Source Of Endogenous DNA Damage Research Articles

VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

What are the DNA lesions underlying formaldehyde toxicity?

Formaldehyde is a highly reactive organic compound. Humans can be exposed to exogenous sources of formaldehyde, but formaldehyde is also produced endogenously as a byproduct of cellular metabolism. Because formaldehyde can react with DNA, it is considered a major endogenous source of DNA damage. However, the nature of the lesions underlying formaldehyde toxicity in cells remains vastly unknown. Here, we review the current knowledge of the different types of nucleic acid lesions that are induced by formaldehyde and describe the repair pathways known to counteract formaldehyde toxicity. Taking this knowledge together, we discuss and speculate on the predominant lesions generated by formaldehyde, which underly its natural toxicity.

The dimeric deubiquitinase USP28 integrates 53BP1 and MYC functions to limit DNA damage.

DNA replication is a major source of endogenous DNA damage in tumor cells and a key target of cellular response to genotoxic stress. DNA replication can be deregulated by oncoproteins, such as transcription factor MYC, aberrantly activated in many human cancers. MYC is stringently regulated by the ubiquitin system - for example, ubiquitination controls recruitment of the elongation factor PAF1c, instrumental in MYC activity. Curiously, a key MYC-targeting deubiquitinase USP28 also controls cellular response to DNA damage via the mediator protein 53BP1. USP28 forms stable dimers, but the biological role of USP28 dimerization is unknown. We show here that dimerization limits USP28 activity and restricts recruitment of PAF1c by MYC. Expression of monomeric USP28 stabilizes MYC and promotes PAF1c recruitment, leading to ectopic DNA synthesis and replication-associated DNA damage. USP28 dimerization is stimulated by 53BP1, which selectively binds USP28 dimers. Genotoxic stress diminishes 53BP1-USP28 interaction, promotes disassembly of USP28 dimers and stimulates PAF1c recruitment by MYC. This triggers firing of DNA replication origins during early response to genotoxins and exacerbates DNA damage. We propose that dimerization of USP28 prevents ectopic DNA replication at transcriptionally active chromatin to maintain genome stability.

Mitochondrial ROS Triggers KIN Pathogenesis in FAN1-Deficient Kidneys.

Karyomegalic interstitial nephritis (KIN) is a genetic adult-onset chronic kidney disease (CKD) characterized by genomic instability and mitotic abnormalities in the tubular epithelial cells. KIN is caused by recessive mutations in the FAN1 DNA repair enzyme. However, the endogenous source of DNA damage in FAN1/KIN kidneys has not been identified. Here we show, using FAN1-deficient human renal tubular epithelial cells (hRTECs) and FAN1-null mice as a model of KIN, that FAN1 kidney pathophysiology is triggered by hypersensitivity to endogenous reactive oxygen species (ROS), which cause chronic oxidative and double-strand DNA damage in the kidney tubular epithelial cells, accompanied by an intrinsic failure to repair DNA damage. Furthermore, persistent oxidative stress in FAN1-deficient RTECs and FAN1 kidneys caused mitochondrial deficiencies in oxidative phosphorylation and fatty acid oxidation. The administration of subclinical, low-dose cisplatin increased oxidative stress and aggravated mitochondrial dysfunction in FAN1-deficient kidneys, thereby exacerbating KIN pathophysiology. In contrast, treatment of FAN1 mice with a mitochondria-targeted ROS scavenger, JP4-039, attenuated oxidative stress and accumulation of DNA damage, mitigated tubular injury, and preserved kidney function in cisplatin-treated FAN1-null mice, demonstrating that endogenous oxygen stress is an important source of DNA damage in FAN1-deficient kidneys and a driver of KIN pathogenesis. Our findings indicate that therapeutic modulation of kidney oxidative stress may be a promising avenue to mitigate FAN1/KIN kidney pathophysiology and disease progression in patients.

Lnc956-TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis.

Replication stress is a major source of endogenous DNA damage. Despite the identification of numerous proteins on replication forks to modulate fork or replication machinery activities, it remains unexplored whether noncoding RNAs can localize on stalled forks and play critical regulatory roles. Here, we identify an uncharacterized long noncoding RNA NONMMUT028956 (Lnc956 for short) predominantly expressed in mouse embryonic stem cells. Lnc956 is accumulated on replication forks to prevent fork collapse and preserve genomic stability and is essential for mouse embryogenesis. Mechanistically, it drives assembly of the Lnc956-TRIM28-HSP90B1 complex on stalled forks in an interdependent manner downstream of ataxia telangiectasia and Rad3-related (ATR) signaling. Lnc956-TRIM28-HSP90B1 complex physically associates with minichromosome maintenance proteins 2 (MCM2) to minichromosome maintenance proteins 7 (MCM7) hexamer via TRIM28 and directly regulates the CDC45-MCM-GINS (CMG) helicase retention on chromatin. The regulation of Lnc956-TRIM28-HSP90B1 on CMG retention is mediated by HSP90B1's chaperoning function. These findings reveal a player that actively regulates replisome retention to prevent fork collapse.

Multi-Faceted Roles of ERCC1-XPF Nuclease in Processing Non-B DNA Structures

Genetic instability can result from increases in DNA damage and/or alterations in DNA repair proteins and can contribute to disease development. Both exogenous and endogenous sources of DNA damage and/or alterations in DNA structure (e.g., non-B DNA) can impact genome stability. Multiple repair mechanisms exist to counteract DNA damage. One key DNA repair protein complex is ERCC1-XPF, a structure-specific endonuclease that participates in a variety of DNA repair processes. ERCC1-XPF is involved in nucleotide excision repair (NER), repair of DNA interstrand crosslinks (ICLs), and DNA double-strand break (DSB) repair via homologous recombination. In addition, ERCC1-XPF contributes to the processing of various alternative (i.e., non-B) DNA structures. This review will focus on the processing of alternative DNA structures by ERCC1-XPF.

Defective repair of topoisomerase I induced chromosomal damage in Huntington\u2019s disease

Topoisomerase1 (TOP1)-mediated chromosomal breaks are endogenous sources of DNA damage that affect neuronal genome stability. Whether TOP1 DNA breaks are sources of genomic instability in Huntington’s disease (HD) is unknown. Here, we report defective 53BP1 recruitment in multiple HD cell models, including striatal neurons derived from HD patients. Defective 53BP1 recruitment is due to reduced H2A ubiquitination caused by the limited RNF168 activity. The reduced availability of RNF168 is caused by an increased interaction with p62, a protein involved in selective autophagy. Depletion of p62 or disruption of the interaction between RNAF168 and p62 was sufficient to restore 53BP1 enrichment and subsequent DNA repair in HD models, providing new opportunities for therapeutic interventions. These findings are reminiscent to what was described for p62 accumulation caused by C9orf72 expansion in ALS/FTD and suggest a common mechanism by which protein aggregation perturb DNA repair signaling.

Aldehyde-driven transcriptional stress triggers an anorexic DNA damage response.

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription1-4. This response is absent in the human disease Cockayne syndrome, which iscaused by loss of the Cockayne syndrome A (CSA) or CSB proteins5-7. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5-/-) and CSB (Csbm/m; Csb is also known as Ercc6) develop cachexiaand neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNAsequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5-/-Csbm/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as wellas chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.

The Loss of ALDH9A1 Is a Significant Source of Endogenous DNA Damage Which May be Reversed By the Inhibition of Polyamine Transport System

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome and is caused by impaired DNA interstrand crosslink repair. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage likely play an important role in the pathogenesis of FA.We previously identified loss of ALDH9A1 as a significant source of endogenous DNA damage using a metabolism-focused CRISPR knockout (KO) screen. This finding was validated using Jurkat cells as well as human hematopoietic stem and progenitor cells. Here, we present updates of our project.To determine whether endogenous DNA damage was induced by the combined loss of FANCD2 and ALDH9A1, we investigated markers of DNA damage in bulk-edited cells. We found that the numbers of chromosomal breaks, 53BP1 foci, and gamma-H2AX foci were increased in FANCD2-/-ALDH9A1-/- cells, compared with single KO or wildtype (WT) controls, in the absence of an exogenous DNA damaging agent. These findings are consistent with spontaneously increased basal levels of DNA damage in FANCD2-/-ALDH9A1-/- cells.To study in vivo BMF and tumorigenesis phenotypes of ALDH9A1 deficiency in the setting of FA, we generated a mouse model. Fanca-/-Aldh9a1-/- mice showed the lowest frequency of long-term hematopoietic stem cells and lineage-negative Sca-1-positive cKit-positive cells, however the differences were not significant compared with control groups. While we did not observe aplastic anemia or leukemia, we found a higher incidence of solid tumors, most notably ovarian tumors and hepatocellular carcinoma in aged Fanca-/-Aldh9a1-/- mice. This suggests that the level of endogenous reactive aldehydes created by ALDH9A1 deficiency in mouse is not high enough to cause full-blown hematopoietic phenotypes, most likely due to redundant detoxification pathways. However, in tissues where ALDH9A1 is active and non-redundant, the low level of endogenous DNA damage accumulating over time causes solid tumors.To identify the specific reactive aldehydes responsible for DNA damage in ALDH9A1 deficiency, we performed a growth selection screen using FANCD2-/-ALDH9A1-/- cells. We found that the loss of ATP13A3 conferred survival advantage to FANCD2-/-ALDH9A1-/- cells. ATP13A3 transports endocytosed polyamines into the cytosol where polyamines can be metabolized by serum amine oxidases into 3-aminopropanal, a reactive aminoaldehyde. 3-aminopropanal also undergoes spontaneous decomposition to acrolein, a well-known reactive aldehyde carcinogen. Finding that the loss of ATP13A3 rescues that FANCD2-/-ALDH9A1-/- cells indicates that 3-aminopropanal and/or acrolein induces endogenous DNA damage requiring the Fanconi anemia pathway function for repair.Finally, to determine the contribution of ALDH9A1 variants to clinical manifestations of FA patients, we performed targeted sequencing of DNA from FA patients. We identified five missense variants, four of which had high CADD scores (>20). ALDH9A1 cDNA containing missense variants with high CADD scores expressed in ALDH9A1-/- Jurkat cells resulted in lower protein expression than the WT cDNA. Cell culture supernatant from cells expressing the variant cDNAs also had increased aldehyde levels as assessed by fluorometric assays, suggesting decreased enzymatic activity of the variant proteins. The patients with ALDH9A1 missense variants with high CADD scores had either early hematologic onset of FA (n=3; two patients before age 1 and one patient before age 4) or AML (n=1).In conclusion, we showed that the loss of ALDH9A1 generates endogenous DNA damage necessitating the FA pathway for its repair. Synthetic lethality caused by the combined loss of FANCD2 and ALDH9A1 was rescued by the loss of ATP13A3, which suggests that 3-aminopropanal is the culprit aminoaldehyde that accumulates in ALDH9A1-deficient cells and results in DNA damage. Functionally deleterious ALDH9A1 variants were observed in some FA patients with early onset of disease suggesting that ALDH9A1 could be a modifier of FA in humans. DisclosuresSridhar: Deciphera Pharmaceuticals: Current Employment; CRISPR Therapeutics: Ended employment in the past 24 months. White: Regeneron Pharmaceuticals: Current Employment. Smogorzewska: Rocket Pharmaceuticals: Research Funding.

Bone Marrow Microenvironment Induces Genomic Instability and Enables Clonal Evolution in Multiple Myeloma

Multiple myeloma (MM) is characterized by ongoing DNA damage and genomic instability, with acquisition of mutations and structural changes during disease progression. Its growth and survival charactersitics are also affected by the bone marrow (BM) microenvironment, influencing its pathogenesis as well as development of drug resistance. However, it is unclear whether the BM microenvironment can affect the ability of the tumor cells to acquire new genomic changes. The susceptibility to genomic changes might come from an increase in the endogenous sources of DNA damage or an impairment or deregulation of the DNA Damage Response (DDR). We hypothesized that the microenvironment may also contribute to ongoing DNA damage and genomic instability in MM.To investigate the potential role of the microenvironment in genomic stability in MM, we evaluated change in frequency of spontaneous DNA damage in myeloma cells when co-cultured with various microenvironment cells (HS-5 stromal cell line and primary patient BM stromal cells [BMSC]). We observed that DNA breaks, already present at baseline as demonstrated by high levels of pH2AX, were significantly increased when MM cell lines were co-cultured with either HS5 cell or primary BMSCs (2-4 folds increase, p <0.05). Moreover, similar results were observed when MM cell lines were cultured in presence of stromal cell supernatant or IL6 alone, also suggesting a possible effect of the cytokine. Immunofluorescence experiments revealed that after co-culture with stromal cells pH2AX recruitment to sites of DNA DSBs was also increased.Since we have shown that elevated HR and nuclease (especially apurinic/apyrimidinic endonuclease) activities are important mechanisms of genomic instability in MM, we next evaluated the impact of microenvironment on nuclease and HR activities using functional assays. Interestingly, we found that MM-BMSC co-culture induced a significant increase in overall nuclease activity, with specific increase in the expression of APEX1, a major apurinic/apyrimidinic (AP) nuclease, leading to increase in AP acitivity as measured by a flourescent-based oliginucleotide assay. MM-BMSC co-culture also induced HR activity demonstrating the impact of BMSC on genomic instability.To more comprehensively evaluate effects of stromal cells on different DNA Damage Response pathways, we performed RNA-sequencing of 3 different MM cell lines cultured alone or in presence of HS-5. We observed that the network of genes involved in DNA Damage Response were significantly altered in MM in the presence of stromal cells. We further confirmed our results in publicly available microarray gene expression data in MM cell lines cultured alone or with HS-5. These results support the idea that microenvironment can impact DNA integrity and stability in MM cells.Finally, we cultured MM cell lines alone or in presence of BMSC from MM patients for 4 weeks and investigated genomic changes using cytoscan HD array to detect effects of co-culture on acquisition of new copy-number alterations. We observed that MM cell line growth in the presence of BMSCs provides a selective environment for clonal selection, demonstrating the role of stromal cells in clonal diversity in MM.In conclusion, our data suggest that BMSC enhance DNA damage and instability in MM. Understanding the mechanisms leading to genomic instability is important to develop new therapeutic strategies aimed at preventing MM evolution. DisclosuresAnderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder.

Evaluation of repair activity by quantification of ribonucleotides in the genome

Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ‐seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ‐seq analysis of cells harboring wild‐type DNA polymerases revealed that ribonucleotides were incorporated nonuniformly in the genome with a 3′‐shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild‐type and repair‐deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ‐seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ‐seq can provide an estimate of indel risk.

A Novel Source of Endogenous DNA Damage That Requires Repair By the Fanconi Anemia Pathway

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome. Impaired DNA interstrand crosslink (ICL) repair is the underlying mechanism for BMF in FA. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage are most likely to play an important role in the pathogenesis of FA. Metabolic by-products, such as reactive aldehydes, have been implicated in the acceleration of BMF or leukemia in both humans and mice lacking the ICL repair pathway. However, the potential contribution of other detoxifying metabolic enzymes to genome maintenance has not been systematically investigated. Identification of all sources of endogenous DNA damage will allow us to develop novel strategies to prevent DNA damage from arising, and possibly prevent BMF and leukemia in FA as well as other BMF syndromes. To determine whether detoxifying enzymes other than ALDH2 or ADH5 play a role in the protection of HSPC, we performed a metabolism-focused CRISPR/Cas9 synthetic lethality screen (3000 metabolism genes, 10 sgRNA per gene), using wild-type and FANCD2-/- Jurkat cells. From the screen, we identified ALDH9A1 as the most significantly depleted gene in FANCD2-/- Jurkat cells compared with wild-type. Eight out of ten sgALDH9A1 were significantly depleted in FANCD2-/- Jurkat cells, indicating robust effect of the ALDH9A1 knockout. ADH5, a known synthetic lethal gene with FA, was also depleted in FANCD2-/- Jurkat cells, but to a lesser degree. In vitro fluorescence-based competition assay confirmed synthetic lethal interaction between the two genes, in two independent FANCD2-/- Jurkat clones. To determine whether ALDH9A1 deficiency also caused cell death in FA-deficient human hematopoietic stem progenitor cells (HSPCs), we performed an in vitro validation assay using human umbilical cord blood (UCB). UCB CD34+ cells were edited by ribonucleoprotein delivery of Cas9 and sgRNA, in which either of sgCTRL, sgFANCD2 or sgALDH9A1, or both sgFANCD2 and sgALDH9A1 were used. Edited cells were grown on methylcellulose for 10 to 14 days, after which individual colonies were scored and harvested for sequencing. While CD34+ cells that were targeted by both sgFANCD2 and sgALDH9A1 (double KO) achieved lower editing efficiency for each gene compared with cells targeted by single guides, they produced the fewest hematopoietic colonies and the lowest frequency of GEMM (Granulocyte, Erythrocyte, Macrophage and Megakaryocyte) colonies. We observed fewer colonies targeted for both genes (biallelic double KO; observed to expected ratio 0.33) as compared to either single gene KO. These results suggest that loss of ALDH9A1 is deleterious in FANCD2-deficient HSPC. Lastly, we generated a Fanca-/-Aldh9a1-/- mouse model to examine the in vivo hematopoietic phenotype due to increased endogenous aldehydes. These mice were born at the Mendelian ratio without significant anomalies except rare cases of eye abnormalities. At three months of life, Fanca-/-Aldh9a1-/- mice had lower platelet counts than wild-type, Fanca-/- or Aldh9a1-/- control mice, but total white blood counts and hemoglobin levels were similar between groups. A follow-up result of this mouse model will be presented at the meeting. In conclusion, we identified that cells with ALDH9A1 deficiency require the FA pathway for survival. ALDH9A1 may protect human and mouse HSPC that are deficient in the FA pathway from DNA damage and cell death. Disclosures No relevant conflicts of interest to declare.

Interactome Analysis of APOBEC3B in Multiple Myeloma

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family proteins restrict retroviruses and retrotransposons by inducing hypermutation or degradation of the replication intermediates through their DNA cytidine deaminase activity. APOBECs can also act as endogenous sources of DNA damage that mutate many human cancers. Accumulation of APOBEC signature mutations is associated with disease progression and poor overall survival in multiple myeloma (Walker et al. Nat Commun, 2015). Among APOBEC3 enzymes, APOBEC3B (A3B) is the only family member that is predominantly located in the nucleus throughout the cell cycle. We previously reported that A3B knockdown decreased cytidine deaminase activity in myeloma cells, suggesting that among APOBECs, A3B plays a major role in cytidine deamination-related mutagenesis in myeloma cells (Yamazaki et al., Sci Rep, 2019). Recent studies showed that cofactors of A3B could affect the functions of A3B: heterogeneous nuclear ribonucleoproteins (hnRNPs) interact with surface hydrophobic residues of the N-terminal domain in order to bind to A3B (Xiao et al., Nuc. Acids Res, 2017; Zhang al., Cell Microbiol, 2008); BORF2, an Epstein-Barr viral protein, interacts with the A3B catalytic domain and inhibits A3B DNA cytidine deaminase activity (Cheng et al., Nat Microbiol, 2018). However, the biological mechanisms of how endogenous A3B induces mutations in genomic DNA are still unclear. In this study, we aim to ascertain the cofactors for nucleic acid binding and elucidate the regulatory mechanisms that prevent APOBEC-mediated genomic mutagenesis. Because of the high homology between APOBEC3 proteins, a specific antibody against A3B is not available, and it is difficult to analyze A3B-interaction at the endogenous expression level. To overcome this technical impediment, we used a lentiCRISPR system to insert a FLAG-tag sequence at the C-terminus of the A3B gene in A3B highly expressing myeloma cell lines (AMO1 and RPMI8226). We then conducted A3B-immunoprecipitation with the anti-FLAG M2 antibody, followed by mass spectrometry (MS) analysis to identify potential A3B interacting proteins. MS analysis identified 40 putative interacting proteins and these proteins were clustered largely into two interaction networks: ribonucleoprotein complex and ribosomal-associated proteins. We also performed Gene Ontology (GO) enrichment analysis and revealed that spliceosome, ribosome, and RNA transport were significantly enriched terms. We confirmed the binding between A3B and selected A3B interacting proteins: hnRNPs, interleukin enhancer-binding factor 2 and 3 (ILF2, ILF3) in myeloma cell lines by co-immunoprecipitation assays. Next, we tested the intracellular colocalization of overexpressed A3B and interacting proteins in Hela cells by immunofluorescence microscopy. We found that ILF2 presents strong colocalization with A3B in the nuclei of cells. We also employed density-gradient sedimentation analysis to test if these proteins form high molecular mass (HMM) complexes with A3B in the nucleus using HEK293T cells expressing FLAG-tagged A3B. We detected that ILF2 is one of the components of HMM A3B complexes. To check whether these putative interacting proteins affect A3B cytidine deaminase activity, we next performed an in vitro luminescence-based screening assay (AlphaScreen)using a FLAG-GST protein library which was produced by the wheat cell-free protein production system. We found that hnRNP A1 and ILF2 decreased A3B cytidine deaminase activity. This study provides for the first time a proteomic characterization of A3B interactome in a myeloma cell context. Our findings reveal putative A3B cofactors in myeloma cell lines which may regulate the catalytic activity of A3B. We discuss how these proteins bind A3B and affect its activity in myeloma cells. Disclosures Takaori-Kondo: Pfizer: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding.

Synthetic lethality between BRCA1 deficiency and poly(ADP-ribose) polymerase inhibition is modulated by processing of endogenous oxidative DNA damage.

Poly(ADP-ribose) polymerases (PARPs) facilitate the repair of DNA single-strand breaks (SSBs). When PARPs are inhibited, unrepaired SSBs colliding with replication forks give rise to cytotoxic double-strand breaks. These are normally rescued by homologous recombination (HR), but, in cells with suboptimal HR, PARP inhibition leads to genomic instability and cell death, a phenomenon currently exploited in the therapy of ovarian cancers in BRCA1/2 mutation carriers. In spite of their promise, resistance to PARP inhibitors (PARPis) has already emerged. In order to identify the possible underlying causes of the resistance, we set out to identify the endogenous source of DNA damage that activates PARPs. We argued that if the toxicity of PARPis is indeed caused by unrepaired SSBs, these breaks must arise spontaneously, because PARPis are used as single agents. We now show that a significant contributor to PARPi toxicity is oxygen metabolism. While BRCA1-depleted or -mutated cells were hypersensitive to the clinically approved PARPi olaparib, its toxicity was significantly attenuated by depletion of OGG1 or MYH DNA glycosylases, as well as by treatment with reactive oxygen species scavengers, growth under hypoxic conditions or chemical OGG1 inhibition. Thus, clinical resistance to PARPi therapy may emerge simply through reduced efficiency of oxidative damage repair.

Somatic mutation load and spectra: A record of DNA damage and repair in healthy human cells.

Somatic genome instability is a hallmark of cancer genomes and has been linked to aging and a variety of other pathologies. Large-scale cancer genome and exome sequencing have revealed that mutation load and spectra in cancers can be influenced by environmental exposures, the anatomical site of exposures, and tissue type. There is now an abundance of data favoring the hypothesis that a substantial portion of the mutations in cancers originate prior to carcinogenesis in stem cells of the healthy individual. Rapid advances in sequencing of noncancer cells from healthy humans have shown that their mutation loads and spectra resemble cancer data. Similar to cancer genomes, mutation profiles of healthy cells show marked intra-individual variation, thus providing a metric of the various factors-environmental and endogenous-involved in mutagenesis in these individuals. This review focuses on the current methodologies to measure mutation loads and to determine mutation signatures for evaluating the environmental and endogenous sources of DNA damage in human somatic cells. We anticipate that in future, such large-scale studies aimed at exploring the landscapes of somatic mutations across different cell types in healthy people would provide a valuable resource for designing personalized preventative strategies against diseases associated with somatic genome instability. Environ. Mol. Mutagen. 59:672-686, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Alcohol and endogenous aldehydes damage chromosomes and mutate stem cells.

Haematopoietic stem cells renew blood. Accumulation of DNA damage in these cells promotes their decline, while misrepair of this damage initiates malignancies. Here we describe the features and mutational landscape of DNA damage caused by acetaldehyde, an endogenous and alcohol-derived metabolite. This damage results in DNA double-stranded breaks that, despite stimulating recombination repair, also cause chromosome rearrangements. We combined transplantation of single haematopoietic stem cells with whole-genome sequencing to show that this damage occurs in stem cells, leading to deletions and rearrangements that are indicative of microhomology-mediated end-joining repair. Moreover, deletion of p53 completely rescues the survival of aldehyde-stressed and mutated haematopoietic stem cells, but does not change the pattern or the intensity of genome instability within individual stem cells. These findings characterize the mutation of the stem-cell genome by an alcohol-derived and endogenous source of DNA damage. Furthermore, we identify how the choice of DNA-repair pathway and a stringent p53 response limit the transmission of aldehyde-induced mutations in stem cells.

A missense MT-ND5 mutation in differentiated Parkinson Disease cytoplasmic hybrid induces ROS-dependent DNA Damage Response amplified by DROSHA

Genome integrity is continuously threatened by endogenous sources of DNA damage including reactive oxygen species (ROS) produced by cell metabolism. Factors of the RNA interference (RNAi) machinery have been recently involved in the cellular response to DNA damage (DDR) in proliferating cells. To investigate the impact of component of RNAi machinery on DDR activation in terminally differentiated cells, we exploited cytoplasmic hybrid (cybrid) cell lines in which mitochondria of sporadic Parkinson’s disease patients repopulate neuroblastoma SH-SY5Y-Rho(0) cells. Upon differentiation into dopaminergic neuron-like cells, PD63 cybrid showed increased intracellular level of ROS and chronic DDR activation, compared to other cybrids with the same nuclear background. Importantly, DDR activation in these cells can be prevented by ROS scavenging treatment suggesting that ROS production is indeed causative of nuclear DNA damage. Sequence analysis of the mitogenomes identified a rare and heteroplasmic missense mutation affecting a highly conserved residue of the ND5-subunit of respiratory complex I, which accounts for ROS increase. We demonstrated that the assembly of nuclear DDR foci elicited by oxidative stress in these cells relies on DROSHA, providing the first evidence that components of RNAi machinery play a crucial role also in the mounting of ROS-induced DDR in non-replicating neuronal cells.

Abstract 1415: SAN1: a novel senataxin associated nuclease required for the repair of interstrand crosslinks

Abstract The DNA damage response (DDR) is a set of complex signaling pathways capable of sensing DNA damage, and activating a large number of enzymes involved in the remodeling and repair of the genome. Mutations in the genes involved in the DDR lead to DNA damage, genomic instability, and various cancers. One particularly dangerous type of DNA damage that can occur is an interstrand crosslink (ICL). ICLs can lead to the development of double strand breaks through the blockage of DNA replication and transcription. Although ICLs can arise endogenously from molecules such as aldehydes, most commonly they are induced from chemotherapeutic drugs such as Cisplatin and Mitomycin C (MMC). These drugs are commonly used in the treatment of breast and ovarian cancers. A better understanding of which proteins are involved in the repair of ICLs is critical for understanding resistance, toxicity, and response in patients treated with ICL inducing agents. The repair of ICLs requires the coordination of several DNA repair pathways including the Fanconi Anemia pathway, homologous recombination (HR), and nucleotide excision repair (NER). The Fanconi Anemia pathway is essential for the repair of these lesions as it is responsible for the recognition of the ICL lesion, as well as the recruitment of several nucleases responsible for unhooking and removal the cross-linked nucleotides. Recently, we identified an uncharacterized 5’ nuclease that interacts with the RNA/DNA helicase Senataxin, which we have named senataxin-associated nuclease 1 (SAN1). Senataxin has been shown to act on R loops, RNA/DNA hybrids that are a source of endogenous DNA damage. Deletion of the SAN1 gene in HeLa cells or in mouse embryonic fibroblasts leads to the sensitization of cells to Cisplatin and Mitomycin C (MMC), but not to ionizing radiation that induces double strand breaks. Importantly, the defect in ICL repair can be restored using WT SAN1 but not with a mutant that is catalytically inactive. Treatment of SAN1 -/- HeLa cells with MMC also leads to radial chromosome formation, a characteristic of cells deficient in ICL repair. Additionally, SAN1 partially colocalizes with Fancd2 MMC induced foci, and treatment with MMC results in increased DNA damage and R loops in SAN1-/- cells. In conclusion, this study highlights the discovery of a novel nuclease involved in the repair ICLs, a process critical for understanding resistance and response to chemotherapies such as Cisplatin and MMC. Future work is aimed at determining the specific step of ICL repair that SAN1 participates in, how and when during repair SAN1 is recruited to ICL sites, and which other DNA repair proteins SAN1 functions with. Citation Format: Alex Andrews, Ian Macara. SAN1: a novel senataxin associated nuclease required for the repair of interstrand crosslinks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1415. doi:10.1158/1538-7445.AM2017-1415

Abstract 1261: Lipid-peroxidation derived DNA damage is prevented in obesity-related hepatocarcinogenesis through CD4+ mediated apoptosis in the livers of mice on a green-tea diet

Abstract Using a C57BL/6J bioassay, we assessed whether formation of a lipid-peroxidation (LPO)-derived DNA adduct would induce mutations during the progression of obesity-induced hepatocarcinogenesis and if its formation could be prevented using a diet infused with Theaphenon E, a mixture of polyphenols extracted from green tea. Obesity is now the leading risk factor for the development of hepatocellular carcinoma in the United States. We have conducted a life-time C57BL/6J tumor bioassay to assess the role of obesity in the formation of γ-hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG), a promutagenic DNA adduct formed endogenously by LPO. Previously, we have shown γ-OHPdG was detected in FFPE liver tissues of patients with different stages of liver disease, demonstrating that its formation is consistent with LPO in the early stages of liver disease and suggesting that it may be a source of endogenous DNA damage in hepatocarcinogenesis. Tea polyphenols, predominately EGCG, are antioxidants that have been shown to prevent obesity and tumorigenesis. For the bioassay, mice were fed either a high fat, low fat, or high fat 2% TE diet starting at 4 weeks of age. Tissue and blood were collected over the course of 80 weeks at 11 separate time points. Health of the mice was assessed using serum concentrations of ALT and AST, gross morphology and immunohistochemistry of TUNEL, Ki67 and CD4+. In contrast to the high-fat and low-fat diet mice, the TE treated mice maintained a healthy body weight, liver to body weight ratio and low levels of AST and ALT enzymes which are elevated in liver disease. Additionally, the health of the liver appears to be maintained through increased CD4+ mediated apoptosis in TE treated mice as demonstrated through decreased Ki67 expression and TUNEL staining showing increased apoptosis in TE treated mice livers in conjunction with increased CD4+ cells. The CD4+ immune response may, therefore, be targeting cells with mutagenic potential. Similar to the trend indicated by the clinical samples, during hepatocarcinogenesis in the high fat diet mice, adduct levels were increased initially and likely fixed into mutation at later time points, where their levels decreased, as quantified by LC-MS/MS. In contrast, in the TE treated mice, the average γ-OHPdG levels remained consistently low. Further studies incorporating whole exome sequencing of the tumors that were formed during the life-time bioassay will indicate whether γ-OHPdG may contribute to the mutations that occured. In conclusion, our study has shown that a robust immune system response mediated by TE may be an underlying mechanism to combat the DNA adduct-induced DNA damage that may lead to HCC. Citation Format: Heidi Coia, Ning Ma, Marcin Dyba, Fung-Lung Chung. Lipid-peroxidation derived DNA damage is prevented in obesity-related hepatocarcinogenesis through CD4+ mediated apoptosis in the livers of mice on a green-tea diet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2017-1261

Efficacy of ATR inhibitors as single agents in Ewing sarcoma.

Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas.