bims-numges 2020-10-11 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2020‒10‒11
37 papers selected by
Sean Rudd
Karolinska Institutet

Modulating pyrimidine ribonucleotide levels for the treatment of cancer.
Exploiting Replication Stress as a Novel Therapeutic Intervention.
Monitoring and modeling of lymphocytic leukemia cell bioenergetics reveals decreased ATP synthesis during cell division.
DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions.
HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.
Ubiquitin Phosphorylation at Thr12 Modulates the DNA Damage Response.
CK2 kinase-mediated PHF8 phosphorylation controls TopBP1 stability to regulate DNA replication.
ATM inhibition synergizes with fenofibrate in high grade serous ovarian cancer cells.
Lactate dehydrogenase inhibition affects homologous recombination repair independently of cell metabolic asset; implications for anticancer treatment.
RHOAming Through the Nucleotide Excision Repair Pathway as a Mechanism of Cellular Response Against the Effects of UV Radiation.
The Roles of SPOP in DNA Damage Response and DNA Replication.
Posttranslational Regulation of DNA Polymerase η, a Connection to Damage-Induced Cohesion in Saccharomyces cerevisiae.
The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion.
Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass.
Inhibition of poly(ADP-ribose) polymerase induces synthetic lethality in BRIP1 deficient ovarian epithelial cells.
The involvement of nucleotide excision repair proteins in the removal of oxidative DNA damage.
When Endoplasmic Reticulum Proteostasis Meets the DNA Damage Response.
Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer.
Alteration of genetic recombination and double-strand break repair in human cells by progerin expression.
Mahogunin Ring Finger 1 Is Required for Genomic Stability and Modulates the Malignant Phenotype of Melanoma Cells.
Phase separation by ssDNA binding protein controlled via protein-protein and protein-DNA interactions.
PARP Inhibitors: Clinical Relevance, Mechanisms of Action and Tumor Resistance.
The roles of nuclear myosin in the DNA damage response.
The ALPK1/TIFA/NF-κB axis links a bacterial carcinogen to R-loop-induced replication stress.
Regulation and pharmacological targeting of RAD51 in cancer.
Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast.
Targeting MALAT1 induces DNA damage and sensitize non-small cell lung cancer cells to cisplatin by repressing BRCA1.
Molecular contribution of BRCA1 and BRCA2 to genome instability in breast cancer patients: review of radiosensitivity assays.
CEP131 Abrogates CHK1 Inhibitor-Induced Replication Defects and Is Associated with Unfavorable Outcome in Neuroblastoma.
Pharmacological Inhibition of WEE1 Potentiates the Antitumoral Effect of the dl922-947 Oncolytic Virus in Malignant Mesothelioma Cell Lines.
Fibroblast growth factor 2 upregulates ecto-5'-nucleotidase and adenosine deaminase via MAPK pathways in cultured rat spinal cord astrocytes.
NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential.
Targeting mitochondrial respiration for the treatment of acute myeloid leukemia.
Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors.
Cac1 WHD and PIP domains have distinct roles in replisome progression and genomic stability.
Enhancing efficacy of gemcitabine in pancreatic patient-derived xenograft mouse models.
Evaluation of sulfonate and sulfamate derivatives possessing benzofuran or benzothiophene nucleus as inhibitors of nucleotide pyrophosphatases/phosphodiesterases and anticancer agents.

Cancer Metab. 2020 ;8 12

Modulating pyrimidine ribonucleotide levels for the treatment of cancer.

Tanzina Mollick, Sonia Laín.

  By providing the necessary building blocks for nucleic acids and precursors for cell membrane synthesis, pyrimidine ribonucleotides are essential for cell growth and proliferation. Therefore, depleting pyrimidine ribonucleotide pools has long been considered as a strategy to reduce cancer cell growth. Here, we review the pharmacological approaches that have been employed to modulate pyrimidine ribonucleotide synthesis and degradation routes and discuss their potential use in cancer therapy. New developments in the treatment of myeloid malignancies with inhibitors of pyrimidine ribonucleotide synthesis justify revisiting the literature as well as discussing whether targeting this metabolic pathway can be effective and sufficiently selective for cancer cells to warrant an acceptable therapeutic index in patients.

Keywords:  CAD; CDA; CTPS; Cancer therapy; DHODH; Nucleoside transporters; Pyrimidine ribonucleotide metabolism; Therapeutic index; UMPS

DOI:  https://doi.org/10.1186/s40170-020-00218-5
Mol Cancer Res. 2020 Oct 05. pii: molcanres.0651.2020. [Epub ahead of print]

Exploiting Replication Stress as a Novel Therapeutic Intervention.

Jeffrey C Martin, Tamara J Hoegel, Miranda L Lynch, Anna Woloszynska, Thomas Melendy, Joyce Ellen Ohm.

Ewing sarcoma is an aggressive pediatric tumor of the bone and soft tissue. The current standard of care is radiation and chemotherapy, and patients generally lack targeted therapies. One of the defining molecular features of this tumor type is the presence of significantly elevated levels of replication stress as compared to both normal cells and many other types of cancers, but the source of this stress is poorly understood. Tumors that harbor elevated levels of replication stress rely on the replication stress and DNA damage-response pathways to retain viability. Understanding the source of the replication stress in Ewing sarcoma may reveal novel therapeutic targets. Ewing sarcomagenesis is complex, and in this review, we discuss the current state of our knowledge regarding elevated replication stress and the DNA damage response in Ewing sarcoma, one contributor to the disease process. We will also describe how these pathways are being successfully targeted therapeutically in other tumor types, and discuss possible novel, evidence based therapeutic interventions in Ewing sarcoma. We hope that this consolidation will spark investigations that uncover new therapeutic targets and lead to the development of better treatment options for Ewing sarcoma patients. Implications: This review uncovers new therapeutic targets in Ewing Sarcoma and highlights replication stress as an exploitable vulnerability across multiple cancers.

DOI: https://doi.org/10.1158/1541-7786.MCR-20-0651
Nat Commun. 2020 10 05. 11(1): 4983

Monitoring and modeling of lymphocytic leukemia cell bioenergetics reveals decreased ATP synthesis during cell division.

Joon Ho Kang, Georgios Katsikis, Zhaoqi Li, Kiera M Sapp, Max A Stockslager, Daniel Lim, Matthew G Vander Heiden, Michael B Yaffe, Scott R Manalis, Teemu P Miettinen.

The energetic demands of a cell are believed to increase during mitosis, but the rates of ATP synthesis and consumption during mitosis have not been quantified. Here, we monitor mitochondrial membrane potential of single lymphocytic leukemia cells and demonstrate that mitochondria hyperpolarize from the G2/M transition until the metaphase-anaphase transition. This hyperpolarization was dependent on cyclin-dependent kinase 1 (CDK1) activity. By using an electrical circuit model of mitochondria, we quantify mitochondrial ATP synthesis rates in mitosis from the single-cell time-dynamics of mitochondrial membrane potential. We find that mitochondrial ATP synthesis decreases by approximately 50% during early mitosis and increases back to G2 levels during cytokinesis. Consistently, ATP levels and ATP synthesis are lower in mitosis than in G2 in synchronized cell populations. Overall, our results provide insights into mitotic bioenergetics and suggest that cell division is not a highly energy demanding process.

DOI: https://doi.org/10.1038/s41467-020-18769-y
NAR Cancer. 2020 Sep;2(3): zcaa028

DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions.

Zhenbao Yu, Sofiane Y Mersaoui, Laure Guitton-Sert, Yan Coulombe, Jingwen Song, Jean-Yves Masson, Stéphane Richard.

R-loops are three-stranded structures consisting of a DNA/RNA hybrid and a displaced DNA strand. The regulatory factors required to process this fundamental genetic structure near double-strand DNA breaks (DSBs) are not well understood. We previously reported that cellular depletion of the ATP-dependent DEAD box RNA helicase DDX5 increases R-loops genome-wide causing genomic instability. In this study, we define a pivotal role for DDX5 in clearing R-loops at or near DSBs enabling proper DNA repair to avoid aberrations such as chromosomal deletions. Remarkably, using the non-homologous end joining reporter gene (EJ5-GFP), we show that DDX5-deficient U2OS cells exhibited asymmetric end deletions on the side of the DSBs where there is overlap with a transcribed gene. Cross-linking and immunoprecipitation showed that DDX5 bound RNA transcripts near DSBs and required its helicase domain and the presence of DDX5 near DSBs was also shown by chromatin immunoprecipitation. DDX5 was excluded from DSBs in a transcription- and ATM activation-dependent manner. Using DNA/RNA immunoprecipitation, we show DDX5-deficient cells had increased R-loops near DSBs. Finally, DDX5 deficiency led to delayed exonuclease 1 and replication protein A recruitment to laser irradiation-induced DNA damage sites, resulting in homologous recombination repair defects. Our findings define a role for DDX5 in facilitating the clearance of RNA transcripts overlapping DSBs to ensure proper DNA repair.

DOI: https://doi.org/10.1093/narcan/zcaa028
Cells. 2020 Oct 04. pii: E2237. [Epub ahead of print]9(10):

HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

Niko Moses, Mu Zhang, Jheng-Yu Wu, Chen Hu, Shengyan Xiang, Xinran Geng, Yue Chen, Wenlong Bai, You-Wei Zhang, Gerold Bepler, Xiaohong Mary Zhang.

  We have previously discovered that HDAC6 regulates the DNA damage response (DDR) via modulating the homeostasis of a DNA mismatch repair protein, MSH2, through HDAC6's ubiquitin E3 ligase activity. Here, we have reported HDAC6's second potential E3 ligase substrate, a critical cell cycle checkpoint protein, Chk1. We have found that HDAC6 and Chk1 directly interact, and that HDAC6 ubiquitinates Chk1 in vivo and in vitro. Specifically, HDAC6 interacts with Chk1 via the DAC1 domain, which contains its ubiquitin E3 ligase activity. During the cell cycle, Chk1 protein levels fluctuate, peaking at the G2 phase, subsequently resolving via the ubiquitin-proteasome pathway, and thereby allowing cells to progress to the M phase. However, in HDAC6 knockdown non-small cell lung cancer (NSCLC) cells, Chk1 is constitutively active and fails to resolve post-ionizing radiation (IR), and this enhanced Chk1 activity leads to preferential G2 arrest in HDAC6 knockdown cells accompanied by a reduction in colony formation capacity and viability. Depletion or pharmacological inhibition of Chk1 in HDAC6 knockdown cells reverses this radiosensitive phenotype, suggesting that the radiosensitivity of HDAC6 knockdown cells is dependent on increased Chk1 kinase activity. Overall, our results highlight a novel mechanism of Chk1 regulation at the post-translational level, and a possible strategy for sensitizing NSCLC to radiation via inhibiting HDAC6's E3 ligase activity.

Keywords:  DNA damage response (DDR); checkpoint kinase 1 (Chk1); histone deacetylase 6 (HDAC6); ionizing radiation (IR); ubiquitin E3 ligase; ubiquitination

DOI:  https://doi.org/10.3390/cells9102237
Mol Cell. 2020 Sep 29. pii: S1097-2765(20)30647-X. [Epub ahead of print]

Ubiquitin Phosphorylation at Thr12 Modulates the DNA Damage Response.

Franziska Walser, Monique P C Mulder, Benoît Bragantini, Sibylle Burger, Tatiana Gubser, Marco Gatti, Maria Victoria Botuyan, Alessandra Villa, Matthias Altmeyer, Dario Neri, Huib Ovaa, Georges Mer, Lorenza Penengo.

  The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.

Keywords:  53BP1; BRCA1/BARD1; DDR; DNA damage response; DNA repair; H2AK15pUbT12; RAD51; RNF168; RNF169; RNF8; USP51; chromatin ubiquitination; genome stability; histone mark H2AK15ub; pUbT12; phospho-ubiquitin Thr12; ubiquitin phosphorylation

DOI:  https://doi.org/10.1016/j.molcel.2020.09.017
Nucleic Acids Res. 2020 Oct 03. pii: gkaa756. [Epub ahead of print]

CK2 kinase-mediated PHF8 phosphorylation controls TopBP1 stability to regulate DNA replication.

Haihua Feng, Jingchen Lu, Xiaotian Song, Angkana Thongkum, Fan Zhang, Lihong Lou, Ofer Reizes, Alexandru Almasan, Zihua Gong.

ATR functions as a master regulator of the DNA-damage response. ATR activation requires the ATR activator, topoisomerase IIβ-binding protein 1 (TopBP1). However, the underlying mechanism of TopBP1 regulation and how its regulation affects DNA replication remain unknown. Here, we report a specific interaction between TopBP1 and the histone demethylase PHF8. The TopBP1/PHF8 interaction is mediated by the BRCT 7+8 domain of TopBP1 and phosphorylation of PHF8 at Ser854. This interaction is cell-cycle regulated and phosphorylation-dependent. PHF8 is phosphorylated by CK2, which regulates binding of PHF8 to TopBP1. Importantly, PHF8 regulates TopBP1 protein level by preventing its ubiquitination and degradation mediated by the E3 ligase UBR5. Interestingly, PHF8pS854 is likely to contribute to regulation of TopBP1 stability and DNA replication checkpoint. Further, both TopBP1 and PHF8 are required for efficient replication fork restart. Together, these data identify PHF8 as a TopBP1-binding protein and provide mechanistic insight into how PHF8 regulates TopBP1 stability to maintain DNA replication.

DOI: https://doi.org/10.1093/nar/gkaa756
Heliyon. 2020 Sep;6(9): e05097

ATM inhibition synergizes with fenofibrate in high grade serous ovarian cancer cells.

Chi-Wei Chen, Raquel Buj, Erika S Dahl, Kelly E Leon, Katherine M Aird.

  While therapies targeting deficiencies in the homologous recombination (HR) pathway are emerging as the standard treatment for high grade serous ovarian cancer (HGSOC) patients, this strategy is limited to the ~50% of patients with a deficiency in this pathway. Therefore, patients with HR-proficient tumors are likely to be resistant to these therapies and require alternative strategies. We found that the HR gene Ataxia Telangiectasia Mutated (ATM) is wildtype and its activity is upregulated in HGSOC compared to normal fallopian tube tissue. Interestingly, multiple pathways related to metabolism are inversely correlated with ATM expression in HGSOC specimens, suggesting that combining ATM inhibition with metabolic drugs would be effective. Analysis of FDA-approved drugs from the Dependency Map demonstrated that ATM-low cells are more sensitive to fenofibrate, a PPARα agonist that affects multiple cellular metabolic pathways. Consistently, PPARα signaling is associated with ATM expression. We validated that combined inhibition of ATM and treatment with fenofibrate is synergistic in multiple HGSOC cell lines by inducing senescence. Together, our results suggest that metabolic changes induced by ATM inhibitors are a potential target for the treatment of HGSOC.

Keywords:  Biochemistry; Bioinformatics; Cancer research; Cell biology; Cellular metabolism; Cellular senescence; Drug combinations; Homologous recombination; Metabolite; Molecular biology; PPARa

DOI:  https://doi.org/10.1016/j.heliyon.2020.e05097
Biochim Biophys Acta Gen Subj. 2020 Oct 06. pii: S0304-4165(20)30271-3. [Epub ahead of print] 129760

Lactate dehydrogenase inhibition affects homologous recombination repair independently of cell metabolic asset; implications for anticancer treatment.

Andrea Balboni, Marzia Govoni, Valentina Rossi, Marinella Roberti, Andrea Cavalli, Giuseppina Di Stefano, Marcella Manerba.

  BACKGROUND: Cancer cells show highly increased glucose utilization which, among other cancer-essential functions, was found to facilitate DNA repair. Lactate dehydrogenase (LDH) activity is pivotal for supporting the high glycolytic flux of cancer cells; to our knowledge, a direct contribution of this enzyme in the control of DNA integrity was never investigated. In this paper, we looked into a possible LDH-mediated regulation of homologous recombination (HR) repair.METHODS: We identified two cancer cell lines with different assets in energy metabolism: either based on glycolytic ATP or on oxidative reactions. In cells with inhibited LDH, we assessed HR function by applying four different procedures.
RESULTS: Our findings revealed an LDH-mediated control of HR, which was observed independently of cell metabolic asset. Since HR inhibition is known to make cancer cells responsive to PARP inhibitors, in both the cellular models we finally explored the effects of a combined inhibition of LDH and PARP.
CONCLUSIONS: The obtained results suggest for LDH a central role in cancer cell biology, not merely linked to the control of energy metabolism. The involvement of LDH in the DNA damage response could suggest new drug combinations to obtain improved antineoplastic effects.
GENERAL SIGNIFICANCE: Several evidences have correlated the metabolic features of cancer cells with drug resistance and LDH inhibition has been repeatedly shown to increase the antineoplastic power of chemotherapeutics. By shedding light on the processes linking cell metabolism to the control of DNA integrity, our findings also give a mechanistic explanation to these data.

Keywords:  Cancer cell metabolism; Glycolysis; Homologous recombination; Lactate; Lactate dehydrogenase; Olaparib

DOI:  https://doi.org/10.1016/j.bbagen.2020.129760
Front Cell Dev Biol. 2020 ;8 816

RHOAming Through the Nucleotide Excision Repair Pathway as a Mechanism of Cellular Response Against the Effects of UV Radiation.

Yuli T Magalhaes, Gisele E T Silva, Juliana H Osaki, Clarissa R R Rocha, Fabio L Forti.

  Typical Rho GTPases include the enzymes RhoA, Rac1, and Cdc42 that act as molecular switches to regulate essential cellular processes in eukaryotic cells such as actomyosin dynamics, cell cycle, adhesion, death and differentiation. Recently, it has been shown that different conditions modulate the activity of these enzymes, but their functions still need to be better understood. Here we examine the interplay between RhoA and the NER (Nucleotide Excision Repair) pathway in human cells exposed to UVA, UVB or UVC radiation. The results show high levels and accumulation of UV-induced DNA lesions (strand breaks and cyclobutane pyrimidine dimers, CPDs) in different cells with RhoA loss of function (LoF), either by stable overexpression of negative dominant RhoA (RhoA-N19 mutant), by inhibition with C3 toxin or by transient silencing with siRNA. Cells under RhoA LoF showed reduced levels of γH2AX, p-Chk1 (Ser345) and p-p53 (Ser15) that reflected causally in their accumulation in G1/S phases, in low survival rates and in reduced cell proliferation, also in accordance with the energy of applied UV light. Even NER-deficient cells (XPA, XPC) or DNA translesion synthesis (TLS)-deficient cells (XPV) showed substantial hypersensitivity to UV effects when previously submitted to RhoA LoF. In contrast, analyses of apoptosis, necrosis, autophagy and senescence revealed that all cells displaying normal levels of active RhoA (RhoA-GTP) are more resistant to UV-promoted cell death. This work reaffirms the role of RhoA protein signaling in protecting cells from damage caused by UV radiation and demonstrates relevant communicating mechanisms between actin cytoskeleton and genomic stability.

Keywords:  DNA damage response pathway; Rho GTPases; UV radiation; cell cycle and proliferation; nucleotide excision repair pathway

DOI:  https://doi.org/10.3389/fcell.2020.00816
Int J Mol Sci. 2020 Oct 02. pii: E7293. [Epub ahead of print]21(19):

The Roles of SPOP in DNA Damage Response and DNA Replication.

Masashi Maekawa, Shigeki Higashiyama.

  Speckle-type BTB/POZ protein (SPOP) is a substrate recognition receptor of the cullin-3 (CUL3)/RING type ubiquitin E3 complex. To date, approximately 30 proteins have been identified as ubiquitinated substrates of the CUL3/SPOP complex. Pathologically, missense mutations in the substrate-binding domain of SPOP have been found in prostate and endometrial cancers. Prostate and endometrial cancer-associated SPOP mutations lose and increase substrate-binding ability, respectively. Expression of these SPOP mutants, thus, causes aberrant turnovers of the substrate proteins, leading to tumor formation. Although the molecular properties of SPOP and its cancer-associated mutants have been intensively elucidated, their cellular functions remain unclear. Recently, a number of studies have uncovered the critical role of SPOP and its mutants in DNA damage response and DNA replication. In this review article, we summarize the physiological functions of SPOP as a "gatekeeper" of genome stability.

Keywords:  DNA damage response; DNA repair; DNA replication; SPOP; cancer; cullin-3; genome instability; topoisomerase

DOI:  https://doi.org/10.3390/ijms21197293
Genetics. 2020 Oct 08. pii: genetics.303494.2020. [Epub ahead of print]

Posttranslational Regulation of DNA Polymerase η, a Connection to Damage-Induced Cohesion in Saccharomyces cerevisiae.

Pei-Shang Wu, Elin Enervald, Angelica Joelsson, Carina Palmberg, Dorothea Rutishauser, B Martin Hällberg, Lena Ström.

  Double-strand breaks that are induced post-replication trigger establishment of damage-induced cohesion in Saccharomyces cerevisiae, locally at the break-site and genome wide on undamaged chromosomes. The translesion synthesis polymerase, polymerase η, is required for generation of damage-induced cohesion genome wide. However, its precise role and regulation in this process is unclear. Here, we investigated the possibility that the cyclin dependent kinase Cdc28 and the acetyltransferase Eco1 modulate polymerase η activity. Through in vitro phosphorylation and structure modeling, we showed that polymerase η is an attractive substrate for Cdc28. Mutation of the putative Cdc28-phosphorylation site Ser14 to Ala, not only affected polymerase η protein level, but also prevented generation of damage-induced cohesion in vivo We also demonstrated that Eco1 acetylated polymerase η in vitro Certain non-acetylatable polymerase η mutants showed reduced protein level, deficient nuclear accumulation and increased ultraviolet irradiation sensitivity. In addition, we found that both Eco1 and subunits of the cohesin network are required for cell survival after ultraviolet irradiation. Our findings support functionally important Cdc28-mediated phosphorylation, as well as posttranslational modifications of multiple lysine residues that modulate polymerase η activity, and provide new insights into understanding the regulation of polymerase η for damage-induced cohesion.

Keywords:  Cdc28/Cdk1; Eco1; cohesin; damage-induced cohesion; polymerase eta

DOI:  https://doi.org/10.1534/genetics.120.303494
DNA Repair (Amst). 2020 Oct 01. pii: S1568-7864(20)30238-X. [Epub ahead of print]96 102985

The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion.

Katie M Feher, Alexander Kolbanovskiy, Alexander Durandin, Yoonjung Shim, Jung-Hyun Min, Yuan Cho Lee, Vladimir Shafirovich, Hong Mu, Suse Broyde, Nicholas E Geacintov.

  The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were -2.5-3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 β-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.

Keywords:  Base sequence effect; DNA lesion; NER-resistance; Nucleotide deletion; Nucleotide excision repair; XPC-Rad23B

DOI:  https://doi.org/10.1016/j.dnarep.2020.102985
Cancers (Basel). 2020 Oct 02. pii: E2848. [Epub ahead of print]12(10):

Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass.

Nicole A Wilkinson, Katherine S Mnuskin, Nicholas W Ashton, Roger Woodgate.

  Many endogenous and exogenous factors can induce genomic instability in human cells, in the form of DNA damage and mutations, that predispose them to cancer development. Normal cells rely on DNA damage bypass pathways such as translesion synthesis (TLS) and template switching (TS) to replicate past lesions that might otherwise result in prolonged replication stress and lethal double-strand breaks (DSBs). However, due to the lower fidelity of the specialized polymerases involved in TLS, the activation and suppression of these pathways must be tightly regulated by post-translational modifications such as ubiquitination in order to limit the risk of mutagenesis. Many cancer cells rely on the deregulation of DNA damage bypass to promote carcinogenesis and tumor formation, often giving them heightened resistance to DNA damage from chemotherapeutic agents. In this review, we discuss the key functions of ubiquitin and ubiquitin-like proteins in regulating DNA damage bypass in human cells, and highlight ways in which these processes are both deregulated in cancer progression and might be targeted in cancer therapy.

Keywords:  DNA damage bypass; DNA damage tolerance; ISGylation; NEDDylation; SUMOylation; carcinogenesis; mutagenesis; template switching; translesion synthesis; ubiquitination

DOI:  https://doi.org/10.3390/cancers12102848
Gynecol Oncol. 2020 Oct 05. pii: S0090-8258(20)33950-0. [Epub ahead of print]

Inhibition of poly(ADP-ribose) polymerase induces synthetic lethality in BRIP1 deficient ovarian epithelial cells.

Marcia A Ciccone, Crystal L Adams, Charles Bowen, Teena Thakur, Charité Ricker, Julie O Culver, Asaf Maoz, Marilena Melas, Gregory E Idos, Anand D Jeyasekharan, Koji Matsuo, Lynda D Roman, Stephen B Gruber, Kevin J McDonnell.

  OBJECTIVE: Pathogenic variations in the homologous recombination (HR) gene, BRCA1 interacting protein C-terminal helicase 1 (BRIP1) increase the risk for ovarian cancer. PARP inhibitors (PARPi) exert a synthetic lethal effect in BRCA-mutated ovarian cancers. Effective HR requires cooperation between BRCA1 and BRIP1; therefore, BRIP1-incompetancy may predict vulnerability to synthetic lethality. Here we investigated the response of ovarian epithelial cells with defective BRIP1 function to PARPi, and compared these cells to those lacking BRCA1 activity.METHODS: We engineered Chinese Hamster ovarian (CHO) epithelial cells to express deficient BRIP1 or BRCA1, and exposed them to olaparib with or without carboplatin or cisplatin. We assessed cellular proliferation and survival; we calculated inhibitory concentrations and combination and reduction drug indices.
RESULTS: BRIP1 and BRCA1 inactivation impedes HR activity, decreases cellular proliferation and compromises DNA damage recovery. Platinum agent exposure impairs cellular survival. Olaparib exposure alone decreases cell viability in BRCA1-deficient cells, although has no effect on BRIP1-deficient cells. Combining carboplatin or cisplatin with olaparib synergistically attenuates cellular survival, consistent with synthetic lethality.
CONCLUSIONS: BRIP1-deficient ovarian epithelial cells exhibit defective HR, resulting in synthetic lethality when exposed to a platinum agent/PARPi combination. PARPi alone had no effect; this lack of effect may result from distinguishing molecular properties of BRIP1and/or consequences of genomic background. Our study identifies altered BRIP1 as a target for precision medicine-based therapies for ovarian cancers. This investigation supports consideration of the use of a platinum agent/PARPi combination in ovarian cancers depending upon genetic profile and genomic background.

Keywords:  BRIP1; Genetic predisposition to cancer; Ovarian cancer; PARP inhibitor; Platinum therapeutics

DOI:  https://doi.org/10.1016/j.ygyno.2020.09.040
Nucleic Acids Res. 2020 Oct 03. pii: gkaa777. [Epub ahead of print]

The involvement of nucleotide excision repair proteins in the removal of oxidative DNA damage.

Namrata Kumar, Sripriya Raja, Bennett Van Houten.

The six major mammalian DNA repair pathways were discovered as independent processes, each dedicated to remove specific types of lesions, but the past two decades have brought into focus the significant interplay between these pathways. In particular, several studies have demonstrated that certain proteins of the nucleotide excision repair (NER) and base excision repair (BER) pathways work in a cooperative manner in the removal of oxidative lesions. This review focuses on recent data showing how the NER proteins, XPA, XPC, XPG, CSA, CSB and UV-DDB, work to stimulate known glycosylases involved in the removal of certain forms of base damage resulting from oxidative processes, and also discusses how some oxidative lesions are probably directly repaired through NER. Finally, since many glycosylases are inhibited from working on damage in the context of chromatin, we detail how we believe UV-DDB may be the first responder in altering the structure of damage containing-nucleosomes, allowing access to BER enzymes.

DOI: https://doi.org/10.1093/nar/gkaa777
Trends Cell Biol. 2020 Oct 06. pii: S0962-8924(20)30171-9. [Epub ahead of print]

When Endoplasmic Reticulum Proteostasis Meets the DNA Damage Response.

Matías González-Quiroz, Alice Blondel, Alfredo Sagredo, Claudio Hetz, Eric Chevet, Rémy Pedeux.

  Sustaining both proteome and genome integrity (GI) requires the integration of a wide range of mechanisms and signaling pathways. These comprise, in particular, the unfolded protein response (UPR) and the DNA damage response (DDR). These adaptive mechanisms take place respectively in the endoplasmic reticulum (ER) and in the nucleus. UPR and DDR alterations are associated with aging and with pathologies such as degenerative diseases, metabolic and inflammatory disorders, and cancer. We discuss the emerging signaling crosstalk between UPR stress sensors and the DDR, as well as their involvement in cancer biology.

Keywords:  ATM; DNA damage response; IRE1α; PERK; proteostasis; unfolded protein response

DOI:  https://doi.org/10.1016/j.tcb.2020.09.002
Cell Rep. 2020 Oct 06. pii: S2211-1247(20)31220-1. [Epub ahead of print]33(1): 108231

Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer.

Kate E R Hollinshead, Seth J Parker, Vinay V Eapen, Joel Encarnacion-Rosado, Albert Sohn, Tugba Oncu, Michael Cammer, Joseph D Mancias, Alec C Kimmelman.

  Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive fibrosis and hypovascularization, resulting in significant intratumoral hypoxia (low oxygen) that contributes to its aggressiveness, therapeutic resistance, and high mortality. Despite oxygen being a fundamental requirement for many cellular and metabolic processes, and the severity of hypoxia in PDAC, the impact of oxygen deprivation on PDAC biology is poorly understood. Investigating how PDAC cells survive in the near absence of oxygen, we find that PDAC cell lines grow robustly in oxygen tensions down to 0.1%, maintaining mitochondrial morphology, membrane potential, and the oxidative metabolic activity required for the synthesis of key metabolites for proliferation. Disrupting electron transfer efficiency by targeting mitochondrial respiratory supercomplex assembly specifically affects hypoxic PDAC proliferation, metabolism, and in vivo tumor growth. Collectively, our results identify a mechanism that enables PDAC cells to thrive in severe, oxygen-limited microenvironments.

Keywords:  COX7A2L; aspartate; electron transport chain; hypoxia; pancreatic cancer; respiration; supercomplexes

DOI:  https://doi.org/10.1016/j.celrep.2020.108231
DNA Repair (Amst). 2020 Sep 28. pii: S1568-7864(20)30224-X. [Epub ahead of print]96 102975

Alteration of genetic recombination and double-strand break repair in human cells by progerin expression.

Celina J Komari, Anne O Guttman, Shelby R Carr, Taylor L Trachtenberg, Elise A Orloff, Ashley V Haas, Andrew R Patrick, Sona Chowdhary, Barbara C Waldman, Alan S Waldman.

  Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare autosomal, dominant genetic condition characterized by many features of accelerated aging. On average, children with HGPS live to about fourteen years of age. The syndrome is commonly caused by a point mutation in the LMNA gene which normally codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The LMNA mutation alters splicing, leading to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at very low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), suggesting corruption of DNA repair. In this work, we investigated the influence of progerin expression on DSB repair in the human genome at the nucleotide level. We used a model system that involves a reporter DNA substrate inserted in the genome of cultured human cells. A DSB could be induced within the substrate through exogenous expression of endonuclease I-SceI, and DSB repair events occurring via either homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recoverable. Additionally, spontaneous HR events were recoverable in the absence of artificial DSB induction. We compared DSB repair and spontaneous HR in cells overexpressing progerin versus cells expressing no progerin. We report that overexpression of progerin correlated with an increase in DSB repair via NHEJ relative to HR, as well as an increased fraction of HR events occurring via gene conversion. Progerin also engendered an apparent increase in spontaneous HR events, with a highly significant shift toward gene conversion events, and an increase in DNA amplification events. Such influences of progerin on DNA transactions may impact genome stability and contribute to aging.

Keywords:  Double-strand break repair; Genomic instability; Homologous recombination; Nonhomologous end-joining; Progerin

DOI:  https://doi.org/10.1016/j.dnarep.2020.102975
Cancers (Basel). 2020 Oct 01. pii: E2840. [Epub ahead of print]12(10):

Mahogunin Ring Finger 1 Is Required for Genomic Stability and Modulates the Malignant Phenotype of Melanoma Cells.

Idoya Martínez-Vicente, Marta Abrisqueta, Cecilia Herraiz, Julia Sirés-Campos, María Castejón-Griñán, Dorothy C Bennett, Conchi Olivares, Jose Carlos García-Borrón, Celia Jiménez-Cervantes.

  The mouse mahoganoid mutation abrogating Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase expression causes hyperpigmentation, congenital heart defects and neurodegeneration. To study the pathophysiology of MGRN1 loss, we compared Mgrn1-knockout melanocytes with genetically matched controls and melan-md1 (mahoganoid) melanocytes. MGRN1 knockout induced a more differentiated and adherent phenotype, decreased motility, increased the percentage of cells in the S phase of the cell cycle and promoted genomic instability, as shown by stronger γH2AX labelling, increased burden of DNA breaks and higher abundance of aneuploid cells. Lack of MGRN1 expression decreased the ability of melanocytes to cope with DNA breaks generated by oxidizing agents or hydroxyurea-induced replicative stress, suggesting a contribution of genomic instability to the mahoganoid phenotype. MGRN1 knockout in B16-F10 melanoma cells also augmented pigmentation, increased cell adhesion to collagen, impaired 2D and 3D motility and caused genomic instability. Tumors formed by Mgrn1-KO B16-F10 cells had lower mitotic indices, fewer Ki67-positive cells and showed a trend towards smaller size. In short-term lung colonization assays Mgrn1-KO cells showed impaired colonization potential. Moreover, lower expression of MGRN1 is significantly associated with better survival of human melanoma patients. Therefore, MGRN1 might be an important phenotypic determinant of melanoma cells.

Keywords:  DNA damage; Mahogunin Ring Finger 1; cell cycle; genomic stability; melanocytes; melanoma

DOI:  https://doi.org/10.3390/cancers12102840
Proc Natl Acad Sci U S A. 2020 Oct 05. pii: 202000761. [Epub ahead of print]

Phase separation by ssDNA binding protein controlled via protein-protein and protein-DNA interactions.

Gábor M Harami, Zoltán J Kovács, Rita Pancsa, János Pálinkás, Veronika Baráth, Krisztián Tárnok, András Málnási-Csizmadia, Mihály Kovács.

  Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid-liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.

Keywords:  DNA repair; SSB; liquid−liquid phase separation; membraneless organelle; phase transition

DOI:  https://doi.org/10.1073/pnas.2000761117
Front Cell Dev Biol. 2020 ;8 564601

PARP Inhibitors: Clinical Relevance, Mechanisms of Action and Tumor Resistance.

Maddison Rose, Joshua T Burgess, Kenneth O'Byrne, Derek J Richard, Emma Bolderson.

  The Poly (ADP-ribose) polymerase (PARP) family has many essential functions in cellular processes, including the regulation of transcription, apoptosis and the DNA damage response. PARP1 possesses Poly (ADP-ribose) activity and when activated by DNA damage, adds branched PAR chains to facilitate the recruitment of other repair proteins to promote the repair of DNA single-strand breaks. PARP inhibitors (PARPi) were the first approved cancer drugs that specifically targeted the DNA damage response in BRCA1/2 mutated breast and ovarian cancers. Since then, there has been significant advances in our understanding of the mechanisms behind sensitization of tumors to PARP inhibitors and expansion of the use of PARPi to treat several other cancer types. Here, we review the recent advances in the proposed mechanisms of action of PARPi, biomarkers of the tumor response to PARPi, clinical advances in PARPi therapy, including the potential of combination therapies and mechanisms of tumor resistance.

Keywords:  BRCA; DNA damage; DNA repair; PARP inhibitors; cancer; targeted therapy

DOI:  https://doi.org/10.3389/fcell.2020.564601
J Biochem. 2020 Oct 09. pii: mvaa113. [Epub ahead of print]

The roles of nuclear myosin in the DNA damage response.

Alexander W Cook, Christopher P Toseland.

  Myosin within the nucleus has often been overlooked due to their importance in cytoplasmic processes and a lack of investigation. However, more recently it has been shown that their nuclear roles are just as fundamental to cell function and survival with roles in transcription, DNA damage and viral replication. Myosins can act as molecular transporters and anchors that rely on their actin binding and ATPase capabilities. Their roles within the DNA damage response can varies from a transcriptional response, moving chromatin and stabilising chromosome contacts. This review aims to highlight their key roles in the DNA damage response and how they impact nuclear organisation and transcription.

Keywords:  Actin; DNA; Damage; Myosin; Nucleus

DOI:  https://doi.org/10.1093/jb/mvaa113
Nat Commun. 2020 Oct 09. 11(1): 5117

The ALPK1/TIFA/NF-κB axis links a bacterial carcinogen to R-loop-induced replication stress.

Michael Bauer, Zuzana Nascakova, Anca-Irina Mihai, Phil F Cheng, Mitchell P Levesque, Simon Lampart, Robert Hurwitz, Lennart Pfannkuch, Jana Dobrovolna, Melanie Jacobs, Sina Bartfeld, Anders Dohlman, Xiling Shen, Alevtina A Gall, Nina R Salama, Antonia Töpfer, Achim Weber, Thomas F Meyer, Pavel Janscak, Anne Müller.

Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate β-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of β-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.

DOI: https://doi.org/10.1038/s41467-020-18857-z
NAR Cancer. 2020 Sep;2(3): zcaa024

Regulation and pharmacological targeting of RAD51 in cancer.

McKenzie K Grundy, Ronald J Buckanovich, Kara A Bernstein.

Regulation of homologous recombination (HR) is central for cancer prevention. However, too little HR can increase cancer incidence, whereas too much HR can drive cancer resistance to therapy. Importantly, therapeutics targeting HR deficiency have demonstrated a profound efficacy in the clinic improving patient outcomes, particularly for breast and ovarian cancer. RAD51 is central to DNA damage repair in the HR pathway. As such, understanding the function and regulation of RAD51 is essential for cancer biology. This review will focus on the role of RAD51 in cancer and beyond and how modulation of its function can be exploited as a cancer therapeutic.

DOI: https://doi.org/10.1093/narcan/zcaa024
Microb Cell. 2020 Jul 20. 7(10): 270-285

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast.

Alissa D Clear, Glenn M Manthey, Olivia Lewis, Isabelle Y Lopez, Rossana Rico, Shannon Owens, M Cristina Negritto, Elise W Wolf, Jason Xu, Nikola Kenjić, J Jefferson P Perry, Aaron W Adamson, Susan L Neuhausen, Adam M Bailis.

  RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers.

Keywords:  DNA double strand breaks; HsRAD52 variants; budding yeast; homologous recombination repair; ionizing radiation; tumorigenesis

DOI:  https://doi.org/10.15698/mic2020.10.732
Cancer Chemother Pharmacol. 2020 Oct 08.

Targeting MALAT1 induces DNA damage and sensitize non-small cell lung cancer cells to cisplatin by repressing BRCA1.

Jinghua Huang, Changxiu Lin, Hai Dong, Zhengri Piao, Chunhua Jin, Hengmin Han, Dongchun Jin.

  PURPOSE: Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA which has been identified to be involved in alternative non-homologous end joining (A-NHEJ) pathways by binding with PARP1 and LIG3 in myeloma cells. This study aims to explore the roles of MALAT1 in DNA repair processes in non-small cell lung cancer (NSCLC).METHODS: The interactions between MALAT1 and proteins were identified by co-immunoprecipitation and RNA pulldown. The interactions between MALAT1 and microRNAs (miRNA) were predicted by bioinformatics tools and confirmed by luciferase assay and RNA pulldown. The DNA damages were quantified by comet assay. The cell viability was examined by MTT assay and the cell apoptosis was determined by flow cytometry.
RESULTS: MALAT1 is identified to be involved in A-NHEJ pathway in NSCLC cells. However, in LIG3-null cells where A-NHEJ pathway is inactivated, targeting MALAT1 still increases DNA damages, suggesting that MALAT1 participates in other DNA repair pathways. Subsequently, MALAT1 is identified to bind with miR-146a and miR-216b, which directly target the 3'UTR of BRCA1. MALAT1 is confirmed to functions as a competing endogenous RNA (ceRNA) absorbing miR-146a and miR-216b, upregulating BRCA1 expression and protecting Homologous Recombination (HR) pathway in NSCLC cells. Finally, overexpression MALAT1 protects NSCLC cells from the cytotoxic effect of cisplatin. While, targeting MALAT1 in NSCLC cells induces DNA damages by repressing HR pathway and sensitizes NSCLC cells to cisplatin which had the potential for NSCLC treatment.
CONCLUSION: MALAT1 is involved in HR pathway by protecting BRCA1 and targeting MALAT1 induces DNA damages in NSCLC.

Keywords:  DNA repair; Long non-coding RNA; MALAT1; Non-small cell lung cancer; microRNA

DOI:  https://doi.org/10.1007/s00280-020-04152-7
Biol Proced Online. 2020 ;22 23

Molecular contribution of BRCA1 and BRCA2 to genome instability in breast cancer patients: review of radiosensitivity assays.

Fatemeh Sadeghi, Marzieh Asgari, Mojdeh Matloubi, Maral Ranjbar, Nahid Karkhaneh Yousefi, Tahereh Azari, Majid Zaki-Dizaji.

  Background: DNA repair pathways, cell cycle arrest checkpoints, and cell death induction are present in cells to process DNA damage and prevent genomic instability caused by various extrinsic and intrinsic ionizing factors. Mutations in the genes involved in these pathways enhances the ionizing radiation sensitivity, reduces the individual's capacity to repair DNA damages, and subsequently increases susceptibility to tumorigenesis.Body: BRCA1 and BRCA2 are two highly penetrant genes involved in the inherited breast cancer and contribute to different DNA damage pathways and cell cycle and apoptosis cascades. Mutations in these genes have been associated with hypersensitivity and genetic instability as well as manifesting severe radiotherapy complications in breast cancer patients. The genomic instability and DNA repair capacity of breast cancer patients with BRCA1/2 mutations have been analyzed in different studies using a variety of assays, including micronucleus assay, comet assay, chromosomal assay, colony-forming assay, γ -H2AX and 53BP1 biomarkers, and fluorescence in situ hybridization. The majority of studies confirmed the enhanced spontaneous & radiation-induced radiosensitivity of breast cancer patients compared to healthy controls. Using G2 micronucleus assay and G2 chromosomal assay, most studies have reported the lymphocyte of healthy carriers with BRCA1 mutation are hypersensitive to invitro ionizing radiation compared to non-carriers without a history of breast cancer. However, it seems this approach is not likely to be useful to distinguish the BRCA carriers from non-carrier with familial history of breast cancer.
Conclusion: In overall, breast cancer patients are more radiosensitive compared to healthy control; however, inconsistent results exist about the ability of current radiosensitive techniques in screening BRCA1/2 carriers or those susceptible to radiotherapy complications. Therefore, developing further radiosensitivity assay is still warranted to evaluate the DNA repair capacity of individuals with BRCA1/2 mutations and serve as a predictive factor for increased risk of cancer mainly in the relatives of breast cancer patients. Moreover, it can provide more evidence about who is susceptible to manifest severe complication after radiotherapy.

Keywords:  BRCA1; BRCA2; DNA repair pathway; apoptosis; breast cancer; cell cycle; genome stability; homologous recombination; non-homologous end joining; radiosensitivity

DOI:  https://doi.org/10.1186/s12575-020-00133-5
J Oncol. 2020 ;2020 2752417

CEP131 Abrogates CHK1 Inhibitor-Induced Replication Defects and Is Associated with Unfavorable Outcome in Neuroblastoma.

Kiyohiro Ando, Verna Cázares-Ordoñez, Makoto Makishima, Atsushi Yokoyama, Yusuke Suenaga, Hiroki Nagase, Shinichi Kobayashi, Takehiko Kamijo, Tsugumichi Koshinaga, Satoshi Wada.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. Recently, we reported that CHK1i, PF-477736, induced a p53-mediated DNA damage response. As a result, the cancer cells were able to repair DNA damage and became less sensitive to CHK1i. In this study, we discovered that PF-477736 increased expression of MDM2 oncogene along with CHK1i-induced replication defects in neuroblastoma NB-39-nu cells. A mass spectrometry analysis of protein binding to MDM2 in the presence of CHK1i identified the centrosome-associated family protein 131 (CEP131), which was correlated with unfavorable prognosis of neuroblastoma patients. We revealed that MDM2 was associated with CEP131 protein degradation, whereas overexpression of CEP131 accelerated neuroblastoma cell growth and exhibited resistance to CHK1i-induced replication defects. Thus, these findings may provide a future therapeutic strategy against centrosome-associated oncogenes involving CEP131 as a target in neuroblastoma.

DOI: https://doi.org/10.1155/2020/2752417
Int J Mol Sci. 2020 Oct 04. pii: E7333. [Epub ahead of print]21(19):

Pharmacological Inhibition of WEE1 Potentiates the Antitumoral Effect of the dl922-947 Oncolytic Virus in Malignant Mesothelioma Cell Lines.

Carmelina Antonella Iannuzzi, Paola Indovina, Iris Maria Forte, Sarah Di Somma, Anna Maria Malfitano, Martina Bruno, Giuseppe Portella, Francesca Pentimalli, Antonio Giordano.

  Malignant mesothelioma (MM) is a very aggressive asbestos-related cancer, for which no therapy proves to be effective. We have recently shown that the oncolytic adenovirus dl922-947 had antitumor effects in MM cell lines and murine xenografts. Previous studies demonstrated that dl922-947-induced host cell cycle checkpoint deregulation and consequent DNA lesions associated with the virus efficacy. However, the cellular DNA damage response (DDR) can counteract this virus action. Therefore, we assessed whether AZD1775, an inhibitor of the G2/M DNA damage checkpoint kinase WEE1, could enhance MM cell sensitivity to dl922-947. Through cell viability assays, we found that AZD1775 synergized with dl922-947 selectively in MM cell lines and increased dl922-947-induced cell death, which showed hallmarks of apoptosis (annexinV-positivity, caspase-dependency, BCL-XL decrease, chromatin condensation). Predictably, dl922-947 and/or AZD1775 activated the DDR, as indicated by increased levels of three main DDR players: phosphorylated histone H2AX (γ-H2AX), phospho-replication protein A (RPA)32, phospho-checkpoint kinase 1 (CHK1). Dl922-947 also increased inactive Tyr-15-phosphorylated cyclin-dependent kinase 1 (CDK1), a key WEE1 substrate, which is indicative of G2/M checkpoint activation. This increase in phospho-CDK1 was effectively suppressed by AZD1775, thus suggesting that this compound could, indeed, abrogate the dl922-947-induced DNA damage checkpoint in MM cells. Overall, our data suggest that the dl922-947-AZD1775 combination could be a feasible strategy against MM.

Keywords:  AZD1775; DNA damage response; G2/M checkpoint; MK-1775; WEE1; adavosertib; apoptosis; dl922-947; malignant mesothelioma; oncolytic adenovirus

DOI:  https://doi.org/10.3390/ijms21197333
Purinergic Signal. 2020 Oct 06.

Fibroblast growth factor 2 upregulates ecto-5'-nucleotidase and adenosine deaminase via MAPK pathways in cultured rat spinal cord astrocytes.

Ryota Eguchi, Taisuke Kitano, Ken-Ichi Otsuguro.

  Adenosine triphosphate (ATP) and adenosine are neurotransmitters and neuromodulators in the central nervous system. Astrocytes regulate extracellular concentration of purines via ATP release and its metabolisms via ecto-enzymes. The expression and activity of purine metabolic enzymes in astrocytes are increased under pathological conditions. We previously showed that fibroblast growth factor 2 (FGF2) upregulates the expression and activity of the enzymes ecto-5'-nucleotidase (CD73) and adenosine deaminase (ADA). Here, we further demonstrate that this occurs in concentration- and time-dependent manners in cultured rat spinal cord astrocytes and is suppressed by inhibitors of the FGF receptor as well as the mitogen-activated protein kinases (MAPKs). We also found that FGF2 increased the phosphorylation of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK, leading to the increased expression and activity of CD73 and ADA. Our findings reveal the involvement of FGF2/MAPK pathways in the regulation of purine metabolic enzymes in astrocytes. These pathways may contribute to the control of extracellular purine concentrations under physiological and pathological conditions.

Keywords:  ATP; Adenosine; Adenosine deaminase; Astrocytes; Ecto-5′-nucleotidase; FGF2

DOI:  https://doi.org/10.1007/s11302-020-09731-0
Signal Transduct Target Ther. 2020 Oct 07. 5(1): 227

NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential.

Na Xie, Lu Zhang, Wei Gao, Canhua Huang, Peter Ernst Huber, Xiaobo Zhou, Changlong Li, Guobo Shen, Bingwen Zou.

Nicotinamide adenine dinucleotide (NAD+) and its metabolites function as critical regulators to maintain physiologic processes, enabling the plastic cells to adapt to environmental changes including nutrient perturbation, genotoxic factors, circadian disorder, infection, inflammation and xenobiotics. These effects are mainly achieved by the driving effect of NAD+ on metabolic pathways as enzyme cofactors transferring hydrogen in oxidation-reduction reactions. Besides, multiple NAD+-dependent enzymes are involved in physiology either by post-synthesis chemical modification of DNA, RNA and proteins, or releasing second messenger cyclic ADP-ribose (cADPR) and NAADP+. Prolonged disequilibrium of NAD+ metabolism disturbs the physiological functions, resulting in diseases including metabolic diseases, cancer, aging and neurodegeneration disorder. In this review, we summarize recent advances in our understanding of the molecular mechanisms of NAD+-regulated physiological responses to stresses, the contribution of NAD+ deficiency to various diseases via manipulating cellular communication networks and the potential new avenues for therapeutic intervention.

DOI: https://doi.org/10.1038/s41392-020-00311-7
Biochem Pharmacol. 2020 Oct 01. pii: S0006-2952(20)30489-5. [Epub ahead of print] 114253

Targeting mitochondrial respiration for the treatment of acute myeloid leukemia.

Jenna L Carter, Katie Hege, Hasini A Kalpage, Holly Edwards, Maik Hüttemann, Jeffrey W Taub, Yubin Ge.

  Acute myeloid leukemia (AML) is a heterogeneous disease with variable presentation, molecular phenotype, and cytogenetic abnormalities and has seen very little improvement in patient survival over the last few decades. This heterogeneity supports poor prognosis partially through the variability in response to the standard chemotherapy. Further understanding of molecular heterogeneity has promoted the development of novel treatments, some of which target mitochondrial metabolism and function. This review discusses the relative dependency that AML cells have on mitochondrial function, and the ability to pivot this reliance to target important subsets of AML cells, including leukemia stem cells (LSCs). LSCs are tumor-initiating cells that are resistant to standard chemotherapy and promote the persistence and relapse of AML. Historically, LSCs have been targeted based on immunophenotype, but recent developments in the understanding of LSC metabolism has demonstrated unique abilities to target LSCs while sparing normal hematopoietic stem cells (HSCs) through inhibition of mitochondrial function. Here we highlight the use of small molecules that have been demonstrated to effectively target mitochondrial function. IACS-010759 and ME-344 target the electron transport chain (ETC) to inhibit oxidative phosphorylation (OXPHOS). The imipridone family (ONC201, ONC206, ONC212) of inhibitors target mitochondria through activation of ClpP mitochondrial protease and reduce function of essential pathways. These molecules offer a new mechanism for developing clinical therapies in AML and support novel strategies to target LSCs in parallel with conventional therapies.

Keywords:  Acute myeloid leukemia; IACS-010759; ME-344; Mitochondria; ONC201; Oxidative phosphorylation

DOI:  https://doi.org/10.1016/j.bcp.2020.114253
Front Pharmacol. 2020 ;11 1294

Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors.

Laura Schäkel, Constanze C Schmies, Riham M Idris, Xihuan Luo, Sang-Yong Lee, Vittoria Lopez, Salahuddin Mirza, The Hung Vu, Julie Pelletier, Jean Sévigny, Vigneshwaran Namasivayam, Christa E Müller.

  Nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) inhibitors have potential as novel drugs for the (immuno)therapy of cancer. They increase the extracellular concentration of immunostimulatory ATP and reduce the formation of AMP, which can be further hydrolyzed by ecto-5'-nucleotidase (CD73) to immunosuppressive, cancer-promoting adenosine. In the present study, we synthesized analogs and derivatives of the standard CD39 inhibitor ARL67156, a nucleotide analog which displays a competitive mechanism of inhibition. Structure-activity relationships were analyzed at the human enzyme with respect to substituents in the N 6- and C8-position of the adenine core, and modifications of the triphosph(on)ate chain. Capillary electrophoresis coupled to laser-induced fluorescence detection employing a fluorescent-labeled ATP derivative was employed to determine the compounds' potency. Selected inhibitors were additionally evaluated in an orthogonal, malachite green assay versus the natural substrate ATP. The most potent CD39 inhibitors of the present series were ARL67156 and its derivatives 31 and 33 with Ki values of around 1 µM. Selectivity studies showed that all three nucleotide analogs additionally blocked CD73 acting as dual-target inhibitors. Docking studies provided plausible binding modes to both targets. The present study provides a full characterization of the frequently applied CD39 inhibitor ARL67156, presents structure-activity relationships, and provides a basis for future optimization towards selective CD39 and dual CD39/CD73 inhibitors.

Keywords:  ARL67156; CD39; CD73; docking; dual-target inhibitors; ecto-5’-nucleotidase; nucleoside triphosphate diphosphohydrolase1 (NTPDase1); nucleotides

DOI:  https://doi.org/10.3389/fphar.2020.01294
Curr Genet. 2020 Oct 06.

Cac1 WHD and PIP domains have distinct roles in replisome progression and genomic stability.

Ioannis Tsirkas, Daniel Dovrat, Yang Lei, Angeliki Kalyva, Diana Lotysh, Qing Li, Amir Aharoni.

  Replication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells to maintain chromatin structure during DNA replication. The deposition of newly-synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. CAF-1 is an important histone chaperone complex and its main subunit, Cac1p, contains a PIP and WHD domain for interaction with PCNA and the DNA, respectively. While Cac1p subunit was extensively studied in different systems much less is known regarding the importance of the PIP and WHD domains in replication fork progression and genome stability. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in these Cac1p domains affect replication fork progression and post-replication characteristics. Our experiments revealed that mutations in the Cac1p WHD domain, which abolished the CAF-1-DNA interaction, slows down replication fork progression. In contrast, mutations in Cac1p PIP domain, abolishing Cac1p-PCNA interaction, lead to extended late-S/Anaphase duration, elevated number of RPA foci and increased spontaneous mutation rate. Our research shows that Cac1p WHD and PIP domains have distinct roles in high replisome progression and maintaining genome stability during cell cycle progression.

Keywords:  Cac1; DNA replication; Histone chaperones

DOI:  https://doi.org/10.1007/s00294-020-01113-8
Int J Pharm X. 2020 Dec;2 100056

Enhancing efficacy of gemcitabine in pancreatic patient-derived xenograft mouse models.

Andriana Inkoom, Nkafu Ndemazie, Kevin Affram, Taylor Smith, Xue Zhu, Patrick Underwood, Sunil Krishnan, Edward Ofori, Bo Han, Jose Trevino, Edward Agyare.

  Gemcitabine (Gem), a nucleoside analog, is a preferred choice of treatment for pancreatic cancer (PCa) and often used in combination therapy against wide range of solid tumors. It is known to be rapidly inactivated in blood by cytidine deaminase. The objective of the study was to improve the systemic stability and anticancer activity of modified Gem termed 4-N-stearoylGem (4NSG) In this study, the IC50 values of 4NSG treated MiaPaCa-2 and primary pancreatic cancer (PPCL-46) cultures were significantly lower when compared with gemcitabine hydrochloride (GemHCl) treated cultures. In acute toxicity study, liver enzyme level of aspartate aminotransferase (AST) of the control mice was not significantly different from AST levels of 4NSG and GemHCl treated mice. However, alanine aminotransferase (ALT) level of control mice (67 ± 5 mUnits/mL) was significantly lower compared with ALT levels of GemHCl (232 ± 28 mUnits/mL) and that of 4NSG (172 ± 22 mUnits/mL) (p < 0.0001). More importantly, ALT level of 4NSG was lower than ALT level of GemHCl (p < 0.05). Although ALT levels were elevated, pathological images of liver and kidney tissues of control, GemHCl and 4NSG treated mice revealed no architectural changes and no significant change in mice weight was observed during treatment. The bioavailability (AUC) of 4NSG was 3-fold high and significantly inhibited the tumor growth as compared with equivalent dose of GemHCl. Immunohistochemical staining revealed that 4NSG significantly inhibited the expression vascular endothelial growth factor (VEGF) receptor. The study is unique because it established, for the first time, enhanced anticancer activity of 4NSG against pancreatic patient-derived xenograft (PDX) mouse model and PPCL-46 cells compared with Gem. 4SGN enhanced pharmacokinetic profile and improved the therapeutic efficacy of the standard-of-care Gem. Lastly, 4GSN showed a remarkable tumor growth inhibition and revealed significant antiangiogenic activity in 4GSN treated pancreatic PDX tumor.

Keywords:  4-N-stearoylGem; Antitumor efficacy; Gemcitabine; PDX; Pancreatic cancer; Patient-derived xenograft

DOI:  https://doi.org/10.1016/j.ijpx.2020.100056
Bioorg Chem. 2020 Sep 24. pii: S0045-2068(20)31603-5. [Epub ahead of print]104 104305

Evaluation of sulfonate and sulfamate derivatives possessing benzofuran or benzothiophene nucleus as inhibitors of nucleotide pyrophosphatases/phosphodiesterases and anticancer agents.

Hanan S Anbar, Randa El-Gamal, Saif Ullah, Seyed-Omar Zaraei, Mariya Al-Rashida, Sumera Zaib, Julie Pelletier, Jean Sévigny, Jamshed Iqbal, Mohammed I El-Gamal.

  Ectonucleotidases are a broad family of ectoenzymes that play a crucial role in purinergic cell signaling. Ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) belong to this group and are important drug targets. In particular, NPP1 and NPP3 are known to be druggable targets for treatment of impaired calcification disorders (including pathological aortic calcification) and cancer, respectively. In this study, we investigated a series of sulfonate and sulfamate derivatives of benzofuran and benzothiophene as potent and selective inhibitors of NPP1 and NPP3. Compounds 1c, 1g, 1n, and 1s are the most active NPP1 inhibitors (IC50 values in the range 0.12-0.95 µM). Moreover, compounds 1e, 1f, 1j, and 1l are the most potent inhibitors of NPP3 (IC50 ranges from 0.12 to 0.95 µM). Compound 1d, 1f and 1t are highly selective inhibitors of NPP1 over NPP3, whereas compounds 1m and 1s are found to be highly selective towards NPP3 over NPP1. Structure-activity relationship (SAR) study has been discussed in detailed. With the aid of molecular docking studies, a common binding mode of these compounds and suramin (the standard inhibitor) was revealed, where the sulfonate group acts as a cation-binding moiety that comes in close contact with the zinc ion of the active site. Moreover, cytotoxic evaluation against MCF-7 and HT-29 cancer cell lines revealed that compound 1r is the most cytotoxic towards MCF-7 cell line with IC50 value of 0.19 µM. Compound 1r is more potent and selective against cancer cells than normal cells (WI-38) as compared to doxorubicin.

Keywords:  Antiproliferative activity; Benzofuran sulfonates and sulfamates; Benzothiophene sulfonates and sulfamates; Enzyme inhibition assay; Human ecto-nucleotide pyrophosphatases/phosphodiesterase-1 and -3; In-silico study

DOI:  https://doi.org/10.1016/j.bioorg.2020.104305