTYH predispose to colorectal cancer (CRC) and other cancers. The possible association of heterozygous variants with broader cancer susceptibility remains uncertain. This study investigated the prevalence and consequences of pathogenic MUTYH variants and MUTYH loss of heterozygosity (LOH) in a large pan-cancer analysis. Data from 354,366 solid tumor biopsies that were sequenced as part of routine clinical care were analyzed using a validated algorithm to distinguish germline from somatic MUTYH variants. Biallelic germline pathogenic MUTYH variants were identified in 119 tissue biopsies. Most were CRCs and showed increased tumor mutational burden (TMB) and a mutational signature consistent with defective BER (COSMIC Signature SBS18). Germline heterozygous pathogenic variants were identified in 5,991 biopsies and their prevalence was modestly elevated in some cancer types. About 12% of these cancers (738 samples: including adrenal gland cancers, pancreatic islet cell tumors, nonglioma CNS tumors, GI stromal tumors, and thyroid cancers) showed somatic LOH for MUTYH, higher rates of chromosome 1p loss (where MUTYH is located), elevated genomic LOH, and higher COSMIC SBS18 signature scores, consistent with BER deficiency. This analysis of MUTYH alterations in a large set of solid cancers suggests that in addition to the established role of biallelic pathogenic MUTYH variants in cancer predisposition, a broader range of cancers may possibly arise in MUTYH heterozygotes via a mechanism involving somatic LOH at the MUTYH locus and defective BER. However, the effect is modest and requires confirmation in additional studies before being clinically actionable.

Deficiency in mammalian STN1 promotes colon cancer development via inhibiting DNA repair.

Despite the high lethality of colorectal cancers (CRCs), only a limited number of genetic risk factors are identified. The mammalian ssDNA-binding protein complex CTC1-STN1-TEN1 protects genome stability, yet its role in tumorigenesis is unknown. Here, we show that attenuated CTC1/STN1 expression is common in CRCs. We generated an inducible STN1 knockout mouse model and found that STN1 deficiency in young adult mice increased CRC incidence, tumor size, and tumor load. CRC tumors exhibited enhanced proliferation, reduced apoptosis, and elevated DNA damage and replication stress. We found that STN1 deficiency down-regulated multiple DNA glycosylases, resulting in defective base excision repair (BER) and accumulation of oxidative damage. Collectively, this study identifies STN1 deficiency as a risk factor for CRC and implicates the previously unknown STN1-BER axis in protecting colon tissues from oxidative damage, therefore providing insights into the CRC tumor-suppressing mechanism.

Abstract LB251: Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma

Abstract Background: In cutaneous melanoma, the burden of chromothripsis is high prior to targeted therapy, and additional chromothripsis appears to be a key evolutionary mechanism by which cancer rapidly generates and accumulates highly dynamic structural variants (SVs). Blocking cancer genomic instability may prevent tumor escape from targeted therapies. Methods: We assembled three cohorts of tissues for WGS-based analysis of SVs. The first cohort consisted of patient-matched normal tissues, BRAF V600MUT melanoma tumors before MAPKi therapy and at disease progression (n=10 normal tissues; n=10 pretreatment tumors; n=17 acquired-resistant tumors; n=10 patients). The second cohort consisted of rapid autopsy melanoma tissues (n=3 normal tissues; n=12 acquired-resistant tumors; n=6 metastatic organ sites). The third cohort consisted of cutaneous PDX tumors. To study acquired MAPKi-resistance at the whole-genome level, we subjected PDXs (n=6 models; 1 BRAF MUT and 5 NRAS MUT models) to MAPKi therapy in NSG mice at doses sufficient to elicit tumor regression, and then generated acquired MAPKi-resistant tumors. In total, we used vehicle-treated tumors (n=6), acquired-resistant tumors (n=12), and patient-matched normal tissues (n=6) to generate WGS data. Results: Analysis of genomic amplicons due to intrachromosomal complex genomic rearrangements (CGRs) and extrachromosomal circular DNAs (ecDNAs) uncovered a significant (unpaired Student’s t-test, p=0.0002) association between acquired-resistant tumors and CGRs and/or ecDNAs harboring bona fide MAPKi-resistance genes and revealed copy number amplification of BRAF (range 4.5-27), NRAS (range 5-13), HRAS (range 13-16), MYC (range 12-15) and EGFR (CN 4.6-5), known to drive acquired MAPKi-resistance. Moreover, we validated a recurrent ecDNA by direct isolation and high-depth sequencing using a new approach referred to as CRISPR-CATCH. This alternative technique confirmed the circularized junctions of a 890 kb, driver ecDNA within this acquired-resistant clinical tumor sample. Additionally, resistance-specific (versus sensitivity-specific) chromothriptic single-base substitutions (SBSs) enriched for signatures of defects in base excision repair (BER) and in DNA mismatch repair (MMR) (Wilcoxon rank sum test, p=0.04 and p=0.005 respectively) in 14 of 31 resistant tumors (10 of 16 patients). Moreover, breakpoint-sequence analysis inferred non-homologous end-joining (NHEJ) as critical, and homologous recombination repair (HRR) as adjunctive, to DNA double-stranded break repair underlying CGR and ecDNA formation harboring MAPK-reactivation or MAPKi resistance-driver genes. Inhibition of DNA-PKc or PARP1/2, even only initially during MAPKi treatment, suppressed acquired MAPKi-resistance in melanoma cell lines (BRAF V600MUT n=3 and NRAS Q61MUT n=3) and blunted the expansion of ecDNA + CGR genomic spans. In vivo, DNA-PKi in combination with MAPKi forestalled resistance, reduced ecDNA and CGRs size, and suppressed the contribution of NHEJ in 5 out of 5 cutaneous melanoma PDXs analyzed. Conclusions: Our findings advance the concepts that preventing—instead of reversing—acquired resistant phenotypes may be more clinically impactful and that targeting DNA-PKCS and NHEJ lies at the center of this approach in stabilizing cancer genomes during oncogene-targeted therapies. Citation Format: Prashanthi Dharanipragada, Xiao Zhang, Sixue Liu, Shirley H. Lomeli, Aayoung Hong, Yan Wang, Zhentao Yang, Agustin Vega-Crespo, Antoni Ribas, Stergios J. Moschos, Gatien Moriceau, Roger S. Lo. Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB251.

Abstract 6114: Race-associated base excision repair defects alter the DNA damage landscape in prostate cancer

Abstract Prostate cancer is the most diagnosed cancer among men in the United States. African American men are diagnosed with and succumb to prostate cancer at higher rates than other demographic groups. However, the biological drivers contributing to poorer outcomes in African American men compared to European American men with prostate cancer are still poorly defined. One understudied area is DNA repair defects, which drive genomic instability in cancers. Defects in homologous recombination are often at the forefront of repair defects examined and exploited for therapeutic intervention. However, defects in other DNA repair pathways may contribute to poor disease outcomes and chemoresistance. We demonstrated that defects in the base excision repair (BER) pathway contribute to chemoresistance and alter growth in breast cancer. Others have also shown chemoresistant effects from dysregulated BER protein expression in gastric, colorectal, and ovarian cancers. Examination of the TCGA database show dysregulation of key BER protein like X-ray cross complementing protein 1 (XRCC1) and DNA polymerase β (POLβ) are common across several cancers. Therefore, we decided to explore DNA damage and repair defects in prostate cancer to explore if altered DNA repair contributes to poorer outcomes in African American men. To measure DNA repair defects in formalin-fixed paraffin-embedded (FFPE) tissues, we employed a novel assay, Repair Assisted Damage Detection (RADD), which measures DNA lesions within the tissues. RADD fluorescently tags DNA lesions for quantification and is compatible with immunohistochemistry detection of DNA repair or other proteins of interest. Using the tissue microarrays, we used RADD to discover that prostate tumors from African American patients have more uracil and pyrimidine lesions, elevated uracil DNA glycosylase (UNG) levels, and reduced XRCC1 levels than European American tumors, which indicate defects in the BER pathway. In addition, these men had higher uracil monophosphate (UMP) and lower expression of folate cycle metabolites, suggesting that metabolic rewiring may also contribute to the dysregulation of BER. Defects in the BER pathway may offer new therapeutic opportunities for African American prostate cancer patients. These defects may be targeted by therapeutics which pressure the BER pathways. This work demonstrates that functional detection of DNA repair defects through RADD offers new insight into race-specific DNA repair defects offering new molecular targets or therapeutic strategies to reduce racial disparities in prostate cancer. Citation Format: Kaveri Goel, Kimiko Krieger, Manoj Sonavane, Arun Sreekumar, Natalie R. Gassman. Race-associated base excision repair defects alter the DNA damage landscape in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6114.

TBIO-04. Comprehensive analysis of mutational signatures in pediatric cancers

Abstract Analysis of mutational signatures can reveal the underlying molecular processes that have caused the somatic mutations found in genomes. We performed an extensive analysis encompassing mutational signatures of single base substitution and, for the first time, signatures of small insertions/deletions in ~800 whole-genome-sequenced tumor-normal pairs from 25 molecularly defined pediatric cancer types. More than half of this cohort consists of pediatric CNS-tumors (n=416), including high- and low-grade gliomas, ependymomas, and embryonal tumors. These were subsequently compared with COSMIC v.3 signatures to identify overlap with the latest set of known mutational signatures. We identified only a small number of mutational signatures active in pediatric cancers when compared to previously analyzed adult cancers. Amongst these, SBS1 and SBS5 were present in nearly all pediatric tumors analyzed, with a significant correlation of signature activity with age at diagnosis, as expected. Further, we found SBS21 activity in a fraction of high-grade gliomas, which is the result of defective DNA mismatch repair, SBS36 in fractions of ETMR and Group4-medulloblastoma, the result of defective base excision repair, and SBS44 in Group4-medulloblastomas, caused by defective DNA mismatch repair. For these signatures, no consistent genetic alteration was identified. We report a significantly smaller proportion of pediatric tumors which show homologous-recombination repair defect signature SBS3 compared to previously published analyses. Additionally, the previous mutational signature analysis of this cohort based on COSMIC v.2 reference signatures had identified a novel substitution signature (Signature.P1), active in the brain tumors ATRT and ependymoma. Our updated results suggest that Signature.P1 is not a pediatric specific mutational signature, but a treatment-associated signature identified in a small fraction of brain tumors that were annotated as treatment-naïve. This analysis provides a systematic overview of mutational signatures in pediatric cancers, which is relevant for understanding tumor biology and future research in defining biomarkers for treatment response.

Activated or Impaired: An Overview of DNA Repair in Neurodegenerative Diseases.

As the population ages, age-related neurodegenerative diseases have become a major challenge in health science. Currently, the pathology of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, is still not fully understood. Remarkably, emerging evidence indicates a role of genomic DNA damage and repair in various neurodegenerative disorders. Here, we summarized the current understanding of the function of DNA damage repair, especially base excision repair and double strand break repair pathways, in a variety of neurodegenerative diseases. We concluded that exacerbation of DNA lesions is found in almost all types of neurodegenerative diseases, whereas the activities of different DNA repair pathways demonstrate distinct trends, depending on disease type and even brain region. Specifically, key enzymes involved in base excision repair are likely impaired in Alzheimer's disease and amyotrophic lateral sclerosis but activated in Parkinson's disease, while nonhomologous end joining is likely downregulated in most types of neurodegenerative diseases. Hence, impairment of nonhomologous end joining is likely a common etiology for most neurodegenerative diseases, while defects in base excision repair are likely involved in the pathology of Alzheimer's disease and amyotrophic lateral sclerosis but are Parkinson's disease, based on current findings. Although there are still discrepancies and further studies are required to completely elucidate the exact roles of DNA repair in neurodegeneration, the current studies summarized here provide crucial insights into the pathology of neurodegenerative diseases and may reveal novel drug targets for corresponding neurodegenerative diseases.

Pan-Cancer Survey of Tumor Mass Dormancy and Underlying Mutational Processes.

Tumor mass dormancy is the key intermediate step between immune surveillance and cancer progression, yet due to its transitory nature it has been difficult to capture and characterize. Little is understood of its prevalence across cancer types and of the mutational background that may favor such a state. While this balance is finely tuned internally by the equilibrium between cell proliferation and cell death, the main external factors contributing to tumor mass dormancy are immunological and angiogenic. To understand the genomic and cellular context in which tumor mass dormancy may develop, we comprehensively profiled signals of immune and angiogenic dormancy in 9,631 cancers from the Cancer Genome Atlas and linked them to tumor mutagenesis. We find evidence for immunological and angiogenic dormancy-like signals in 16.5% of bulk sequenced tumors, with a frequency of up to 33% in certain tissues. Mutations in the CASP8 and HRAS oncogenes were positively selected in dormant tumors, suggesting an evolutionary pressure for controlling cell growth/apoptosis signals. By surveying the mutational damage patterns left in the genome by known cancer risk factors, we found that aging-induced mutations were relatively depleted in these tumors, while patterns of smoking and defective base excision repair were linked with increased tumor mass dormancy. Furthermore, we identified a link between APOBEC mutagenesis and dormancy, which comes in conjunction with immune exhaustion and may partly depend on the expression of the angiogenesis regulator PLG as well as interferon and chemokine signals. Tumor mass dormancy also appeared to be impaired in hypoxic conditions in the majority of cancers. The microenvironment of dormant cancers was enriched in cytotoxic and regulatory T cells, as expected, but also in macrophages and showed a reduction in inflammatory Th17 signals. Finally, tumor mass dormancy was linked with improved patient survival outcomes. Our analysis sheds light onto the complex interplay between dormancy, exhaustion, APOBEC activity and hypoxia, and sets directions for future mechanistic explorations.

8‐oxoguanine glycosylase (OGG1) is a determinant of endothelial dysfunction via calcium and nitric oxide signaling

Diabetes mellitus is a chronic disease that hinders the body's ability to produce or respond to the hormone insulin, resulting in abnormal metabolism and elevated glucose levels. Type II diabetes has been characterized by a two-to-four-fold increase in cardiovascular disease, such as carotid artery stenosis, generally attributed to the adverse effects of hyperglycemia and oxidative stress on vascular biology. With it being a very common and significant health complication, understanding the pathology and the mechanisms by which diabetes-induced oxidative stress serves as a precursor to several notable vascular diseases is paramount, and is the overall objective of this study. Endothelial dysfunction (ED) is a vascular phenomenon attributed to a reduction in nitric oxide (NO) bioactivity and an increase in oxygen free radical formation with subsequent oxidative stress, thus leading to impaired vasodilation, and compromised vascular function. ED tends to be a consistent finding in diabetes. A major source of oxidative stress is the abnormal production of reactive oxygen species (ROS) in the context of diabetic inflammation. Increased ROS production leads to mitochondrial DNA (mt DNA) damage and aberrant cell signaling. Typically, mt DNA damage is repaired through the base excision repair (BER) pathway. However, during chronic inflammation in diabetes, this repairing capability is ineffective due to downregulation of key BER enzymes, namely 8-oxoguanine glycosylase (OGG1). Given this rational, we hypothesized that the BER pathway is an important determinant of endothelial dysfunction via impaired calcium and NO signaling. To test this hypothesis, we examined the effect of BER defects on endothelial calcium signaling and NO production using carotid arteries isolated from mice deficient in OGG1. Opened artery measurements of endothelial calcium and NO signaling were performed using a custom-designed parallel plate flow chamber. Briefly, segments of carotid arteries (~200 µm in diameter) were isolated from OGG1 deficient (KO) and wild type (WT) mice and cut open lengthwise to expose the endothelial surface. The isolated vessels were then stained with a loading solution composed of Pluronic acid (Fischer), Cal-520 (AAT bio) calcium indicator dye and DAR-4M (Calbiochem), a fluorescent probe for NO. Time lapse signal measurements were collected via confocal microscopy and were then analyzed and processed with S8, a custom automated solution to signal analysis based on noise filtering and object tracking, which computed dynamic amplitude, frequency, duration, and spatial spread. We discovered that although the number of calcium signals detected within the measurement interval were similar between KO and WT (10 ± 3 vs 9 ± 3, µ ± SEM, respectively), the spatial spread of signals within the KO (19.95 ± 3.78 µm2, µ ± SEM) was greater than WT (9.65 ± 2.7 µm2, p = 0.057 via Welch's unpaired t-test). These data suggest that OGG1 may be a determinant of normal endothelial calcium signaling and might serve as a therapeutic target for preventing the adverse effects of endothelial dysfunction present in diabetes.

Base Excision Repair in the Immune System: Small DNA Lesions With Big Consequences.

The integrity of the genome is under constant threat of environmental and endogenous agents that cause DNA damage. Endogenous damage is particularly pervasive, occurring at an estimated rate of 10,000–30,000 per cell/per day, and mostly involves chemical DNA base lesions caused by oxidation, depurination, alkylation, and deamination. The base excision repair (BER) pathway is primary responsible for removing and repairing these small base lesions that would otherwise lead to mutations or DNA breaks during replication. Next to preventing DNA mutations and damage, the BER pathway is also involved in mutagenic processes in B cells during immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM), which are instigated by uracil (U) lesions derived from activation-induced cytidine deaminase (AID) activity. BER is required for the processing of AID-induced lesions into DNA double strand breaks (DSB) that are required for CSR, and is of pivotal importance for determining the mutagenic outcome of uracil lesions during SHM. Although uracils are generally efficiently repaired by error-free BER, this process is surprisingly error-prone at the Ig loci in proliferating B cells. Breakdown of this high-fidelity process outside of the Ig loci has been linked to mutations observed in B-cell tumors and DNA breaks and chromosomal translocations in activated B cells. Next to its role in preventing cancer, BER has also been implicated in immune tolerance. Several defects in BER components have been associated with autoimmune diseases, and animal models have shown that BER defects can cause autoimmunity in a B-cell intrinsic and extrinsic fashion. In this review we discuss the contribution of BER to genomic integrity in the context of immune receptor diversification, cancer and autoimmune diseases.

DNA Oxidation and Excision Repair Pathways.

The physiological impact of the aberrant oxidation products on genomic DNA were demonstrated by embryonic lethality or the cancer susceptibility and/or neurological symptoms of animal impaired in the base excision repair (BER); the major pathway to maintain genomic integrity against non-bulky DNA oxidation. However, growing evidence suggests that other DNA repair pathways or factors that are not primarily associated with the classical BER pathway are also actively involved in the mitigation of oxidative assaults on the genomic DNA, according to the corresponding types of DNA oxidation. Among others, factors dedicated to lesion recognition in the nucleotide excision repair (NER) pathway have been shown to play eminent roles in the process of lesion recognition and stimulation of the enzyme activity of some sets of BER factors. Besides, substantial bulky DNA oxidation can be preferentially removed by a canonical NER mechanism; therefore, loss of function in the NER pathway shares common features arising from BER defects, including cancer predisposition and neurological disorders, although NER defects generally are nonlethal. Here we discuss recent achievements for delineating newly arising roles of NER lesion recognition factors to facilitate the BER process, and cooperative works of BER and NER pathways in response to the genotoxic oxidative stress.

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer.

DNA repair defects have been increasingly focused on as therapeutic targets. In hormone-positive breast cancer, XRCC1-deficient tumors have been identified and proposed as targets for combination therapies that damage DNA and inhibit DNA repair pathways. XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) polymerase 1, DNA polymerase β (POL β), and DNA ligases. Loss of XRCC1 confers BER defects and hypersensitivity to DNA damaging agents. BER defects have not been evaluated in triple negative breast cancers (TNBC), for which new therapeutic targets and therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects in TNBC, we examined XRCC1 expression in the TCGA database and its expression and localization in TNBC cell lines. The TCGA database revealed high XRCC1 expression in TNBC tumors and TNBC cell lines show variable, but mostly high expression of XRCC1. XRCC1 localized outside of the nucleus in some TNBC cell lines, altering their ability to repair base lesions and single-strand breaks. Subcellular localization of POL β also varied and did not correlate with XRCC1 localization. Basal levels of DNA damage correlated with observed changes in XRCC1 expression, localization, and measure repair capacity. The results confirmed that XRCC1 expression changes indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with protein localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and tumors, XRCC1 expression levels should be assessed when evaluating treatment responses of TNBC preclinical model cells.

Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells.

Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here, we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase β and Proliferating Cell Nuclear Antigen (PCNA), together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells.

Defective Base Excision Repair Associated with NEIL1

Defective Base Excision Repair Associated with NEIL1

Defective Base Excision Repair Associated with NEIL3

Defective Base Excision Repair Associated with NEIL3

Defective Base Excision Repair Associated with NTHL1

Defective Base Excision Repair Associated with NTHL1

Defective Base Excision Repair of Oxidative DNA Damage in Vascular Smooth Muscle Cells Promotes Atherosclerosis

Atherosclerotic plaques demonstrate extensive accumulation of oxidative DNA damage, predominantly as 8-oxoguanine (8oxoG) lesions. 8oxoG is repaired by base excision repair enzymes; however, the mechanisms regulating 8oxoG accumulation in vascular smooth muscle cells (VSMCs) and its effects on their function and in atherosclerosis are unknown. We studied levels of 8oxoG and its regulatory enzymes in human atherosclerosis, the mechanisms regulating 8oxoG repair and the base excision repair enzyme 8oxoG DNA glycosylase I (OGG1) in VSMCs in vitro, and the effects of reducing 8oxoG in VSMCs in atherosclerosis in ApoE-/- mice. Human plaque VSMCs showed defective nuclear 8oxoG repair, associated with reduced acetylation of OGG1. OGG1 was a key regulatory enzyme of 8oxoG repair in VSMCs, and its acetylation was crucial to its repair function through regulation of protein stability and expression. p300 and sirtuin 1 were identified as the OGG1 acetyltransferase and deacetylase regulators, respectively, and both proteins interacted with OGG1 and regulated OGG1 acetylation at endogenous levels. However, p300 levels were decreased in human plaque VSMCs and in response to oxidative stress, suggesting that reactive oxygen species-induced regulation of OGG1 acetylation could be caused by reactive oxygen species-induced decrease in p300 expression. We generated mice that express VSMC-restricted OGG1 or an acetylation defective version (SM22α-OGG1 and SM22α-OGG1K-R mice) and crossed them with ApoE-/- mice. We also studied ApoE-/- mice deficient in OGG1 (OGG1-/-). OGG1-/- mice showed increased 8oxoG in vivo and increased atherosclerosis, whereas mice expressing VSMC-specific OGG1 but not the acetylation mutant OGG1K-R showed markedly reduced intracellular 8oxoG and reduced atherosclerosis. VSMC OGG1 reduced telomere 8oxoG accumulation, DNA strand breaks, cell death and senescence after oxidant stress, and activation of proinflammatory pathways. We identify defective 8oxoG base excision repair in human atherosclerotic plaque VSMCs, OGG1 as a major 8oxoG repair enzyme in VSMCs, and p300/sirtuin 1 as major regulators of OGG1 through acetylation/deacetylation. Reducing oxidative damage by rescuing OGG1 activity reduces plaque development, indicating the detrimental effects of 8oxoG on VSMC function.

DNA polymerase β deficiency in the p53 null cerebellum leads to medulloblastoma formation

Defects in DNA damage response or repair mechanisms during neurogenesis result in genomic instability, which is causative for several neural defects. These include brain tumors, particularly medulloblastoma, which occurs in the cerebellum with a high incidence in children. We generated an animal model with defective base excision repair during brain development through selective inactivation of DNA polymerase β (Polb) in neuroprogenitor cells. All of Polb conditional knockout mice developed medulloblastoma in a p53 null background, similar to the Xrcc1 and p53 double deficient animal model. XRCC1 is a scaffolding protein which is involved in DNA damage repair and binds to POLB. In both animal models, the histopathological characteristics of the medulloblastoma were similar to those of human classic medulloblastoma. Brain tumor development was slower in the Polb and p53 double null animals than in the Xrcc1 and p53 double knockout animals. Molecular marker analysis suggested that Polb- and Xrcc1-deficient medulloblastomas belonged to the SHHα subtype, underscoring the important role of genomic stability in preventing this devastating pediatric cerebellar tumor.

Defective Base Excision Repair Associated with MUTYH

Defective Base Excision Repair Associated with MUTYH

Abstract 4846: Targeting BRCA1-mutated cancer cells with elesclomol

Abstract The breast cancer susceptibility gene 1 (BRCA1) encodes a tumor-suppressor protein that maintains genetic stability through DNA damage response pathways. Specifically, BRCA1 has a well-established role in double-strand DNA break repair, and emerging evidence supports a role for BRCA1 in repair of oxidative DNA damage via the base-excision repair (BER) pathway. We and others have shown that cells with mutant/deficient BRCA1 harbor defective BER of oxidative DNA damage. Interestingly, defective BER of oxidative DNA damage in breast cancer cells rendered sensitivity to elesclomol, an experimental therapeutic that induces reactive oxygen species to levels beyond a viable threshold. Given that BRCA1-mutated cancers associate with a poor prognosis and are in need of targeted treatment options, we hypothesized that breast or ovarian cancer cells with mutant BRCA1 would exhibit selective sensitivity to elesclomol or treatment regimens containing elesclomol. First, we determined the effect of BRCA1 on the cellular response to oxidative stress and elesclomol using human ovarian cancer cells isogenic for BRCA1. Compared to mutant BRCA1 cells (UWB1.289), wild-type BRCA1 cells (UWB1.289+BRCA1) produced significantly greater levels of BRCA1 mRNA (p=0.009), expressed markedly higher levels of BRCA1 protein in the nucleus, and showed significantly greater sensitivity to hydrogen peroxide (p=0.002) as evidenced by RTqPCR, Western blot analysis, and MTT assay, respectively. Importantly, mutant BRCA1 cells displayed an approximate 100-fold greater sensitivity to elesclomol compared to wild-type BRCA1 cells as determined by MTT assay. These data suggest that the oxidative stress induced by elesclomol may be used to selectively target cancer cells with mutant BRCA1 due to their defective BER phenotype. We next asked whether elesclomol may be combined with other agents used clinically or experimentally for the treatment of BRCA1-mutated cancers, including standard chemotherapy drugs and PARP inhibitors. Elesclomol exhibited synergistic sensitivity when combined with DNA-damaging agents, including doxorubicin or cisplatin, in breast cancer cells with mutant BRCA1 (SUM149) as determined by combination index and isobologram analysis. On the other hand, a synergistic effect was not observed with the anti-microtubule agent paclitaxel, which is consistent with a phase III study that evaluated this combination for the treatment of advanced melanoma. However, elesclomol in combination with PARP inhibitors, including rucaparib or talazoparib, indeed showed synergism in breast cancer cells with mutant BRCA1. These data suggest that elesclomol may be used in combination with other anticancer agents to further exploit defects in DNA repair due to mutant BRCA1. In conclusion, elesclomol may be explored as a novel component of treatment regimens for targeting BRCA1-mutated breast and ovarian cancers. Citation Format: Maria Kammire, Emily Fannin, James M. Ford, Elizabeth Alli. Targeting BRCA1-mutated cancer cells with elesclomol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4846.

Defective Base Excision Repair of Oxidative DNA Damage in Vascular Smooth Muscle Cells Promotes Atherosclerosis

Defective Base Excision Repair of Oxidative DNA Damage in Vascular Smooth Muscle Cells Promotes Atherosclerosis