Defects In Nucleotide Excision Repair Research Articles

Abstract 5614: Mechanical regulation of cisplatin-DNA adduct repair in mesenchymal stem cells

Abstract This study examined whether loss of mechanotransduction in pluripotent mesenchymal stem cells (MSCs) disrupted repair of bulky DNA adducts, such as those induced by ultraviolet light or cisplatin. MSCs are important for the maintenance and responsiveness of skeletal tissues to environmental mechanical challenges. Beyond regulating proliferative and differentiative capacity of MSCs, emerging evidence suggests that physical activity is associated with improved recovery from DNA damage caused by environmental genotoxic stressors associated with stress and aging. Highlighting the possible crosstalk between DNA repair and bone health, defects in nucleotide excision repair (NER), which removes bulky DNA adducts, are associated with myelosuppression and progeroid aging phenotypes that result in bone defects and lipidodystrophies in mice. Cancer, early aging syndromes, and certain musculoskeletal diseases are associated with disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that transduces environmental mechanical signals from the extracellular matrix into the nucleus of a cell. Loss of LINC complex elements is associated with breast cancer progression and poorer prognosis. We hypothesize that LINC-mediated mechanical stimulation into the nucleus is critical for repair of bulky DNA adducts. Our pilot project showed that mechanical stimulation applied in the form of low intensity vibration (LIV) improved NER of cisplatin-induced DNA adducts in cultured MSCs. Our findings show a 20% increase in repair of cisplatin-induced DNA adducts in LIV treated samples compared to non-vibrated samples at 24 hours (P<0.005). We also quantified the effect of LINC complex disruption on cisplatin-induced DNA damage removal. Intact MSCs showed significant adduct removal after 24 hours, while LINC disruption significantly reduced DNA repair (P<0.001). Furthermore, initial experiments suggest that LINC disrupted cells do not arrest at DNA repair checkpoints, therefore, loss of LINC complex may reduce entry into senescence increasing DNA mutagenesis. In conclusion, these findings suggest a novel interaction between mechanical stimulation, the DNA damage response, and LINC complex in DNA repair in multipotent mesenchymal stem cells. Furthermore, we suggest that LINC-mediated mechanical changes associated with LIV treatment may lead to novel therapeutic approaches to combat deleterious musculoskeletal conditions associated with anti-cancer treatments. Citation Format: Anamaria Gabriella Zavala, Nina Nikitina, Crystal Cantu, Dalia DeLaCruz, Gunes Uzer. Mechanical regulation of cisplatin-DNA adduct repair in mesenchymal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5614.

XPA tumor variant leads to defects in NER that sensitize cells to cisplatin.

Nucleotide excision repair (NER) reduces efficacy of treatment with platinum (Pt)-based chemotherapy by removing Pt lesions from DNA. Previous study has identified that missense mutation or loss of the NER genes Excision Repair Cross Complementation Group 1 and 2 (ERCC1 and ERCC2) leads to improved patient outcomes after treatment with Pt-based chemotherapies. Although most NER gene alterations found in patient tumors are missense mutations, the impact of mutations in the remaining nearly 20 NER genes is unknown. Towards this goal, we previously developed a machine learning strategy to predict genetic variants in an essential NER protein, Xeroderma Pigmentosum Complementation Group A (XPA), that disrupt repair. In this study, we report in-depth analyses of a subset of the predicted variants, including in vitro analyses of purified recombinant protein and cell-based assays to test Pt agent sensitivity in cells and determine mechanisms of NER dysfunction. The most NER deficient variant Y148D had reduced protein stability, weaker DNA binding, disrupted recruitment to damage, and degradation. Our findings demonstrate that tumor mutations in XPA impact cell survival after cisplatin treatment and provide valuable mechanistic insights to improve variant effect prediction. Broadly, these findings suggest XPA tumor variants should be considered when predicting chemotherapy response.

Preclinical Studies Support the Clinical Development of LP-284 in Relapsed and Refractory B-Cell Lymphomas

Genomic instability, a hallmark of many cancers, often results from corrupted DNA damage response/repair (DDR) pathways. Targeting DDR deficiencies in cancer therapy has been successful in solid tumors with dysfunctional homologous recombination repair (HR). However, while less recognized, lymphomas also carry DDR defects such as HR deficiency (HRD) and can account for approximately 18% of all lymphomas. LP-284 is a fully synthetic DNA-damaging agent that induces double-stranded DNA breaks and displays elevated potency in cancer cells with HRD or transcription-coupled nucleotide excision repair defects, including multiple advanced B-cell lymphomas. In preclinical pharmacology studies, the mantle cell lymphoma (MCL) xenograft tumor mouse model derived from JeKo-1, which has mutated TP53 and checkpoint kinase 2 (CHEK2), an HRD score of 46, and was refractory to ibrutinib and bortezomib, showed near complete response after LP-284 treatment. It is noteworthy that 40~50% of MCL patients may be HRD due to the presence of mutations in the ataxia-telangiectasia mutated (ATM) gene. HRD is also likely to be prevalent in high-grade lymphomas, as IGH/MYC translocation or BCL2 expression has been reported to be associated with HR inhibition. Studies of LP-284 in the MYC/BCL2 dual-translocated OCILY1 tumor xenograft model have shown a robust efficacy profile with an average of 99% tumor growth inhibition. Key pharmacokinetic studies demonstrated that LP-284 is neither a substrate nor modulator of major CYP-450 enzymes, multi-drug resistance transporters, renal drug transporters, or hepatic drug transporters. Safety pharmacology studies indicated that LP-284 does not inhibit hERG potassium channel and has a low potential to affect cardiovascular functions. Toxicology repeat dose studies of LP-284 in Sprague Dawley rats and Beagle dogs, using intravenous infusion on Days 1, 8, and 15 in a 28-day cycle, were generally well tolerated with notable adverse effects in Beagle dogs consisting mainly of reversible hematologic toxicity and testicular toxicity. The highest non-severely toxic dose (HNSTD) in dogs was considered to be 0.4 mg/kg/dose. Overall, based on its impressive preclinical anti-tumor efficacy profile, low drug-drug interaction liability, and findings from toxicological studies, a Phase 1 study is currently planned with LP-284 to assess its safety, tolerability, pharmacokinetics, and clinical activity in patients with relapsed or refractory B-cell lymphoma. Clinical development will be informed by an emphasis on patient selection strategy based on DDR/HR biomarkers.

DNAR-08. BASE EXCISION REPAIR AS A THERAPEUTIC TARGET FOR DIFFUSE HEMISPHERIC GLIOMA, H3.3G34-MUTANT

Abstract Diffuse hemispheric gliomas with H3.3G34R/V mutations are primarily found in the cerebral hemisphere in adolescents and young adults, with a median age of 16-19 years. H3.3G34R frequently co-occurs with mutations in ATRX and TP53 as well as PDGFRα. We performed mutational signature analysis on a cohort of 28 cases of Diffuse hemispheric glioma, H3G34-mutant (7 WGS and 21 WES). Mutational signatures of defects in mismatch repair (MMR), homologous recombination (HR), and nucleotide excision repair (NER) pathways were found in 86% cases whereas only two (7%) had signatures of impaired base excision repair (BER). Analysis of H3.3G34R interactome data showed loss of association with MMR, HR, and NER pathways, with gains in association with proteins in the BER pathway compared to wildtype Histone H3. We thus hypothesise the Diffuse hemispheric glioma, H3G34-mutant may be dependent on BER over other DNA damage repair pathways. To test if BER could be a therapeutic vulnerability, we knocked down integral members of the BER pathway (UNG, APEX1, MPG, and XRCC1) via shRNA in 4 patient-derived H3.3G34R-mutant cell lines (two primary and two matched recurrent), and found that knockdown of XRCC1 and MPG, led to 99% (p< 0.0001) and 96% (p< 0.0001) cell death, respectively, after 8 days, compared to scrambled control. We next tested Triptolide, a BER inhibitor, and it led to cell death in vitro with IC50s of 1.1 to 1.7 nM. Taken together, these results indicate that inhibition of the BER pathway, specifically of the scaffolding-protein XRCC1, could be used as a novel therapeutic approach for diffuse hemispheric glioma, H3.3G34-mutant.

The Spectrum of MORC2-Related Disorders: A Potential Link to Cockayne Syndrome

BackgroundCockayne syndrome (CS) is a DNA repair disorder primarily associated with pathogenic variants in ERCC6 and ERCC8. As in other Mendelian disorders, there are a number of genetically unsolved CS cases. MethodsWe ascertained five individuals with monoallelic pathogenic variants in MORC2, previously associated with three dominantly inherited phenotypes: an axonal form of Charcot-Marie-Tooth disease type 2Z; a syndrome of developmental delay, impaired growth, dysmorphic facies, and axonal neuropathy; and a rare form of spinal muscular atrophy. ResultsOne of these individuals bore a strong phenotypic resemblance to CS. We then identified monoallelic pathogenic MORC2 variants in three of five genetically unsolved individuals with a clinical diagnosis of CS. In total, we identified eight individuals with MORC2-related disorder, four of whom had clinical features strongly suggestive of CS. ConclusionsOur findings indicate that some forms of MORC2-related disorder have phenotypic similarities to CS, including features of accelerated aging. Unlike classic DNA repair disorders, MORC2-related disorder does not appear to be associated with a defect in transcription-coupled nucleotide excision repair and follows a dominant pattern of inheritance with variants typically arising de novo. Such de novo pathogenic variants present particular challenges with regard to both initial gene discovery and diagnostic evaluations. MORC2 should be included in diagnostic genetic test panels targeting the evaluation of microcephaly and/or suspected DNA repair disorders. Future studies of MORC2 and its protein product, coupled with further phenotypic characterization, will help to optimize the diagnosis, understanding, and therapy of the associated disorders.

Abstract A006: LINC00261 acts as a surrogate biomarker for tumor DNA mutational burden and immunotherapeutic response in lung adenocarcinoma

Abstract Defects in DNA repair allow for the age-dependent accumulation of mutations, which is significantly linked to the age-dependent increase in the development of neoplasia. Our lab has identified the long non-coding RNA LINC00261 as a critical activator of DNA damage signaling whose expression increases with age in normal lung tissue but is lost in the majority of Lung Adenocarcinomas (LUAD). We therefore undertook a mechanistic investigation on the relationship between LINC00261 function in lung and the accumulation of mutations by utilizing expression and mutational load data from The Cancer Genome Atlas, as well as in vitro mechanistic investigation into the functional relationship between LINC00261 and tumor mutational burden (TMB). Here we report (1) the observation of an age-dependent increase in LINC00261 expression in normal lung tissue within the TCGA adjacent non-tumor lung dataset; (2) a significant correlation between TMB and LINC00261 expression, where elevated LINC00261 levels are associated with decreased TMB; (3) LINC00261 effects on TMB were driven primarily by transition mutations, characteristic of defects in nucleotide excision repair. Standard of care treatment for disseminated LUAD often includes systemic platinum-based chemotherapies, including cisplatin, that act by inducing lethal levels of DNA damage. We observed that the presence of LINC00261 in LUAD causes cellular resistance to cisplatin-mediated DNA-damage in vitro. Our results suggest that patients with tumors expressing LINC00261 should be considered for non-platinum based therapeutic strategies. High mutational loads are also associated with the creation of neoantigens, allowing for immune recognition and when combined with PD-L1 expression levels can serve as an indicator for successful immunotherapy. Testing for PD-L1 expression is now standard of care, however exome sequencing to determine TMB is not often pursued outside academic medical centers due to expense, extensive bioinformatic involvement, and difficulty interpreting results. In addition, high levels of TMB does not always confer active neoantigen presentation as MHC Class II genes are often dysregulated in cancer. We therefore sought to determine if LINC00261 can function as a surrogate biomarker for TMB and provide complimentary indications to PD-L1 testing for successful immunotherapeutic response. Reintroduction of ectopic LINC00261 was able to activate expression of MHC Class II genes involved in neoantigen presentation. Conversely, Interferon gamma addition was able to induce endogenous expression of LINC00261 in vitro. Our results suggest there is a mechanistic relationship between LINC00261 expression and functional MHC Class II neoantigen presentation in LUAD. This was supported by observations that LINC00261 expression is significantly correlated to MHC Class II expression within TCGA LUAD cohort. Taken together, our results suggest LINC00261 shows promise as a biomarker of mutational burden and MHC Class II expression. Citation Format: Jonathan Castillo, Tianchun Xue, Mayela Norwood, Samantha Joseph, Crystal N. Marconett. LINC00261 acts as a surrogate biomarker for tumor DNA mutational burden and immunotherapeutic response in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A006.

Deregulated DNA damage response network in Behcet's disease

Behcet's disease (BD) is a chronic, relapsing systemic vasculitis of unknown etiology. Since the DNA repair enzyme NEIL1 has been identified as one of the two genetic risk factors for BD by whole exome study, we examined the potential involvement of the DNA damage response (DDR) network in BD. Peripheral blood mononuclear cells from 26 patients and 26 age−/sex-matched healthy controls were studied. Endogenous DNA damage levels were increased in active BD patients compared to controls or patients in remission. In parallel, BD patients had defective nucleotide excision repair capacity. RNA-sequencing revealed reduced expression of NEIL1 that negatively correlated with DNA damage accumulation. On the other hand, expression of genes involved in senescence and senescence-associated secretory phenotype positively correlated with individual endogenous DNA damage levels. We conclude that deregulated DDR contributes to the proinflammatory environment in BD.

Homozygous CRISPR/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells.

Defects in DNA repair pathways have been associated with an improved response to immune checkpoint inhibition (ICI). In particular, patients with the nucleotide excision repair (NER) defect disease Xeroderma pigmentosum (XP) responded impressively well to ICI treatment. Recently, in melanoma patients, pretherapeutic XP gene expression was predictive for anti-programmed cell death-1 (PD-1) ICI response. The underlying mechanisms of this finding are still to be revealed. Therefore, we used CRISPR/Cas9 to disrupt XPA in A375 melanoma cells. The resulting subclonal cell lines were investigated by Sanger sequencing. Based on their genetic sequence, candidates from XPA exon 1 and 2 were selected and further analyzed by immunoblotting, immunofluorescence, HCR and MTT assays. In XPA exon 1, we established a homozygous (c.19delG; p.A7Lfs*8) and a compound heterozygous (c.19delG/c.19_20insG; p.A7Lfs*8/p.A7Gfs*55) cell line. In XPA exon 2, we generated a compound heterozygous mutated cell line (c.206_208delTTG/c.208_209delGA; p.I69_D70delinsN/p.D70Hfs*31). The better performance of the homozygous than the heterozygous mutated exon 1 cells in DNA damage repair (HCR) and post-UV-C cell survival (MTT), was associated with the expression of a novel XPA protein variant. The results of our study serve as the fundamental basis for the investigation of the immunological consequences of XPA disruption in melanoma.

A stable XPG protein is required for proper ribosome biogenesis: Insights on the phenotype of combinate Xeroderma Pigmentosum/Cockayne Syndrome patients.

Nucleotide Excision Repair is one of the five DNA repair systems. More than 30 proteins are involved in this process, including the seven XP proteins. When mutated, the genes coding for these proteins are provoking the rare disease Xeroderma Pigmentosum, which causes cutaneous defects and a high prevalence of skin cancers in patients. The CSA and CSB proteins are also involved in Nucleotide Excision Repair, and their mutation leads to Cockayne Syndrome, another rare disease, causing dwarfism, neurodegeneration, and ultimately early death, but without high skin cancer incidence. Some mutations of ERCC5, the gene coding for XPG, may give rise to a combined Xeroderma Pigmentosum and Cockayne Syndrome. A defect in Nucleotide Excision Repair alone cannot explain all these phenotypes. XPG has been located in the nucleolus, where ribosome biogenesis happens. This energy-consuming process starts with the transcription of the ribosomal DNA in a long ribosomal RNA, the pre-rRNA 47S, by RNA Polymerase 1. 47S pre-rRNA undergoes several cleavages and modifications to form three mature products: the ribosomal RNAs 18S, 5.8S and 28S. In the cytoplasm, these three products will enter the ribosomes’ composition, the producers of protein in our cells. Our work aimed to observe ribosome biogenesis in presence of an unstable XPG protein. By working on Xeroderma Pigmentosum/Cockayne Syndrome cell lines, meaning in the absence of XPG, we uncovered that the binding of UBF, as well as the number of unresolved R-loops, is increased along the ribosomal DNA gene body and flanking regions. Furthermore, ribosomal RNA maturation is impaired, with increased use of alternative pathways of maturation as well as an increase of immature precursors. These defective processes may explain the neurodegeneration observed when the XPG protein is heavily truncated, as ribosomal homeostasis and R-loops resolution are critical for proper neuronal development.

A Preclinical Study to Repurpose Spironolactone for Enhancing Chemotherapy Response in Bladder Cancer.

Neoadjuvant chemotherapy (NAC) followed by radical cystectomy is the standard-of-care for patients with muscle-invasive bladder cancer (MIBC). Defects in nucleotide excision repair (NER) are associated with improved responses to NAC. Excision Repair Cross-Complementation group 3 (ERCC3) is a key component of NER process. No NER inhibitors are available for treating patients with bladder cancer. We have developed an ex vivo cell-based assay of 6-4 pyrimidine-pyrimidinone (6-4PP) removal as a surrogate measure of NER capacity in human bladder cancer cell lines. The protein expression of ERCC3 was examined in human MIBC specimens and cell lines. Small molecule inhibitors were screened for NER inhibition in bladder cancer cell lines. Spironolactone was identified as a potent NER inhibitor. Combined effects of spironolactone with chemo-drugs were evaluated in vitro and in vivo. The efficacy between platinum and spironolactone on cytotoxicity was determined by combination index. A correlation between NER capacity and cisplatin sensitivity was demonstrated in a series of bladder cancer cell lines. Further, siRNA-mediated knockdown of ERCC3 abrogated NER capacity and enhanced cisplatin cytotoxicity. Spironolactone inhibited ERCC3 protein expression, abrogated NER capacity, and increased platinum-induced cytotoxicity in bladder cancer cells in vivo and in patient-derived organoids. Moreover, spironolactone exhibited the potential synergism effects with other clinical chemotherapy regimens in bladder cancer cell lines. Our data support the notion of repurposing spironolactone for improving the chemotherapy response of NAC in patients with MIBC. Further clinical trials are warranted to determine the safety and efficacy of spironolactone in combination with chemotherapy.

Differences in peripheral neuropathy in xeroderma pigmentosum complementation groups A and D as evaluated by nerve conduction studies

BackgroundXeroderma pigmentosum (XP) is a rare autosomal recessive genetic disorder with defective DNA nucleotide excision repair and associated with a high frequency of skin cancer. Approximately 25% of patients develop progressive neurological degeneration. Complementation groups XP-A and XP-D are most frequently associated with neurological disorders.Design/methodsThis is a retrospective review of patients with XP who were evaluated at NIH from 1986 to 2015 and had nerve conduction studies (NCS). In the complementation groups with peripheral neuropathy, further comparisons of the NCS were made with audiological, brain imaging, neuropsychological assessments that were also performed on most of the patients. Limited neuropathology of XP-A and XP-D patients were examined..ResultsThe 33 patients had NCS: XP-A (9 patients), XP-C (7 patients), XP-D (10 patients), XP-E (1 patient), XP-V (4 patients), and XP-unknown (2 patients). Peripheral neuropathy based on nerve conduction studies was documented only in two complementation groups: 78% (7/9) of XP-A patients had a sensorimotor neuropathy while 50% (5/10) of XP-D patients had a sensory neuropathy only. Analysis of sural sensory nerve amplitude in both complementation groups XP-A and XP-D correlated with sensorineural hearing loss (SNHL), MRI/CT severity, and Full-scale Intelligence Quotient (IQ). Analysis of fibular motor nerve amplitude in complementation XP-A correlated with SNHL and MRI/CT severity. Limited follow-up studies showed gradual loss of NCS responses compared to an earlier and more rapid progression of the hearing loss.ConclusionsDespite similar brain imaging and audiological findings patients, XP-A and XP-D complementation groups differ in the type of neuropathy, sensorimotor versus sensory alone. A few cases suggest that sensorineural hearing loss may precede abnormal NCS in XP and therefore serve as valuable clinical indicators of XP patients that will later develop peripheral neuropathy.

Giant Squamous Cell Carcinoma in Xeroderma Pigmentosum: A Case Report and Literature Review

Xeroderma pigmentosum is a rare autosomal recessive genodermatosis characterized by photosensitivity and the development of cutaneous and internal malignancies at an early age. The clinical manifestations basic defect is a nucleotide excision repair defect, leading to deficient repair of DNA damaged by ultraviolet radiation. These patients exhibit increased sensitivity to ionizing radiation Patients with xeroderma pigmentosum who are younger than 20 years old have a significantly higher than a 1000-fold increased risk of developing skin cancer. Early diagnosis of these tumors is crucial because they are fast-growing, early metastasizing, and lead to death. However, early detection and treatment of cutaneous malignancies will decrease morbidity and mortality. Genetic counselling remains the most crucial measure for preventing xeroderma pigmentosum. We report a case of xeroderma pigmentosum in a 17-year-old girl presenting with a giant malignant tumor on her face, emphasizing the importance of early diagnosis and management.

Nucleotide Excision Repair: From Molecular Defects to Neurological Abnormalities.

Nucleotide excision repair (NER) is the most versatile DNA repair pathway, which can remove diverse bulky DNA lesions destabilizing a DNA duplex. NER defects cause several autosomal recessive genetic disorders. Xeroderma pigmentosum (XP) is one of the NER-associated syndromes characterized by low efficiency of the removal of bulky DNA adducts generated by ultraviolet radiation. XP patients have extremely high ultraviolet-light sensitivity of sun-exposed tissues, often resulting in multiple skin and eye cancers. Some XP patients develop characteristic neurodegeneration that is believed to derive from their inability to repair neuronal DNA damaged by endogenous metabolites. A specific class of oxidatively induced DNA lesions, 8,5′-cyclopurine-2′-deoxynucleosides, is considered endogenous DNA lesions mainly responsible for neurological problems in XP. Growing evidence suggests that XP is accompanied by defective mitophagy, as in primary mitochondrial disorders. Moreover, NER pathway is absent in mitochondria, implying that the mitochondrial dysfunction is secondary to nuclear NER defects. In this review, we discuss the current understanding of the NER molecular mechanism and focuses on the NER linkage with the neurological degeneration in patients with XP. We also present recent research advances regarding NER involvement in oxidative DNA lesion repair. Finally, we highlight how mitochondrial dysfunction may be associated with XP.

Distinct roles for RSC and SWI/SNF chromatin remodelers in genomic excision repair.

Nucleosomes are a significant barrier to the repair of UV damage because they impede damage recognition by nucleotide excision repair (NER). The RSC and SWI/SNF chromatin remodelers function in cells to promote DNA access by moving or evicting nucleosomes, and both have been linked to NER in yeast. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking RSC or SWI/SNF activity. Our data indicate that SWI/SNF is not generally required for NER but instead promotes repair of CPD lesions at specific yeast genes. In contrast, mutation or depletion of RSC subunits causes a general defect in NER across the yeast genome. Our data indicate that RSC is required for repair not only in nucleosomal DNA but also in neighboring linker DNA and nucleosome-free regions (NFRs). Although depletion of the RSC catalytic subunit also affects base excision repair (BER) of N-methylpurine (NMP) lesions, RSC activity is less important for BER in linker DNA and NFRs. Furthermore, our data indicate that RSC plays a direct role in transcription-coupled NER (TC-NER) of transcribed DNA. These findings help to define the specific genomic and chromatin contexts in which each chromatin remodeler functions in DNA repair, and indicate that RSC plays a unique function in facilitating repair by both NER subpathways.

A Gene Expression Signature to Predict Nucleotide Excision Repair Defects and Novel Therapeutic Approaches.

Nucleotide excision repair (NER) resolves DNA adducts, such as those caused by ultraviolet light. Deficient NER (dNER) results in a higher mutation rate that can predispose to cancer development and premature ageing phenotypes. Here, we used isogenic dNER model cell lines to establish a gene expression signature that can accurately predict functional NER capacity in both cell lines and patient samples. Critically, none of the identified NER deficient cell lines harbored mutations in any NER genes, suggesting that the prevalence of NER defects may currently be underestimated. Identification of compounds that induce the dNER gene expression signature led to the discovery that NER can be functionally impaired by GSK3 inhibition, leading to synergy when combined with cisplatin treatment. Furthermore, we predicted and validated multiple novel drugs that are synthetically lethal with NER defects using the dNER gene signature as a drug discovery platform. Taken together, our work provides a dynamic predictor of NER function that may be applied for therapeutic stratification as well as development of novel biological insights in human tumors.

Targeting Germline- and Tumor-Associated Nucleotide Excision Repair Defects in Cancer.

Nucleotide excision repair (NER) gene alterations constitute potential cancer therapeutic targets. We explored the prevalence of NER gene alterations across cancers and putative therapeutic strategies targeting these vulnerabilities. We interrogated our institutional dataset with mutational data from more than 40,000 patients with cancer to assess the frequency of putative deleterious alterations in four key NER genes. Gene-edited isogenic pairs of wild-type and mutant ERCC2 or ERCC3 cell lines were created and used to assess response to several candidate drugs. We found that putative damaging germline and somatic alterations in NER genes were present with frequencies up to 10% across multiple cancer types. Both in vitro and in vivo studies showed significantly enhanced sensitivity to the sesquiterpene irofulven in cells harboring specific clinically observed heterozygous mutations in ERCC2 or ERCC3. Sensitivity of NER mutants to irofulven was greater than to a current standard-of-care agent, cisplatin. Hypomorphic ERCC2/3-mutant cells had impaired ability to repair irofulven-induced DNA damage. Transcriptomic profiling of tumor tissues suggested codependencies between DNA repair pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies. These findings provide novel insights into a synthetic lethal relationship between clinically observed NER gene deficiencies and sensitivity to irofulven and its potential synergistic combination with other drugs.See related commentary by Jiang and Greenberg, p. 1833.

Autophagy induction on impaired spermatogenesis of xeroderma pigmentosum group A gene-deficient mice

Xeroderma pigmentosum (XP) involves a defect in the initial step of nucleotide excision repair (NER) and consists of eight genetic complementation groups (groups A-G and a variant). XP group A (XPA) patients have a high incidence of UV-induced skin tumors, immature testicular development, and neurological symptoms. In an earlier study, we have shown that XP group A (Xpa) gene-knockout mice (Xpa-/- mice) were highly sensitive to UV-induced skin carcinogenesis with a defect in NER and were highly susceptibility to spontaneous tumorigenesis with impaired spermatogenesis. However, the pathology of impaired spermatogenesis in Xpa-/- mice is unknown. To unravel the underlying pathology, we made a concerted effort using the testis of 3-month-old Xpa-/- mice. We found many large vacuoles in the seminiferous tubules of 3-month old Xpa-/- mice, while there were no large vacuoles in that of Xpa+/+ mice. Immunohistochemistry of microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, showed degenerating cells with intense signal of LC3 in the seminiferous tubules, and immunoblotting revealed induction of LC3-II in the 3-month-old Xpa-/- mice. The results of the present study suggest autophagy induction as the possible mechanism underlying the impaired spermatogenesis in Xpa-/- mice. Therefore, Xpa-/- mice could be a useful model for investigating aging and male infertility with low expression of XPA.

Rare occurrence of coexistent squamous cell carcinoma and basal cell carcinoma in a case of Xeroderma Pigmentosum

Xeroderma Pigmentosum is an inherited disorder of DNA repair characterised by defective nucleotide excision repair, which is involved in repairing ultraviolet rays induced cross linking of pyramidine residues. Affected individuals are at increased risk of development of mucocutaneous cancers at a much earlier age than normal. This genodermatosis affects both sexes and all races. The incidence in India is still unknown. We report one such case of a nine year old boy with co-existent squamous cell carcinoma and basal cell carcinoma of the face.

Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells.

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

Malignant Melanoma in Child with Xeroderma Pigmentosum: A Rare Case

Background: Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder characterized by photosensitivity, cutaneous pigmentary changes, and malignant tumor development at an early age. The basic defect underlying the clinical manifestations is nucleotide excision repair defect, leading to defective repair of ultraviolet (UV)-induced DNA. XP patients who are younger than 20 years of age have more than 1000-fold increased risk of developing malignant neoplasms of the skin, which commonly include squamous cell carcinoma, basal cell carcinoma, fibrosarcoma, and malignant melanoma. Malignant melanoma arises in only about 3% of XP patients. Purpose: To report a case of malignant melanoma in a child with XP. Case: A 7-years-old girl presented with multiple hypopigmentation and hyperpigmentation macules since age of two, throughout the body, more on sun-exposed areas. The physical examination showed solitary tumor extensive ulcero- proliferative surface with areas of hemorrhage and blackish pigmentation on the vertex region. Histological examination revealed a feature of nodular malignant melanoma, and the condition became worse after she underwent two cycles of chemotherapy. Discussion: Despite the rare occurrence, the nodular type of malignant melanoma in XP patients is the most aggressive and responsible for the fatal condition. Conclusion: Early detection of XP is necessary due to its fast-growing nature and high metastatic possibility as well as mortality index.