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Unsuccessful processing of Okazaki fragments leads to the accumulation of DNA breaks which are associated with many human diseases including cancer and neurodegenerative disorders. Recently, Okazaki fragment maturation (OFM) has received renewed attention regarding how unprocessed Okazaki fragments are sensed and repaired, and how inappropriate OFM impacts on genome stability and cell viability, especially in cancer cells. We provide an overview of the highly efficient and faithful canonical OFM pathways and their regulation of genomic integrity and cell survival. We also discuss how cells induce alternative error-prone OFM processes to promote cell survival in response to environmental stresses. Such stress-induced OFM processes may be important mechanisms driving mutagenesis, cellular evolution, and resistance to radio/chemotherapy and targeted therapeutics in human cancers.

The hetero-trimeric PP2A serine/threonine phosphatases containing the regulatory subunit B56, and in particular B56γ, can function as tumor suppressors. In response to DNA damage, the B56γ subunit complexes with the PP2A AC core (B56γ-PP2A) and binds p53. This event promotes PP2A-mediated dephosphorylation of p53 at Thr55, which induces expression of p21, and the subsequent inhibition of cell proliferation and transformation. In addition to dephosphorylation of p53, B56γ-PP2A also inhibits cell proliferation and transformation by a second, as yet unknown, p53-independent mechanism. Here, we interrogated a panel of B56γ mutations found in human cancer samples and cell lines and showed that these mutations lost B56γ tumor-suppressive activity by two distinct mechanisms: one is by disrupting interactions with the PP2A AC core and the other with B56γ-PP2A substrates (p53 and unknown proteins). For the first mechanism, due to the absence of the C catalytic subunit in the complex, the mutants are unable to mediate dephosphorylation of any substrate and thus failed to promote both the p53-dependent and -independent tumor-suppressive functions of B56γ-PP2A. For the second mechanism, the mutants lacked specific substrate interactions and thus partially lost tumor-suppressive function, i.e., either the p53-dependent or p53-independent contingent upon which substrate binding was affected. Overall, these data provide new insight into the mechanisms of tumor suppression by B56γ.Implications: This study further indicates the importance of B56γ-PP2A in tumorigenesis. Mol Cancer Res; 11(9); 995–1003. ©2013 AACR.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease best known for its critical roles in Okazaki fragment maturation, DNA repair, and apoptosis-induced DNA fragmentation. Functional deficiencies in FEN1, in the forms of somatic mutations and polymorphisms, have recently been shown to lead to autoimmunity, chronic inflammation, and predisposition to and progression of cancer. To explore how FEN1 contributes to cancer progression, we examined FEN1 expression using 241 matched pairs of cancer and corresponding normal tissues on a gene expression profiling array and validated differential expression by quantitative real-time PCR and immunohistochemistry. Furthermore, we defined the minimum promoter of human FEN1 and examined the methylation statuses of the 5′ region of the gene in paired breast cancer tissues. We show that FEN1 is significantly up-regulated in multiple cancers and the aberrant expression of FEN1 is associated with hypomethylation of the CpG island within the FEN1 promoter in tumor cells. The overexpression and promoter hypomethylation of FEN1 may serve as biomarkers for monitoring the progression of cancers. (Mol Cancer Res 2008;6(11):1710–7)

Supplementary Tables S1-S3 from Overexpression and Hypomethylation of Flap Endonuclease 1 Gene in Breast and Other Cancers

PDF file - 76K, Supplementary Figure 1. Cell proliferation assay was carried out using a tetracycline-inducible U2OS stable cell line that overexpresses wild type B56γ upon induction.

Germline RB1 mutations strongly predispose humans to cone precursor–derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type–specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type–restricted thyroid hormone receptor isoform TRβ2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor-suppressive TRβ1. TRβ2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5–dependent inhibition of SKP2 degradation. In RB1 wild-type neuroblastoma cells, endogenous Rb or ectopic TRβ2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRβ1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRβ2 counteracts TRβ1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies. Cancer Res; 77(24); 6838–50. ©2017 AACR.

Supplementary Information. This file contains Extended Experimental Procedures including Table S1 (pLKO lentiviral shRNAs), Table S2 (Antibodies used in this study), and Table S3 (qPCR primers).

How cells with DNA replication defects acquire mutations that allow them to escape apoptosis under environmental stress is a long-standing question. Here, we report that an error-prone Okazaki fragment maturation (OFM) pathway is activated at restrictive temperatures in rad27Δ yeast cells. Restrictive temperature stress activated Dun1, facilitating transformation of unprocessed 5′ flaps into 3′ flaps, which were removed by 3′ nucleases, including DNA polymerase δ (Polδ). However, at certain regions, 3′ flaps formed secondary structures that facilitated 3′ end extension rather than degradation, producing alternative duplications with short spacer sequences, such as pol3 internal tandem duplications. Consequently, little 5′ flap was formed, suppressing rad27Δ-induced lethality at restrictive temperatures. We define a stress-induced, error-prone OFM pathway that generates mutations that counteract replication defects and drive cellular evolution and survival.

Double-strand break (DSB) repair choice is greatly influenced by the initial processing of DNA ends. 53BP1 limits the formation of recombinogenic single-strand DNA (ssDNA) in BRCA1-deficient cells, leading to defects in homologous recombination (HR). However, the exact mechanisms by which 53BP1 inhibits DSB resection remain unclear. Previous studies have identified two potential pathways: protection against DNA2/EXO1 exonucleases presumably through the Shieldin (SHLD) complex binding to ssDNA, and localized DNA synthesis through the CTC1-STN1-TEN1 (CST) and DNA polymerase α (Polα) to counteract resection. Using a combinatorial approach of END-seq, SAR-seq, and RPA ChIP-seq, we directly assessed the extent of resection, DNA synthesis, and ssDNA, respectively, at restriction enzyme-induced DSBs. We show that, in the presence of 53BP1, Polα-dependent DNA synthesis reduces the fraction of resected DSBs and the resection lengths in G0/G1, supporting a previous model that fill-in synthesis can limit the extent of resection. However, in the absence of 53BP1, Polα activity is sustained on ssDNA yet does not substantially counter resection. In contrast, EXO1 nuclease activity is essential for hyperresection in the absence of 53BP1. Thus, Polα-mediated fill-in partially limits resection in the presence of 53BP1 but cannot counter extensive hyperresection due to the loss of 53BP1 exonuclease blockade. These data provide the first nucleotide mapping of DNA synthesis at resected DSBs and provide insight into the relationship between fill-in polymerases and resection exonucleases.

RNABy analyzing The Cancer Genome Atlas (TCGA) dataset, we found that elevated expression ofWe show that YTHDC1 plays a critical oncogenic role in TNBC metastasis through promoting the nuclear export and expression of

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors., PCNA is essential for DNA replication and cellular proliferation. Here, the authors reveal that PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation via regulation of Okazaki fragment maturation and chromatin assembly.

DNA2 nuclease/helicase is a structure-specific nuclease, 5′-to-3′ helicase, and DNA-dependent ATPase. It is involved in multiple DNA metabolic pathways, including Okazaki fragment maturation, replication of ‘difficult-to-replicate’ DNA regions, end resection, stalled replication fork processing, and mitochondrial genome maintenance. The participation of DNA2 in these different pathways is regulated by its interactions with distinct groups of DNA replication and repair proteins and by post-translational modifications. These regulatory mechanisms induce its recruitment to specific DNA replication or repair complexes, such as DNA replication and end resection machinery, and stimulate its efficient cleavage of various structures, for example, to remove RNA primers or to produce 3′ overhangs at telomeres or double-strand breaks. Through these versatile activities at replication forks and DNA damage sites, DNA2 functions as both a tumor suppressor and promoter. In normal cells, it suppresses tumorigenesis by maintaining the genomic integrity. Thus, DNA2 mutations or functional deficiency may lead to cancer initiation. However, DNA2 may also function as a tumor promoter, supporting cancer cell survival by counteracting replication stress. Therefore, it may serve as an ideal target to sensitize advanced DNA2-overexpressing cancers to current chemo- and radiotherapy regimens.

Germline mutations of DNA double-strand break (DSB) response and repair genes that drive tumorigenesis could be a major cause of prostate cancer (PCa) heritability. In this study, we demonstrated the role of novel exonuclease 5 (EXO5) gene in androgen-induced double strand breaks repair via homology-directed repair pathway and prostate tumorigenesis. Using whole-exome sequencing of samples from 20 PCa families, with three or more siblings diagnosed with metastatic PCa, we identified mutations in 31 genes involved in DSB response and repair. Among them, the L151P mutation in the exonuclease 5 (EXO5) gene was present in all affected siblings in three PCa families. We found two other EXO5 SNPs significantly associated with risk of PCa in cases-controls study from databases of genotype and phenotype (dbGaP), which are in linkage disequilibrium (D' = 1) with Exo5 L151P found in PCa family. The L151 residue is conserved across diverse species and its mutation is deleterious for protein functions, as demonstrated by our bioinformatics analyses. The L151P mutation impairs the DNA repair function of EXO5 due to loss of nuclease activity, as well as failure of nuclear localization. CRISPR elimination of EXO5 in a PCa cell line impaired homology-directed recombination repair (HDR) and caused androgen-induced genomic instability, as indicated by frequent occurrence of the oncogenic fusion transcript TMPRSS2-ERG. Genetic and functional validation of the EXO5 mutations indicated that EXO5 is a risk gene for prostate tumorigenesis, likely due to its functions in HDR.

The maintenance of mitochondrial DNA (mtDNA) relies on proteins encoded by nuclear genes. Mutations in their coding sequences result in heterogenous clinical presentations featuring mtDNA instability in affected tissues. DNA2 is a multi‐catalytic protein involved in the removal of single strand DNA during mtDNA replication or Long Patch Base Excision Repair pathway. We have previously described DNA2 mutations in adult patients affected with familial and sporadic forms of mitochondrial myopathy. Here we describe four novel probands presenting with limb weakness associated with novel DNA2 molecular defects. Biochemical assays were established to investigate the functional effects of these variants.

SummaryFANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We showed that FANCD2 inhibits fork degradation in two ways: 1) the N-terminal domain of FANCD2 inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11, and EXO1. Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts as a “RAD51 modulator” to stimulate the strand exchange activity of RAD51. Our work biochemically explains non-canonical mechanisms by which FANCD2 and RAD51 protect stalled forks. We propose a model in which the strand exchange activity of FANCD2 provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator in the FA/BRCA pathway of fork protection

The downregulation of the DNA damage response (DDR) enables aggressive tumors to achieve uncontrolled proliferation against replication stress, but the mechanisms underlying this process in tumors are relatively complex. Here, we demonstrate a mechanism through which a distinct E3 ubiquitin ligase, ITCH, modulates DDR machinery in triple-negative breast cancer (TNBC). We found that expression of a nuclear form of ITCH was significantly increased in human TNBC cell lines and tumor specimens. Phosphorylation of ITCH at Ser257 by AKT led to the nuclear localization of ITCH and ubiquitination of H1.2. The ITCH-mediated polyubiquitination of H1.2 suppressed RNF8/RNF168-dependent formation of 53BP1 foci, which plays important roles in DDR. Consistent with these findings, impaired ITCH nuclear translocation and H1.2 polyubiquitination sensitized cells to replication stress and limited cell growth and migration. AKT activation of ITCH-H1.2 axis may confer TNBC cells with a DDR repression to counteract the replication stress and increase cancer cell survivorship and growth potential.

In the last decade, it has become clear that epigenetic changes act together with genetic mutations to promote virtually every stage of tumorigenesis and cancer progression. This knowledge has triggered searches for “epigenetic drugs” that can be developed into new cancer therapies. Here we report that triptolide reduced lung cancer incidence from 70% to 10% in a Fen1 E160D transgenic mouse model and effectively inhibited cancer growth and metastasis in A549 and H460 mouse xenografts. We found that triptolide induced lung cancer cell apoptosis that was associated with global epigenetic changes to histone 3 (H3). These global epigenetic changes in H3 are correlated with an increase in protein expression of five Wnt inhibitory factors that include WIF1, FRZB, SFRP1, ENY2, and DKK1. Triptolide had no effect on DNA methylation status at any of the CpG islands located in the promoter regions of all five Wnt inhibitory factors. Wnt expression is implicated in promoting the development and progression of many lung cancers. Because of this, the potential to target Wnt signaling with drugs that induce epigenetic modifications provides a new avenue for developing novel therapies for patients with these tumor types.

C57Bl6 (B6) mice devoid of glutathione peroxidases 1 and 2 (Gpx1/2‐DKO) develop ileitis after weaning. We previously showed germ‐free Gpx1/2‐DKO mice of mixed B6.129 background did not develop ileocolitis. Here, we examine the composition of the ileitis provoking microbiota in B6 Gpx1/2‐DKO mice. DNA was isolated from the ileum fecal stream and subjected to high‐throughput sequencing of the V3 and V4 regions of the 16S rRNA gene to determine the abundance of operational taxonomic units (OTUs). We analyzed the role of bacteria by comparing the microbiomes of the DKO and pathology‐free non‐DKO mice. Mice were treated with metronidazole, streptomycin, and vancomycin to alter pathology and correlate the OTU abundances with pathology levels. Principal component analysis based on Jaccard distance of abundance showed 3 distinct outcomes relative to the source Gpx1/2‐DKO microbiome. Association analyses of pathology and abundance of OTUs served to rule out 7–11 of 24 OTUs for involvement in the ileitis. Collections of OTUs were identified that appeared to be linked to ileitis in this animal model and would be classified as commensals. In Gpx1/2‐DKO mice, host oxidant generation from NOX1 and DUOX2 in response to commensals may compromise the ileum epithelial barrier, a role generally ascribed to oxidants generated from mitochondria, NOX2 and endoplasmic reticulum stress in response to presumptive pathogens in IBD. Elevated oxidant levels may contribute to epithelial cell shedding, which is strongly associated with progress toward inflammation in Gpx1/2‐DKO mice and predictive of relapse in IBD by allowing leakage of microbial components into the submucosa., Hydrogen peroxide (H2O2) and superoxide are generated by some bacterial genera and by the intestinal mucosa in response to bacteria. Gpx1/2‐double knockout mice are sensitive to intestinal H2O2 production, which causes crypt apoptosis and anoikis leading to inflammation. We investigate the normal ileum pathology inducing microbiota and use antibiotics to narrow down potential candidates. The principal candidate, Lactobacillus, stands out while not demonstrating unambiguous domination in provoking the pathology.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential multifunctional protein in mammals that plays critical roles in DNA repair and redox signaling within the cell. Impaired APE1 function or dysregulation is associated with disease susceptibility and poor cancer prognosis. Orchestrated regulatory mechanisms are crucial to ensure its function in a specific subcellular location at specific time. Here, we report arginine methylation as a post-translational modification (PTM) that regulates APE1 translocation to mitochondria in HeLa and HEK-293 cells. Protein arginine methyl-transferase 1 (PRMT1) was shown to methylate APE1 in vitro. Site-directed mutagenesis identified R301 as the major methylation site. We confirmed that APE1 is methylated in cells and that the R301K mutation significantly reduces its methylation. Baseline mitochondrial APE1 levels were low under standard culture conditions, but they could be induced by oxidative agents. Methylation-deficient APE1 showed reduced mitochondrial translocation. Methylation affected the interaction of APE1 with Tom20, translocase of the outer mitochondrial membrane. Methylation-deficient APE1 resulted in increased mitochondrial DNA damage and increased cytochrome c release after stimuli. These data suggest that methylation of APE1 promotes its mitochondrial translocation and protects cells from oxidative damage. This work describes a novel PTM regulation model of APE1 subcellular distribution through arginine methylation.

During DNA synthesis, DNA replication and transcription machinery can collide, and the replication fork may temporarily dislodge RNA polymerase II (RNAPII) to resolve the transcription-replication conflict (TRC), a major source of endogenous DNA double-strand breaks (DSBs) and common fragile site (CFS) instability. However, the mechanism of TRC resolution remains unclear. Here, we show that conjugation of SUMO2, but not SUMO1 or SUMO3, to the essential replication factor PCNA is induced on transcribed chromatin by the RNAPII-bound helicase RECQ5. Proteomic analysis reveals that SUMO2-PCNA enriches histone chaperones CAF1 and FACT in the replication complex via interactions with their SUMO-interacting motifs. SUMO2-PCNA enhances CAF1-dependent histone deposition, which correlates with increased histone H3.1 at CFSs and repressive histone marks in the chromatin to reduce chromatin accessibility. Hence, SUMO2-PCNA dislodges RNAPII at CFSs, and overexpressing either SUMO2-PCNA or CAF1 reduces the incidence of DSBs in TRC-prone RECQ5-deficient cells., Transcription-replication conflicts need to be resolved to minimize genome instability. Here the authors show that SUMO2-conjugated PCNA destabilizes RNAPII from chromatin, enhances replication progression and limits transcription-induced DNA damage at common fragile sites.

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing ubiquitination-deficient 293T and RPE1 cells generated through CRISPR/Cas9 genome editing, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-mediated but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork, and incomplete Okazaki fragment synthesis and maturation, thus interfering with efficient PCNA unloading by ATAD5 and subsequent nucleosomal deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and BRCA2 results in a synergistic increase in nascent DNA degradation and sensitivity to PARP-inhibitors. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.

SUMMARY The arginine methylation status of histones dynamically changes during many cellular processes, including hematopoietic stem/progenitor cell (HSPC) development. The arginine methyltransferases and the readers that transduce the histone codes have been defined. However, whether arginine demethylation actively occurs in cells and what enzyme demethylates the methylarginine residues during various cellular processes are unknown. We report that JMJD1B, previously identified as a lysine demethylase for H3K9me2, mediates arginine demethylation of H4R3me2s and its intermediate, H4R3me1. We show that demethylation of H4R3me2s and H3K9me2s in promoter regions is correlated with active gene expression. Furthermore, knockout of JMJD1B blocks demethylation of H4R3me2s and/or H3K9me2 at distinct clusters of genes and impairs the activation of genes important for HSPC differentiation and development. Consequently, JMJD1B−/− mice show defects in hematopoiesis. Altogether, our study demonstrates that demethylase-mediated active arginine demethylation process exists in eukaryotes and that JMJD1B demethylates both H4R3me2s and H3K9me2 for epigenetic programming during hematopoiesis., In Brief Li et al. identify the arginine demethylase (RDM) activity of JMJD1B, a known lysine demethylase (KDM). They reveal that JMJD1B actively mediates demethylation of histone markers H4R3me2s and H3K9me2 in hematopoietic stem/progenitor cells (HSPCs).

Germline RB1 mutations strongly predispose humans to cone precursor–derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type–specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type–restricted thyroid hormone receptor isoform TRβ2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor-suppressive TRβ1. TRβ2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5–dependent inhibition of SKP2 degradation. In RB1 wild-type neuroblastoma cells, endogenous Rb or ectopic TRβ2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRβ1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRβ2 counteracts TRβ1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies. Cancer Res; 77(24); 6838–50. ©2017 AACR.

Mice deficient in glutathione peroxidase (GPx)-1 and -2 (GPx1-/-GPx2-/- double knockout or DKO mice) develop very-early-onset (VEO) ileocolitis, suggesting that lack of defense against reactive oxygen species (ROS) renders susceptibility to intestinal inflammation. Two members of ROS-generating NADPH oxidase family, NOX1 and DUOX2, are highly inducible in the intestinal epithelium. Previously, we reported that Nox1 deficiency ameliorated the pathology in DKO mice (Nox1-TKO). The role of Duox2 in ileocolitis of the DKO mice is evaluated here in Duoxa-TKO mice by breeding DKO mice with Duoxa-/- mice (Duoxa-TKO), which do not have Duox2 activity. Similar to Nox1-TKO mice, Duoxa-TKO mice no longer have growth retardation, shortened intestine, exfoliation of crypt epithelium, crypt abscesses and depletion of goblet cells manifested in DKO mice by 35 days of age. Unlike Nox1-TKO mice, Duoxa-TKO mice still have rampant crypt apoptosis, elevated proliferation, partial loss of Paneth cells and diminished crypt density. Treating DKO mice with NOX inhibitors (di-2-thienyliodonium/DTI and thioridazine/THZ) and an antioxidant (mitoquinone/MitoQ) significantly reduced gut pathology. Furthermore, in the inflamed human colon, DUOX protein expression is highly elevated in the apical, lateral and perinuclear membrane along the whole length of gland. Taken together, we conclude that exfoliation of crypt epithelium, but not crypt apoptosis, is a major contributor to inflammation. Both Nox1 and Duox2 induce exfoliation of crypt epithelium, but only Nox1 induces apoptosis. NOX1 and DUOX2 may be potential therapeutic targets for treating ileocolitis in human patients suffering inflammatory bowel disease (IBD)., Highlights • Glutathione peroxidase-1/2-double knockout mice have very-early-onset ileocolitis. • By deletion of Nox1 gene expression, the triple knockout mice are without pathology. • By deletion of Duoxa, the mice have milder pathology without crypt exfoliation. • The Duoxa triple knock mice still have rampant crypt epithelium apoptosis. • Several antioxidants and NOX inhibitors reduce gut inflammation in the DKO mice. • DKO mice are an excellent animal model for preclinical testing of NOX inhibitors.

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 in DNA repair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1-phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen-induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1-phase arrest due to activation of the p53-mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50% of newborns with the S187A mutant died in the first week due to failure to undergo the peroxisome proliferator-activated receptor signaling-dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen-induced stress in the heart during the fetal-to-neonatal transition.—Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

// Shirong Zhang 1, * , Kan Wu 2, 3, * , Jianguo Feng 4, * , Zhibing Wu 3 , Qinghua Deng 3 , Chao Guo 5 , Bing Xia 3 , Jing Zhang 1 , Haixiu Huang 1 , Lucheng Zhu 1 , Ke Zhang 3 , Binghui Shen 5 , Xufeng Chen 6 , Shenglin Ma 1 1 Department of Oncology, Hangzhou First People’s Hospital, Nanjing Medical University, Hangzhou, China 2 Department of Oncology, Affiliated Hangzhou First People’s Hospital of Zhejiang Chinese Medical University, Hangzhou, China 3 Department of Oncology, Hangzhou Cancer Hospital, Hangzhou, China 4 Cancer Research institute, Zhejiang Cancer Hospital, Hangzhou, China 5 Department of Cancer Genetics and Epigenetics, City of Hope National Medical Center, Duarte, CA, USA 6 Department of Pathology and Laboratory Medicine, University of California at Los Angeles, Los Angeles, CA, USA * These authors have contributed equally to this work Correspondence to: Shenglin Ma, email: mashenglin@medmail.com.cn Keywords: non-small cell lung cancer, invasion, epigenetics, suberoylanilide hydroxamic acid, epigenetic therapy Received: March 29, 2016 Accepted: August 24, 2016 Published: September 12, 2016 ABSTRACT Metastasis is the reason for most cancer death, and a crucial primary step for cancer metastasis is invasion of the surrounding tissue, which may be initiated by some rare tumor cells that escape the heterogeneous primary tumor. In this study, we isolated invasive subpopulations of cancer cells from human non-small cell lung cancer (NSCLC) H460 and H1299 cell lines, and determined the gene expression profiles and the responses of these invasive cancer cells to treatments of ionizing radiation and chemotherapeutic agents. The subpopulation of highly invasive NSCLC cells showed epigenetic signatures of epithelial-mesenchymal transition, cancer cell stemness, increased DNA damage repair and cell survival signaling. We also investigated the epigenetic therapy potential of suberoylanilide hydroxamic acid (SAHA) on invasive cancer cells, and found that SAHA suppresses cancer cell invasiveness and sensitizes cancer cells to treatments of IR and chemotherapeutic agents. Our results provide guidelines for identification of metastatic predictors and for clinical management of NSCLC. This study also suggests a beneficial clinical potential of SAHA as a chemotherapeutic agent for NSCLC patients.

The multifunctional human DNA2 (hDNA2) nuclease/helicase is required to process DNA ends for homology-directed recombination repair (HDR) and to counteract replication stress. To participate in these processes, hDNA2 must localize to the nucleus and be recruited to the replication or repair sites. However, because hDNA2 lacks the nuclear localization signal that is found in its yeast homolog, it is unclear how its migration into the nucleus is regulated during replication or in response to DNA damage. Here, we report that the E3 ligase TRAF6 binds to and mediates the K63-linked polyubiquitination of hDNA2, increasing the stability of hDNA2 and promoting its nuclear localization. Inhibiting TRAF6-mediated polyubiquitination abolishes the nuclear localization of hDNA2, consequently impairing DNA end resection and HDR. Thus, the current study reveals a mechanism for the regulation of hDNA2 localization and establishes that TRAF6-mediated hDNA2 ubiquitination activates DNA repair pathways to maintain nuclear genome integrity.

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD+ leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD+ AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD+ AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation–dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD+ AML cells.

Retinoblastoma is a childhood retinal tumor that develops from cone photoreceptor precursors in response to inactivating RB1 mutations and loss of functional RB protein. The cone precursor's response to RB loss involves cell type-specific signaling circuitry that helps to drive tumorigenesis. One component of the cone precursor circuitry, the thyroid hormone receptor β2 (TRβ2), enables the aberrant proliferation of diverse RB-deficient cells in part by opposing the down-regulation of S-phase kinase-associated protein 2 (SKP2) by the more widely expressed and tumor-suppressive TRβ1. However, it is unclear how TRβ2 opposes TRβ1 to enable SKP2 expression and cell proliferation. Here, we show that in human retinoblastoma cells TRβ2 mRNA encodes two TRβ2 protein isoforms: a predominantly cytoplasmic 54-kDa protein (TRβ2-54) corresponding to the well-characterized full-length murine Trβ2 and an N-terminally truncated and exclusively cytoplasmic 46-kDa protein (TRβ2-46) that starts at Met-79. Whereas TRβ2 knockdown decreased SKP2 expression and impaired retinoblastoma cell cycle progression, re-expression of TRβ2-46 but not TRβ2-54 stabilized SKP2 and restored proliferation to an extent similar to that of ectopic SKP2 restoration. We conclude that TRβ2-46 is an oncogenic thyroid hormone receptor isoform that promotes SKP2 expression and SKP2-dependent retinoblastoma cell proliferation.

Although the Oct4/Sox2 complex is crucial for maintaining the pluripotency of stem cells, the molecular basis underlying its regulation during lineage-specific differentiation remains unknown. Here, we revealed that the highly conserved Oct4/Lys-156 is important for maintaining the stability of the Oct4 protein and the intermolecular salt bridge between Oct4/Lys-151 and Sox2/Asp-107 that contributes to the Oct4/Sox2 interaction. Post-translational modifications at Lys-156 and K156N, a somatic mutation detected in bladder cancer patients, both impaired the Lys-151-Asp-107 salt bridge and the Oct4/Sox2 interaction. When produced as a recombinant protein or overexpressed in pluripotent stem cells, Oct4/K156N, with reduced binding to Sox2, significantly down-regulated the stemness genes that are cooperatively controlled by the Oct4/Sox2 complex and specifically up-regulated the mesendodermal genes and the SNAIL family genes that promote the epithelial-mesenchymal transition. Thus, we conclude that Oct4/Lys-156-modulated Oct4/Sox2 interaction coordinately controls the epithelial-mesenchymal transition and mesendoderm specification induced by specific differentiation signals.

Flap endonuclease-1 (FEN1) is a multifunctional, structure-specific nuclease that has a critical role in maintaining human genome stability. FEN1 mutations have been detected in human cancer specimens and have been suggested to cause genomic instability and cancer predisposition. However, the exact relationship between FEN1 deficiency and cancer susceptibility remains unclear. In the current work, we report a novel colorectal cancer-associated FEN1 mutation, L209P. This mutant protein lacks the FEN, exonuclease (EXO) and gap endonuclease (GEN) activities of FEN1 but retains DNA-binding affinity. The L209P FEN1 variant interferes with the function of the wild-type FEN1 enzyme in a dominant-negative manner and impairs long-patch base excision repair in vitro and in vivo. Expression of L209P FEN1 sensitizes cells to DNA damage, resulting in endogenous genomic instability and cellular transformation, as well as tumor growth in a mouse xenograft model. These data indicate that human cancer-associated genetic alterations in the FEN1 gene can contribute substantially to cancer development.

Cancer cells frequently up-regulate DNA replication and repair proteins such as the multifunctional DNA2 nuclease/helicase, counteracting DNA damage due to replication stress and promoting survival. Therefore, we hypothesized that blocking both DNA replication and repair by inhibiting the bifunctional DNA2 could be a potent strategy to sensitize cancer cells to stresses from radiation or chemotherapeutic agents. We show that homozygous deletion of DNA2 sensitizes cells to ionizing radiation and camptothecin (CPT). Using a virtual high throughput screen, we identify 4-hydroxy-8-nitroquinoline-3-carboxylic acid (C5) as an effective and selective inhibitor of DNA2. Mutagenesis and biochemical analysis define the C5 binding pocket at a DNA-binding motif that is shared by the nuclease and helicase activities, consistent with structural studies that suggest that DNA binding to the helicase domain is necessary for nuclease activity. C5 targets the known functions of DNA2 in vivo: C5 inhibits resection at stalled forks as well as reducing recombination. C5 is an even more potent inhibitor of restart of stalled DNA replication forks and over-resection of nascent DNA in cells defective in replication fork protection, including BRCA2 and BOD1L. C5 sensitizes cells to CPT and synergizes with PARP inhibitors., Highlights • C5 binds to the helicase domain of DNA2 and inhibits its nuclease, ATPase, and helicase activities. • C5 inhibits DNA end resection for DNA double strand break repair and restart of stalled replication forks. • C5 suppresses over-resection of nascent DNA in cells defective in fork protection. • C5 sensitizes cancer cells to chemotherapeutic agents. Most chemotherapeutics introduce DNA lesions that block DNA replication to kill cancer cells. Upregulation of DNA repair proteins in cancer cells is a major reason for human cancer to become resistant to chemotherapeutics. DNA repair proteins such as DNA2 nuclease/helicase have long been proposed as targets for sensitization of cancer cells to chemotherapy. We identify a selective DNA2 inhibitor (C5) and demonstrate that DNA2 inhibition by C5 suppresses cancer cells to rescue stalled replication forks. Consequently, it sensitizes cancer cells to replication fork stalling agents such as camptothecin. Thus, C5 is a promising lead compound for developing new anticancer drugs.

DNA polymerase β (Pol β) is a key enzyme in DNA base excision repair (BER), a pathway that maintains genome integrity and stability. Pol β mutations have been detected in various types of cancers, suggesting a possible linkage between Pol β mutations and cancer. However, it is not clear whether and how Pol β mutations cause cancer onset and progression. In the current work, we show that a substitution mutation, R152C, impairs Pol β polymerase activity and BER efficiency. Cells harboring Pol β R152C are sensitive to the DNA damaging agents methyl methanesulfonate (MMS) and H2O2. Moreover, the mutant cells display a high frequency of chromatid breakages and aneuploidy and also form foci. Taken together, our data indicate that Pol β R152C can drive cellular transformation.