bims-numges 2021-04-25 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021‒04‒25
thirty-four papers selected by
Sean Rudd
Karolinska Institutet

DDX11 loss causes replication stress and pharmacologically exploitable DNA repair defects.
PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer.
FANCM regulates repair pathway choice at stalled replication forks.
Transcriptional silencing of ALDH2 confers a dependency on Fanconi anemia proteins in acute myeloid leukemia.
Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells.
Telomere Replication: Solving Multiple End Replication Problems.
Chemotherapy-induced CDA expression renders resistant non-small cell lung cancer cells sensitive to 5'-deoxy-5-fluorocytidine (5'-DFCR).
Interfaces between cellular responses to DNA damage and cancer immunotherapy.
ATR kinase activity promotes antibody class switch recombination in B cells through cell cycle regulation without suppressing DSB resection and microhomology usage.
Expanding roles of cell cycle checkpoint inhibitors in radiation oncology.
Drug and molecular radiotherapy combinations for metastatic castration resistant prostate cancer.
DNA damage response and repair in pancreatic cancer development and therapy.
The emerging relationship between metabolism and DNA repair.
Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer.
Genome instability and loss of protein homeostasis: converging paths to neurodegeneration?
Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.
Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance.
Preferential CEBP binding to T:G mismatches and increased C-to-T human somatic mutations.
Mechanisms of Vertebrate DNA Interstrand Cross-Link Repair.
Decreased expression of the translation factor eIF3e induces senescence in breast cancer cells via suppression of PARP1 and activation of mTORC1.
Co-regulation and function of FOXM1/RHNO1 bidirectional genes in cancer.
DNA repair and cholesterol-mediated drug efflux induce dose-dependent chemoresistance in nutrient-deprived neuroblastoma cells.
COMMD4 functions with the histone H2A-H2B dimer for the timely repair of DNA double-strand breaks.
CDK7 Inhibitor THZ1 Induces the Cell Apoptosis of B-Cell Acute Lymphocytic Leukemia by Perturbing Cellular Metabolism.
A novel and highly effective mitochondrial uncoupling drug in T-cell leukemia.
Ectonucleotidases in Inflammation, Immunity, and Cancer.
Olaparib-mediated enhancement of 5-fluorouracil cytotoxicity in mismatch repair deficient colorectal cancer cells.
Crystal structure and molecular dynamics of human POLDIP2, a multifaceted adaptor protein in metabolism and genome stability.
RRM2B Is Frequently Amplified Across Multiple Tumor Types: Implications for DNA Repair, Cellular Survival, and Cancer Therapy.
HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation.
Replication timing maintains the global epigenetic state in human cells.
STING enhances cell death through regulation of reactive oxygen species and DNA damage.
BRCA1 degradation in response to mitochondrial damage in breast cancer cells.
Purine synthesis as a target for radiation resistance in molecular glioblastoma.

Proc Natl Acad Sci U S A. 2021 Apr 27. pii: e2024258118. [Epub ahead of print]118(17):

DDX11 loss causes replication stress and pharmacologically exploitable DNA repair defects.

Nanda Kumar Jegadesan, Dana Branzei.

  DDX11 encodes an iron-sulfur cluster DNA helicase required for development, mutated, and overexpressed in cancers. Here, we show that loss of DDX11 causes replication stress and sensitizes cancer cells to DNA damaging agents, including poly ADP ribose polymerase (PARP) inhibitors and platinum drugs. We find that DDX11 helicase activity prevents chemotherapy drug hypersensitivity and accumulation of DNA damage. Mechanistically, DDX11 acts downstream of 53BP1 to mediate homology-directed repair and RAD51 focus formation in manners nonredundant with BRCA1 and BRCA2. As a result, DDX11 down-regulation aggravates the chemotherapeutic sensitivity of BRCA1/2-mutated cancers and resensitizes chemotherapy drug-resistant BRCA1/2-mutated cancer cells that regained homologous recombination proficiency. The results further indicate that DDX11 facilitates recombination repair by assisting double strand break resection and the loading of both RPA and RAD51 on single-stranded DNA substrates. We propose DDX11 as a potential target in cancers by creating pharmacologically exploitable DNA repair vulnerabilities.

Keywords:  BRCA1/2; DDX11; chemotherapy; homologous recombination; replication stress

DOI:  https://doi.org/10.1073/pnas.2024258118
Cancer Res. 2021 Apr 22.

PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer.

Roman M Chabanon, Daphné Morel, Thomas Eychenne, Léo Colmet-Daage, Ilirjana Bajrami, Nicolas Dorvault, Marlène Garrido, Cornelia Meisenberg, Andrew Lamb, Carine Ngo, Suzanna R Hopkins, Theodoros I Roumeliotis, Samuel Jouny, Clémence Hénon, Asuka Kawai-Kawachi, Clémence Astier, Asha Konde, Elaine Del Nery, Christophe Massard, Stephen J Pettitt, Raphaël Margueron, Jyoti S Choudhary, Geneviève Almouzni, Jean-Charles Soria, Eric Deutsch, Jessica A Downs, Christopher J Lord, Sophie Postel-Vinay.

Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. SIGNIFICANCE: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/00/0/000/F1.large.jpg.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-0628
Mol Cell. 2021 Apr 16. pii: S1097-2765(21)00265-3. [Epub ahead of print]

FANCM regulates repair pathway choice at stalled replication forks.

Arvind Panday, Nicholas A Willis, Rajula Elango, Francesca Menghi, Erin E Duffey, Edison T Liu, Ralph Scully.

  Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.

Keywords:  BRCA1; Bloom’s syndrome helicase; FANCM; Fanconi anemia; break-induced replication; genomic instability; homologous recombination; replication restart; synthetic lethality; tandem duplication

DOI:  https://doi.org/10.1016/j.molcel.2021.03.044
Cancer Discov. 2021 Apr 23. pii: candisc.1542.2020. [Epub ahead of print]

Transcriptional silencing of ALDH2 confers a dependency on Fanconi anemia proteins in acute myeloid leukemia.

Zhaolin Yang, Xiaoli S Wu, Yiliang Wei, Sofya A Polyanskaya, Shruti V Iyer, Moonjung Jung, Francis P Lach, Emmalee R Adelman, Olaf Klingbeil, Joseph P Milazzo, Melissa Kramer, Osama E Demerdash, Kenneth Chang, Sara Goodwin, Emily Hodges, W Richard McCombie, Maria E Figueroa, Agata Smogorzewska, Christopher R Vakoc.

Hundreds of genes become aberrantly silenced in acute myeloid leukemia (AML), with most of these epigenetic changes being of unknown functional consequence. Here, we demonstrate how gene silencing can lead to an acquired dependency on the DNA repair machinery in AML. We make this observation by profiling the essentiality of the ubiquitination machinery in cancer cell lines using domain-focused CRISPR screening, which revealed Fanconi anemia (FA) proteins UBE2T and FANCL as unique dependencies in AML. We demonstrate that these dependencies are due to a synthetic lethal interaction between FA proteins and Aldehyde Dehydrogenase 2 (ALDH2), which function in parallel pathways to counteract the genotoxicity of endogenous aldehydes. We show that DNA hypermethylation and silencing of ALDH2 occur in a recurrent manner in human AML, which is sufficient to confer FA pathway dependency. Our study suggests that targeting of the ubiquitination reaction catalyzed by FA proteins can eliminate ALDH2-deficient AML.

DOI: https://doi.org/10.1158/2159-8290.CD-20-1542
NAR Cancer. 2021 Jun;3(2): zcab014

Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells.

Megan R Reed, Leena Maddukuri, Amit Ketkar, Stephanie D Byrum, Maroof K Zafar, April C L Bostian, Alan J Tackett, Robert L Eoff.

Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced-indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation-a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.

DOI: https://doi.org/10.1093/narcan/zcab014
Front Cell Dev Biol. 2021 ;9 668171

Telomere Replication: Solving Multiple End Replication Problems.

Erin Bonnell, Emeline Pasquier, Raymund J Wellinger.

  Eukaryotic genomes are highly complex and divided into linear chromosomes that require end protection from unwarranted fusions, recombination, and degradation in order to maintain genomic stability. This is accomplished through the conserved specialized nucleoprotein structure of telomeres. Due to the repetitive nature of telomeric DNA, and the unusual terminal structure, namely a protruding single stranded 3' DNA end, completing telomeric DNA replication in a timely and efficient manner is a challenge. For example, the end replication problem causes a progressive shortening of telomeric DNA at each round of DNA replication, thus telomeres eventually lose their protective capacity. This phenomenon is counteracted by the recruitment and the activation at telomeres of the specialized reverse transcriptase telomerase. Despite the importance of telomerase in providing a mechanism for complete replication of telomeric ends, the majority of telomere replication is in fact carried out by the conventional DNA replication machinery. There is significant evidence demonstrating that progression of replication forks is hampered at chromosomal ends due to telomeric sequences prone to form secondary structures, tightly DNA-bound proteins, and the heterochromatic nature of telomeres. The telomeric loop (t-loop) formed by invasion of the 3'-end into telomeric duplex sequences may also impede the passage of replication fork. Replication fork stalling can lead to fork collapse and DNA breaks, a major cause of genomic instability triggered notably by unwanted repair events. Moreover, at chromosomal ends, unreplicated DNA distal to a stalled fork cannot be rescued by a fork coming from the opposite direction. This highlights the importance of the multiple mechanisms involved in overcoming fork progression obstacles at telomeres. Consequently, numerous factors participate in efficient telomeric DNA duplication by preventing replication fork stalling or promoting the restart of a stalled replication fork at telomeres. In this review, we will discuss difficulties associated with the passage of the replication fork through telomeres in both fission and budding yeasts as well as mammals, highlighting conserved mechanisms implicated in maintaining telomere integrity during replication, thus preserving a stable genome.

Keywords:  DNA replication; genome stability; replication fork stability; telomeres; telomeric chromatin

DOI:  https://doi.org/10.3389/fcell.2021.668171
J Exp Clin Cancer Res. 2021 Apr 19. 40(1): 138

Chemotherapy-induced CDA expression renders resistant non-small cell lung cancer cells sensitive to 5'-deoxy-5-fluorocytidine (5'-DFCR).

Yanyun Gao, Philipp Zens, Min Su, Camila Anna Gemperli, Haitang Yang, Haibin Deng, Zhang Yang, Duo Xu, Sean R R Hall, Sabina Berezowska, Patrick Dorn, Ren-Wang Peng, Ralph Alexander Schmid, Wenxiang Wang, Thomas Michael Marti.

  BACKGROUND: Pemetrexed (MTA) plus cisplatin combination therapy is considered the standard of care for patients with advanced non-small-cell lung cancer (NSCLC). However, in advanced NSCLC, the 5-year survival rate is below 10%, mainly due to resistance to therapy. We have previously shown that the fraction of mesenchymal-like, chemotherapy-resistant paraclone cells increased after MTA and cisplatin combination therapy in the NSCLC cell line A549. Cytidine deaminase (CDA) and thymidine phosphorylase (TYMP) are key enzymes of the pyrimidine salvage pathway. 5'-deoxy-5-fluorocytidine (5'-DFCR) is a cytidine analogue (metabolite of capecitabine), which is converted by CDA and subsequently by TYMP into 5-fluorouracil, a chemotherapeutic agent frequently used to treat solid tumors. The aim of this study was to identify and exploit chemotherapy-induced metabolic adaptations to target resistant cancer cells.METHODS: Cell viability and colony formation assays were used to quantify the efficacy of MTA and cisplatin treatment in combination with schedule-dependent addition of 5'-DFCR on growth and survival of A549 paraclone cells and NSCLC cell lines. CDA and TYMP protein expression were monitored by Western blot. Finally, flow cytometry was used to analyze the EMT phenotype, DNA damage response activation and cell cycle distribution over time after treatment. CDA expression was measured by immunohistochemistry in tumor tissues of patients before and after neoadjuvant chemotherapy.
RESULTS: We performed a small-scale screen of mitochondrial metabolism inhibitors, which revealed that 5'-DFCR selectively targets chemotherapy-resistant A549 paraclone cells characterized by high CDA and TYMP expression. In the cell line A549, CDA and TYMP expression was further increased by chemotherapy in a time-dependent manner, which was also observed in the KRAS-addicted NSCLC cell lines H358 and H411. The addition of 5'-DFCR on the second day after MTA and cisplatin combination therapy was the most efficient treatment to eradicate chemotherapy-resistant NSCLC cells. Moreover, recovery from treatment-induced DNA damage was delayed and accompanied by senescence induction and acquisition of a hybrid-EMT phenotype. In a subset of patient tumors, CDA expression was also increased after treatment with neoadjuvant chemotherapy.
CONCLUSIONS: Chemotherapy increases CDA and TYMP expression thereby rendering resistant lung cancer cells susceptible to subsequent 5'-DFCR treatment.

Keywords:  5′-DFCR; Chemotherapy resistant; Cisplatin; Cytidine deaminase (CDA); DNA damage; Non-small cell lung cancer; Pemetrexed; Thymidine phosphorylase (TYMP)

DOI:  https://doi.org/10.1186/s13046-021-01938-2
Genes Dev. 2021 Apr 22.

Interfaces between cellular responses to DNA damage and cancer immunotherapy.

Domenic Pilger, Leonard W Seymour, Stephen P Jackson.

  The DNA damage response (DDR) fulfils essential roles to preserve genome integrity. Targeting the DDR in tumors has had remarkable success over the last decade, exemplified by the licensing of PARP inhibitors for cancer therapy. Recent studies suggest that the application of DDR inhibitors impacts on cellular innate and adaptive immune responses, wherein key DNA repair factors have roles in limiting chronic inflammatory signaling. Antitumor immunity plays an emerging part in cancer therapy, and extensive efforts have led to the development of immune checkpoint inhibitors overcoming immune suppressive signals in tumors. Here, we review the current understanding of the molecular mechanisms underlying DNA damage-triggered immune responses, including cytosolic DNA sensing via the cGAS/STING pathway. We highlight the implications of DDR components for therapeutic outcomes of immune checkpoint inhibitors or their use as biomarkers. Finally, we discuss the rationale for novel combinations of DDR inhibitors with antagonists of immune checkpoints and current hindrances limiting their broader therapeutic applications.

Keywords:  DNA damage response; DNA repair; PARP inhibitors; PD-1; PD-L1; STING; cGAS; immunotherapy

DOI:  https://doi.org/10.1101/gad.348314.121
J Leukoc Biol. 2021 Apr 22.

ATR kinase activity promotes antibody class switch recombination in B cells through cell cycle regulation without suppressing DSB resection and microhomology usage.

Xikui Sun, Meiling Liu, Jingning Bai, Jiejie Xu, Chengming Zhu, Junchao Dong, Chun Chen.

  Class switch recombination (CSR) changes the effector functions of antibodies and is carried out by classical and alternative nonhomologous end joining (c-NHEJ and A-EJ) of repetitive switch (S) region double-strand breaks (DSBs). The master DNA damage response (DDR) kinase ataxia-telangiectasia mutated (ATM) is critical for CSR in part by suppressing S region DSB resection. However, whether another related DDR kinase ATM- and Rad3-related (ATR) plays similar role in CSR remains elusive. In this study, we investigated the requirement for ATR kinase activity on CSR in both c-NHEJ competent and deficient B cell lines with high-throughput sequencing of S-S junctions. We found that ATR kinase inhibition efficiently blocked both c-NHEJ- and A-EJ-mediated CSR without affecting germline transcription and activation-induced cytosine deaminase expression. In contrast to ATM, ATR does not suppress S region DSB resection and microhomology usage. In addition, ATR kinase inhibition did not affect Cas9-generated DSB end joining by either c-NHEJ and A-EJ. ATR kinase-inhibited stimulated B cells proliferate much slower than controls and exhibited altered cell cycle profile with increased G1 and G2/M phase cells. In summary, our data revealed a role for ATR in promoting both c-NHEJ- and A-EJ-mediated CSR through regulating cell proliferation upon damage without negatively influencing DSB end-joining features.

Keywords:  ATR; class switch recombination; microhomology; nonhomologous end joining

DOI:  https://doi.org/10.1002/JLB.2MA0321-064R
Int J Radiat Biol. 2021 Apr 20. 1-10

Expanding roles of cell cycle checkpoint inhibitors in radiation oncology.

Sissel Hauge, Adrian Eek Mariampillai, Gro Elise Rødland, Lilli T E Bay, Helga B Landsverk, Randi G Syljuåsen.

  PURPOSE: Radiation-induced activation of cell cycle checkpoints have been of long-standing interest. The WEE1, CHK1 and ATR kinases are key factors in cell cycle checkpoint regulation and are essential for the S and G2 checkpoints. Here, we review the rationale for why inhibitors of WEE1, CHK1 and ATR could be beneficial in combination with radiation.CONCLUSIONS: Combined treatment with radiation and inhibitors of these kinases results in checkpoint abrogation and subsequent mitotic catastrophe. This might selectively radiosensitize tumor cells, as they often lack the p53-dependent G1 checkpoint and therefore rely more on the G2 checkpoint to repair DNA damage. Further affecting the repair of radiation damage, inhibition of WEE1, CHK1 or ATR also specifically suppresses the homologous recombination repair pathway. Moreover, inhibition of these kinases can induce massive replication stress during S phase of the cell cycle, likely contributing to eliminate radioresistant S phase cells. Intriguingly, recent findings suggest that cell cycle checkpoint inhibitors in combination with radiation can also enhance anti-tumor immune effects. Altogether, the expanding knowledge about the functional roles of WEE1, CHK1 and ATR inhibitors support that they are promising candidates for use in combination with radiation treatment.

Keywords:  ATR inhibitor; CHK1 inhibitor; DNA damage; Radiation; WEE1 inhibitor; anti-tumor immune response; cell cycle checkpoints; repair

DOI:  https://doi.org/10.1080/09553002.2021.1913529
Nucl Med Biol. 2021 Apr 09. pii: S0969-8051(21)00052-4. [Epub ahead of print]96-97 101-111

Drug and molecular radiotherapy combinations for metastatic castration resistant prostate cancer.

Magdalena Staniszewska, Janette Iking, Katharina Lückerath, Boris Hadaschik, Ken Herrmann, Justin Ferdinandus, Wolfgang P Fendler.

  Metastatic castration resistant prostate cancer (mCRPC) is a highly lethal disease. Several novel therapies have been assessed in the past years. Targeting DNA damage response (DDR) pathways in prostate cancer became a promising treatment strategy and olaparib and rucaparib, Poly(ADP-ribose) polymerase (PARP) inhibitors, have been approved for patients carrying mutations in homologous recombination (HR) repair pathways. Other DDR inhibitor targets, such as ATM, ATR, CHK1, CHK2, and WEE1 are under extensive investigation. Additionally, molecular radiotherapy (MRT) including [177Lu]Lu-PSMA, [225Ac]Ac-PSMA, [223Ra]Ra-dichloride, [153Sm]-EDTMP, [188Re]Re-HDMP and GRPR-targeted MRT treat cancer through internal ionizing radiation causing DNA damage and demonstrate promising efficacy in clinical trials. In the field of immunotherapy, checkpoint inhibition as well as sipuleucel-T and PROSTVAC demonstrated only limited efficacy in mCRPC when used as monotherapy. This review discusses recent therapeutic strategies for mCRPC highlighting the need for rational combination of treatment options.

Keywords:  Combination therapy; DNA damage response pathways; Radionuclide therapy; mCRPC

DOI:  https://doi.org/10.1016/j.nucmedbio.2021.03.009
DNA Repair (Amst). 2021 Apr 15. pii: S1568-7864(21)00072-0. [Epub ahead of print]103 103116

DNA damage response and repair in pancreatic cancer development and therapy.

Parnia Rahnamay Farnood, Romina Danesh Pazhooh, Zatollah Asemi, Bahman Yousefi.

  Pancreatic cancer (PC) is among fatal malignancies, with a dismal prognosis and a low survival rate of 5-10%. In both sporadic and inherited PC, gene alterations, such as BRCA1/2, PALB2, and ATM, can occur frequently. Currently, surgery, chemo- and radio-therapy are the most common therapeutic strategies for treating this cancer. DNA damage response (DDR) establishes multiple pathways that eliminate DNA damage sites to maintain genomic integrity. Various types of cancers and age-related diseases are associated with DDR machinery defects. According to the severity of the damage, DDR pathways respond appropriately to lesions through repairing damage, arresting the cell cycle, or apoptosis. Recently, novel agents, particularly those targeting DDR pathways, are being utilized to improve the response of many cancers to chemotherapy and radiotherapy. In this paper, we briefly reviewed DDR processes and their components, including DDR sensors, DDR mediators, and DDR transducers in the progression, prognosis, and treatment of PC.

Keywords:  DNA damage response; DNA repair; Malignancy; Pancreatic cancer

DOI:  https://doi.org/10.1016/j.dnarep.2021.103116
Cell Cycle. 2021 Apr 20. 1-17

The emerging relationship between metabolism and DNA repair.

Danilo Cucchi, Amy Gibson, Sarah A Martin.

  The DNA damage response (DDR) consists of multiple specialized pathways that recognize different insults sustained by DNA and repairs them where possible to avoid the accumulation of mutations. While loss of activity of genes in the DDR has been extensively associated with cancer predisposition and progression, in recent years it has become evident that there is a relationship between the DDR and cellular metabolism. The activity of the metabolic pathways can influence the DDR by regulating the availability of substrates required for the repair process and the function of its players. Additionally, proteins of the DDR can regulate the metabolic flux through the major pathways such as glycolysis, tricarboxylic acid cycle (TCA) and pentose phosphate pathway (PPP) and the production of reactive oxygen species (ROS). This newly discovered connection bears great importance in the biology of cancer and represents a new therapeutic opportunity. Here we describe the nature of the relationship between DDR and metabolism and its potential application in the treatment of cancer. Keywords: DNA repair, metabolism, mitochondria.

Keywords:  DNA repair; metabolism; mitochondria

DOI:  https://doi.org/10.1080/15384101.2021.1912889
Gynecol Oncol. 2021 Apr 15. pii: S0090-8258(21)00325-5. [Epub ahead of print]

Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer.

Vijayalaxmi G Gupta, Jeff Hirst, Shariska Petersen, Katherine F Roby, Meghan Kusch, Helen Zhou, Makena L Clive, Andrea Jewell, Harsh B Pathak, Andrew K Godwin, Andrew J Wilson, Marta A Crispens, Emily Cybulla, Alessandro Vindigni, Katherine C Fuh, Dineo Khabele.

  OBJECTIVE: Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have showndhown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer.==.METHODS: We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model.
RESULTS: Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability.
CONCLUSION: Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

Keywords:  Entinostat; Olaparib HR-proficient ovarian cancer

DOI:  https://doi.org/10.1016/j.ygyno.2021.04.015
Open Biol. 2021 Apr;11(4): 200296

Genome instability and loss of protein homeostasis: converging paths to neurodegeneration?

Anna Ainslie, Wouter Huiting, Lara Barazzuol, Steven Bergink.

  Genome instability and loss of protein homeostasis are hallmark events of age-related diseases that include neurodegeneration. Several neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis are characterized by protein aggregation, while an impaired DNA damage response (DDR) as in many genetic DNA repair disorders leads to pronounced neuropathological features. It remains unclear to what degree these cellular events interconnect with each other in the development of neurological diseases. This review highlights how the loss of protein homeostasis and genome instability influence one other. We will discuss studies that illustrate this connection. DNA damage contributes to many neurodegenerative diseases, as shown by an increased level of DNA damage in patients, possibly due to the effects of protein aggregates on chromatin, the sequestration of DNA repair proteins and novel putative DNA repair functions. Conversely, genome stability is also important for protein homeostasis. For example, gene copy number variations and the loss of key DDR components can lead to marked proteotoxic stress. An improved understanding of how protein homeostasis and genome stability are mechanistically connected is needed and promises to lead to the development of novel therapeutic interventions.

Keywords:  DNA damage; genome stability; neurodegeneration; protein aggregation; protein homeostasis

DOI:  https://doi.org/10.1098/rsob.200296
Nucleic Acids Res. 2021 Apr 20. pii: gkab262. [Epub ahead of print]

Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

Suleman S Hussain, Rahul Majumdar, Grace M Moore, Himanshi Narang, Erika S Buechelmaier, Maximilian J Bazil, Pavithran T Ravindran, Jonathan E Leeman, Yi Li, Manisha Jalan, Kyrie S Anderson, Andrea Farina, Rekha Soni, Neeman Mohibullah, Edin Hamzic, Xiaoqing Rong-Mullins, Christopher Sifuentes, Rama R Damerla, Agnes Viale, Simon N Powell, Daniel S Higginson.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

DOI: https://doi.org/10.1093/nar/gkab262
Nat Commun. 2021 Apr 23. 12(1): 2428

Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance.

Anna Fortuny, Audrey Chansard, Pierre Caron, Odile Chevallier, Olivier Leroy, Olivier Renaud, Sophie E Polo.

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV. SETDB1 coordinates histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

DOI: https://doi.org/10.1038/s41467-021-22575-5
Nucleic Acids Res. 2021 Apr 20. pii: gkab276. [Epub ahead of print]

Preferential CEBP binding to T:G mismatches and increased C-to-T human somatic mutations.

Jie Yang, John R Horton, Kadir C Akdemir, Jia Li, Yun Huang, Janani Kumar, Robert M Blumenthal, Xing Zhang, Xiaodong Cheng.

DNA cytosine methylation in mammals modulates gene expression and chromatin accessibility. It also impacts mutation rates, via spontaneous oxidative deamination of 5-methylcytosine (5mC) to thymine. In most cases the resulting T:G mismatches are repaired, following T excision by one of the thymine DNA glycosylases, TDG or MBD4. We found that C-to-T mutations are enriched in the binding sites of CCAAT/enhancer binding proteins (CEBP). Within a CEBP site, the presence of a T:G mismatch increased CEBPβ binding affinity by a factor of >60 relative to the normal C:G base pair. This enhanced binding to a mismatch inhibits its repair by both TDG and MBD4 in vitro. Furthermore, repair of the deamination product of unmethylated cytosine, which yields a U:G DNA mismatch that is normally repaired via uracil DNA glycosylase, is also inhibited by CEBPβ binding. Passage of a replication fork over either a T:G or U:G mismatch, before repair can occur, results in a C-to-T mutation in one of the daughter duplexes. Our study thus provides a plausible mechanism for accumulation of C-to-T human somatic mutations.

DOI: https://doi.org/10.1093/nar/gkab276
Annu Rev Biochem. 2021 Apr 21.

Mechanisms of Vertebrate DNA Interstrand Cross-Link Repair.

Daniel R Semlow, Johannes C Walter.

DNA interstrand cross-links (ICLs) covalently connect the two strands of the double helix and are extremely cytotoxic. Defective ICL repair causes the bone marrow failure and cancer predisposition syndrome, Fanconi anemia, and upregulation of repair causes chemotherapy resistance in cancer. The central event in ICL repair involves resolving the cross-link (unhooking). In this review, we discuss the chemical diversity of ICLs generated by exogenous and endogenous agents. We then describe how proliferating and nonproliferating vertebrate cells unhook ICLs. We emphasize fundamentally new unhooking strategies, dramatic progress in the structural analysis of the Fanconi anemia pathway, and insights into how cells govern the choice between different ICL repair pathways. Throughout, we highlight the many gaps that remain in our knowledge of these fascinating DNA repair pathways. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

DOI: https://doi.org/10.1146/annurev-biochem-080320-112510
Oncotarget. 2021 Mar 30. 12(7): 649-664

Decreased expression of the translation factor eIF3e induces senescence in breast cancer cells via suppression of PARP1 and activation of mTORC1.

Christelle Morris, Sébastien Durand, Pierre Jalinot.

  Altered expression of the translation factor eIF3e is associated with breast cancer occurrence. We have previously shown that eIF3e deficiency leads to an impaired DNA damage response with a marked decrease in DNA repair by homologous recombination. Here, we explored the possibility to exploit this DNA repair defect in targeted cancer therapy using PARP inhibitors. Surprisingly, eIF3e-deficient breast cancer cells are resistant to these drugs, in contrast to BRCA1-deficient cells. Studying this, we found that eIF3e-depleted cells synthesize lowered amounts of PARP1 protein, due to a weakened translation of the corresponding mRNA, associated with a strong decrease in cellular poly(ADP-ribosyl)ation. Additionally, we discovered that the mTORC1 signaling pathway is aberrantly activated in response to eIF3e suppression. Together, these PARP1 and mTORC1 dysfunctions upon eIF3e depletion are causally linked to induction of cellular senescence associated with a pro-inflammatory secretory phenotype. This study provides mechanistic insights into how eIF3e protects against breast cancer, with potential novel cancer therapeutic opportunities. While PARP inhibitors appear as inappropriate drugs for eIF3e-deficient breast tumors, our findings suggest that such cancers may benefit from senolytic drugs or mTORC1 inhibitors.

Keywords:  PARP1; breast cancer; eIF3; mTORC1; senescence-associated secretory phenotype

DOI:  https://doi.org/10.18632/oncotarget.27923
Elife. 2021 Apr 23. pii: e55070. [Epub ahead of print]10

Co-regulation and function of FOXM1/RHNO1 bidirectional genes in cancer.

Carter J Barger, Linda Chee, Mustafa Albahrani, Catalina Munoz-Trujillo, Lidia Boghean, Connor Branick, Kunle Odunsi, Ronny Drapkin, Lee Zou, Adam R Karpf.

  The FOXM1 transcription factor is an oncoprotein and a top biomarker of poor prognosis in human cancer. Overexpression and activation of FOXM1 is frequent in high-grade serous carcinoma (HGSC), the most common and lethal form of human ovarian cancer, and is linked to copy number gains at chromosome 12p13.33. We show that FOXM1 is co-amplified and co-expressed with RHNO1, a gene involved in the ATR-Chk1 signaling pathway that functions in the DNA replication stress (RS) response. We demonstrate that FOXM1 and RHNO1 are head-to-head (i.e. bidirectional) genes (BDG) regulated by a bidirectional promoter (BDP) (named F/R-BDP). FOXM1 and RHNO1 each promote oncogenic phenotypes in HGSC cells, including clonogenic growth, DNA homologous recombination repair (HR), and poly-ADP ribosylase (PARP) inhibitor resistance. FOXM1 and RHNO1 are one of the first examples of oncogenic BDG, and therapeutic targeting of FOXM1/RHNO1 BDG is a potential therapeutic approach for ovarian and other cancers.

Keywords:  cancer biology; chromosomes; gene expression; human

DOI:  https://doi.org/10.7554/eLife.55070
iScience. 2021 Apr 23. 24(4): 102325

DNA repair and cholesterol-mediated drug efflux induce dose-dependent chemoresistance in nutrient-deprived neuroblastoma cells.

Soo Yeon Chae, Dowoon Nam, Do Young Hyeon, Areum Hong, Timothy Dain Lee, Sujin Kim, Dongjoon Im, Jiwon Hong, Chaewon Kang, Ji Won Lee, Daehee Hwang, Sang-Won Lee, Hugh I Kim.

  Neuroblastoma is a solid, heterogeneous pediatric tumor. Chemotherapy is widely used to treat neuroblastoma. However, dose-dependent responses and chemoresistance mechanisms of neuroblastoma cells to anticancer drugs remain challenging. Here, we investigated the dose-dependent effects of topotecan on human neuroblastoma cells (SK-N-SH, SH-SY5Y, and SK-N-BE) under various nutrient supply conditions. Serum-starved human neuroblastoma cells showed reduced toxicity. Their survival rate increased upon treatment with a high concentration (1 μM) of topotecan. Quantitative profiling of global and phosphoproteome identified 12,959 proteins and 48,812 phosphosites, respectively, from SK-N-SH cells. Network analysis revealed that topotecan upregulated DNA repair and cholesterol-mediated topotecan efflux, resulting in topotecan resistance. Results of DNA damage assay, cell cycle, and quantitative analyses of membrane cholesterol supported the validity of these resistance factors and their applicability to all neuroblastoma cells. Our results provide a model for high dose-dependent chemoresistance in neuroblastoma cells that could enable a patient-dependent chemotherapy screening strategy.

Keywords:  Cancer; Molecular Biology; Proteomics

DOI:  https://doi.org/10.1016/j.isci.2021.102325
Commun Biol. 2021 Apr 19. 4(1): 484

COMMD4 functions with the histone H2A-H2B dimer for the timely repair of DNA double-strand breaks.

Amila Suraweera, Neha S Gandhi, Sam Beard, Joshua T Burgess, Laura V Croft, Emma Bolderson, Ali Naqi, Nicholas W Ashton, Mark N Adams, Kienan I Savage, Shu-Dong Zhang, Kenneth J O'Byrne, Derek J Richard.

Genomic stability is critical for normal cellular function and its deregulation is a universal hallmark of cancer. Here we outline a previously undescribed role of COMMD4 in maintaining genomic stability, by regulation of chromatin remodelling at sites of DNA double-strand breaks. At break-sites, COMMD4 binds to and protects histone H2B from monoubiquitination by RNF20/RNF40. DNA damage-induced phosphorylation of the H2A-H2B heterodimer disrupts the dimer allowing COMMD4 to preferentially bind H2A. Displacement of COMMD4 from H2B allows RNF20/40 to monoubiquitinate H2B and for remodelling of the break-site. Consistent with this critical function, COMMD4-deficient cells show excessive elongation of remodelled chromatin and failure of both non-homologous-end-joining and homologous recombination. We present peptide-mapping and mutagenesis data for the potential molecular mechanisms governing COMMD4-mediated chromatin regulation at DNA double-strand breaks.

DOI: https://doi.org/10.1038/s42003-021-01998-2
Front Oncol. 2021 ;11 663360

CDK7 Inhibitor THZ1 Induces the Cell Apoptosis of B-Cell Acute Lymphocytic Leukemia by Perturbing Cellular Metabolism.

Tuersunayi Abudureheman, Jing Xia, Ming-Hao Li, Hang Zhou, Wei-Wei Zheng, Neng Zhou, Rong-Yi Shi, Jian-Min Zhu, Li-Ting Yang, Li Chen, Liang Zheng, Kai Xue, Kai Qing, Cai-Wen Duan.

  B-cell acute lymphocytic leukemia (B-ALL) is a malignant blood cancer that develops in children and adults and leads to high mortality. THZ1, a covalent cyclin-dependent kinase 7 (CDK7) inhibitor, shows anti-tumor effects in various cancers by inhibiting cell proliferation and inducing apoptosis. However, whether THZ1 has an inhibitory effect on B-ALL cells and the underlying mechanism remains obscure. In this study, we showed that THZ1 arrested the cell cycle of B-ALL cells in vitro in a low concentration, while inducing the apoptosis of B-ALL cells in vitro in a high concentration by activating the apoptotic pathways. In addition, RNA-SEQ results revealed that THZ1 disrupted the cellular metabolic pathways of B-ALL cells. Moreover, THZ1 suppressed the cellular metabolism and blocked the production of cellular metabolic intermediates in B-ALL cells. Mechanistically, THZ1 inhibited the cellular metabolism of B-ALL by downregulating the expression of c-MYC-mediated metabolic enzymes. However, THZ1 treatment enhanced cell apoptosis in over-expressed c-MYC B-ALL cells, which was involved in the upregulation of p53 expression. Collectively, our data demonstrated that CDK7 inhibitor THZ1 induced the apoptosis of B-ALL cells by perturbing c-MYC-mediated cellular metabolism, thereby providing a novel treatment option for B-ALL.

Keywords:  B-cell acute lymphocytic leukemia; CDK7 inhibitor; c-MYC; cell apoptosis; metabolism

DOI:  https://doi.org/10.3389/fonc.2021.663360
Blood. 2021 Apr 19. pii: blood.2020008955. [Epub ahead of print]

A novel and highly effective mitochondrial uncoupling drug in T-cell leukemia.

Victoria da Silva-Diz, Bin Cao, Olga Lancho, Eric Chiles, Amer Alasadi, Maya Aleksandrova, Shirley Luo, Amartya Singh, Hanlin Tao, David John Augeri, Sonia Minuzzo, Stefano Indraccolo, Hossein Khiabanian, Xiaoyang Su, Shengkan Jin, Daniel Herranz.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted AMPK activation and downregulation of mTOR signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL PDXs led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47-driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease.

DOI: https://doi.org/10.1182/blood.2020008955
J Immunol. 2021 May 01. 206(9): 1983-1990

Ectonucleotidases in Inflammation, Immunity, and Cancer.

Clarissa Branco Haas, Marianna Lovászi, Elizandra Braganhol, Pál Pacher, György Haskó.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes that hydrolyze nucleotides such as ATP, UTP, ADP, and UDP to monophosphates derivates such as AMP and UMP. The NTPDase family consists of eight enzymes, of which NTPDases 1, 2, 3, and 8 are expressed on cell membranes thereby hydrolyzing extracellular nucleotides. Cell membrane NTPDases are expressed in all tissues, in which they regulate essential physiological tissue functions such as development, blood flow, hormone secretion, and neurotransmitter release. They do so by modulating nucleotide-mediated purinergic signaling through P2 purinergic receptors. NTPDases 1, 2, 3, and 8 also play a key role during infection, inflammation, injury, and cancer. Under these conditions, NTPDases can contribute and control the pathophysiology of infectious, inflammatory diseases and cancer. In this review, we discuss the role of NTPDases, focusing on the less understood NTPDases 2-8, in regulating inflammation and immunity during infectious, inflammatory diseases, and cancer.

DOI: https://doi.org/10.4049/jimmunol.2001342
BMC Cancer. 2021 Apr 22. 21(1): 448

Olaparib-mediated enhancement of 5-fluorouracil cytotoxicity in mismatch repair deficient colorectal cancer cells.

Helena de Castro E Gloria, Laura Jesuíno Nogueira, Patrícia Bencke Grudzinski, Paola Victória da Costa Ghignatti, Temenouga Nikolova Guecheva, Natalia Motta Leguisamo, Jenifer Saffi.

  BACKGROUND: The advances in colorectal cancer (CRC) treatment include the identification of deficiencies in Mismatch Repair (MMR) pathway to predict the benefit of adjuvant 5-fluorouracil (5-FU) and oxaliplatin for stage II CRC and immunotherapy. Defective MMR contributes to chemoresistance in CRC. A growing body of evidence supports the role of Poly-(ADP-ribose) polymerase (PARP) inhibitors, such as Olaparib, in the treatment of different subsets of cancer beyond the tumors with homologous recombination deficiencies. In this work we evaluated the effect of Olaparib on 5-FU cytotoxicity in MMR-deficient and proficient CRC cells and the mechanisms involved.METHODS: Human colon cancer cell lines, proficient (HT29) and deficient (HCT116) in MMR, were treated with 5-FU and Olaparib. Cytotoxicity was assessed by MTT and clonogenic assays, apoptosis induction and cell cycle progression by flow cytometry, DNA damage by comet assay. Adhesion and transwell migration assays were also performed.
RESULTS: Our results showed enhancement of the 5-FU citotoxicity by Olaparib in MMR-deficient HCT116 colon cancer cells. Moreover, the combined treatment with Olaparib and 5-FU induced G2/M arrest, apoptosis and polyploidy in these cells. In MMR proficient HT29 cells, the Olaparib alone reduced clonogenic survival, induced DNA damage accumulation and decreased the adhesion and migration capacities.
CONCLUSION: Our results suggest benefits of Olaparib inclusion in CRC treatment, as combination with 5-FU for MMR deficient CRC and as monotherapy for MMR proficient CRC. Thus, combined therapy with Olaparib could be a strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC.

Keywords:  5-fluorouracil; Colorectal cancer; Mismatch repair; Olaparib; PARP

DOI:  https://doi.org/10.1186/s12885-021-08188-7
Protein Sci. 2021 Apr 21.

Crystal structure and molecular dynamics of human POLDIP2, a multifaceted adaptor protein in metabolism and genome stability.

Anastasija A Kulik, Klaudia K Maruszczak, Dana C Thomas, Naomi L A Nabi, Martin Carr, Richard J Bingham, Christopher D O Cooper.

  Polymerase δ-interacting protein 2 (POLDIP2, PDIP38) is a multifaceted, 'moonlighting' protein, involved in binding protein partners from many different cellular processes, including mitochondrial metabolism and DNA replication and repair. How POLDIP2 interacts with many different proteins is unknown. Towards this goal, we present the crystal structure of POLDIP2 to 2.8 å, which exhibited a compact two-domain β-strand-rich globular structure, confirmed by circular dichroism and small angle X-ray scattering approaches. POLDIP2 comprised canonical DUF525 and YccV domains, but with a conserved domain linker packed tightly, resulting in an 'extended' YccV module. A central channel was observed, which we hypothesise could influence structural changes potentially mediated by redox conditions, following observation of a modified cysteine residue in the channel. Unstructured regions were rebuilt by ab initio modelling to generate a model of full-length POLDIP2. Molecular dynamics simulations revealed a highly dynamic N-terminal region tethered to the YccV-domain by an extended linker, potentially facilitating interactions with distal binding partners. Models of POLDIP2 complexed with two of its partners, PrimPol and PCNA, indicated that dynamic flexibility of the POLDIP2 N-terminus and loop regions likely mediate protein interactions. This article is protected by copyright. All rights reserved.

Keywords:  DNA polymerase; PCNA; PDIP38; POLDIP2; PrimPol; protein structure

DOI:  https://doi.org/10.1002/pro.4085
Front Genet. 2021 ;12 628758

RRM2B Is Frequently Amplified Across Multiple Tumor Types: Implications for DNA Repair, Cellular Survival, and Cancer Therapy.

Waleed Iqbal, Elena V Demidova, Samantha Serrao, Taha ValizadehAslani, Gail Rosen, Sanjeevani Arora.

  RRM2B plays a crucial role in DNA replication, repair and oxidative stress. While germline RRM2B mutations have been implicated in mitochondrial disorders, its relevance to cancer has not been established. Here, using TCGA studies, we investigated RRM2B alterations in cancer. We found that RRM2B is highly amplified in multiple tumor types, particularly in MYC-amplified tumors, and is associated with increased RRM2B mRNA expression. We also observed that the chromosomal region 8q22.3-8q24, is amplified in multiple tumors, and includes RRM2B, MYC along with several other cancer-associated genes. An analysis of genes within this 8q-amplicon showed that cancers that have both RRM2B-amplified along with MYC have a distinct pattern of amplification compared to cancers that are unaltered or those that have amplifications in RRM2B or MYC only. Investigation of curated biological interactions revealed that gene products of the amplified 8q22.3-8q24 region have important roles in DNA repair, DNA damage response, oxygen sensing, and apoptosis pathways and interact functionally. Notably, RRM2B-amplified cancers are characterized by mutation signatures of defective DNA repair and oxidative stress, and at least RRM2B-amplified breast cancers are associated with poor clinical outcome. These data suggest alterations in RR2MB and possibly the interacting 8q-proteins could have a profound effect on regulatory pathways such as DNA repair and cellular survival, highlighting therapeutic opportunities in these cancers.

Keywords:  8q-amplicon; MYC; RRM2B; cancer; chromosome 8

DOI:  https://doi.org/10.3389/fgene.2021.628758
Nucleic Acids Res. 2021 Apr 19. pii: gkab269. [Epub ahead of print]

HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation.

Soichiro Kumamoto, Atsuya Nishiyama, Yoshie Chiba, Ryota Miyashita, Chieko Konishi, Yoshiaki Azuma, Makoto Nakanishi.

DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.

DOI: https://doi.org/10.1093/nar/gkab269
Science. 2021 Apr 23. 372(6540): 371-378

Replication timing maintains the global epigenetic state in human cells.

Kyle N Klein, Peiyao A Zhao, Xiaowen Lyu, Takayo Sasaki, Daniel A Bartlett, Amar M Singh, Ipek Tasan, Meng Zhang, Lotte P Watts, Shin-Ichiro Hiraga, Toyoaki Natsume, Xuemeng Zhou, Timour Baslan, Danny Leung, Masato T Kanemaki, Anne D Donaldson, Huimin Zhao, Stephen Dalton, Victor G Corces, David M Gilbert.

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.

DOI: https://doi.org/10.1126/science.aba5545
Nat Commun. 2021 Apr 19. 12(1): 2327

STING enhances cell death through regulation of reactive oxygen species and DNA damage.

Thomas J Hayman, Marta Baro, Tyler MacNeil, Chatchai Phoomak, Thazin Nwe Aung, Wei Cui, Kevin Leach, Radhakrishnan Iyer, Sreerupa Challa, Teresa Sandoval-Schaefer, Barbara A Burtness, David L Rimm, Joseph N Contessa.

Resistance to DNA-damaging agents is a significant cause of treatment failure and poor outcomes in oncology. To identify unrecognized regulators of cell survival we performed a whole-genome CRISPR-Cas9 screen using treatment with ionizing radiation as a selective pressure, and identified STING (stimulator of interferon genes) as an intrinsic regulator of tumor cell survival. We show that STING regulates a transcriptional program that controls the generation of reactive oxygen species (ROS), and that STING loss alters ROS homeostasis to reduce DNA damage and to cause therapeutic resistance. In agreement with these data, analysis of tumors from head and neck squamous cell carcinoma patient specimens show that low STING expression is associated with worse outcomes. We also demonstrate that pharmacologic activation of STING enhances the effects of ionizing radiation in vivo, providing a rationale for therapeutic combinations of STING agonists and DNA-damaging agents. These results highlight a role for STING that is beyond its canonical function in cyclic dinucleotide and DNA damage sensing, and identify STING as a regulator of cellular ROS homeostasis and tumor cell susceptibility to reactive oxygen dependent, DNA damaging agents.

DOI: https://doi.org/10.1038/s41467-021-22572-8
Sci Rep. 2021 Apr 22. 11(1): 8735

BRCA1 degradation in response to mitochondrial damage in breast cancer cells.

Kana Miyahara, Naoharu Takano, Yumiko Yamada, Hiromi Kazama, Mayumi Tokuhisa, Hirotsugu Hino, Koji Fujita, Edward Barroga, Masaki Hiramoto, Hiroshi Handa, Masahiko Kuroda, Takashi Ishikawa, Keisuke Miyazawa.

BRCA1 is a well-studied tumor suppressor involved in the homologous repair of DNA damage, whereas PINK1, a mitochondrial serine/threonine kinase, is known to be involved in mitochondrial quality control. Genetic mutations of PINK1 and Parkin cause autosomal recessive early-onset Parkinson's disease. We found that in breast cancer cells, the mitochondrial targeting reagents, which all induce mitochondrial depolarization along with PINK1 upregulation, induced proteasomal BRCA1 degradation. This BRCA1 degradation was dependent on PINK1, and BRCA1 downregulation upon mitochondrial damage caused DNA double-strand breaks. BRCA1 degradation was mediated through the direct interaction with the E3 ligase Parkin. Strikingly, BRCA1 and PINK1/Parkin expression were inversely correlated in cancerous mammary glands from breast cancer patients. BRCA1 knockdown repressed cancer cell growth, and high BRCA1 expression predicted poor relapse-free survival in breast cancer patients. These observations indicate a novel mechanism by which mitochondrial damage is transmitted to the nucleus, leading to BRCA1 degradation.

DOI: https://doi.org/10.1038/s41598-021-87698-7
J Neurol Sci. 2021 Apr 08. pii: S0022-510X(21)00133-7. [Epub ahead of print]425 117439

Purine synthesis as a target for radiation resistance in molecular glioblastoma.

Connor J Kinslow, Kunal R Chaudhary, Pavan S Upadhyayula, Tony J C Wang, Ramon C Sun, Simon K Cheng.



Keywords:  Glioblastoma; Metabolism; Purine; Radiation; Resistance

DOI:  https://doi.org/10.1016/j.jns.2021.117439