bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒06‒13
thirty-nine papers selected by
Sean Rudd
Karolinska Institutet
  1. XRCC1 prevents toxic PARP1 trapping during DNA base excision repair.
  2. RADX prevents genome instability by confining replication fork reversal to stalled forks.
  3. Dual covalent inhibition of PKM and IMPDH targets metabolism in cutaneous metastatic melanoma.
  4. Unchecked nick ligation can promote localized genome re-replication.
  5. Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability.
  6. ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation.
  7. Transcription shapes DNA replication initiation to preserve genome integrity.
  8. mTOR pathway and DNA damage response: A therapeutic strategy in cancer therapy.
  9. Rhodium Complexes Targeting DNA Mismatches as a Basis for New Therapeutics in Cancers Deficient in Mismatch Repair.
  10. DNAscent v2: detecting replication forks in nanopore sequencing data with deep learning.
  11. Mind the replication gap.
  12. Catalysis-dependent and redundant roles of Dma1 and Dma2 in maintenance of genome stability in Saccharomyces cerevisiae.
  13. CDK-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity.
  14. LEO1 is a partner for Cockayne syndrome protein B (CSB) in response to transcription-blocking DNA damage.
  15. The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes.
  16. Fanconi anemia proteins participate in a break-induced-replication-like pathway to counter replication stress.
  17. Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin.
  18. Recent Advances in Therapeutic Application of DNA Damage Response Inhibitors against Cancer.
  19. Perspectives on formaldehyde dysregulation: Mitochondrial DNA damage and repair in mammalian cells.
  20. Dynamics of replication origin over-activation.
  21. RAD51AP1 loss attenuates colorectal cancer stem cell renewal and sensitizes to chemotherapy.
  22. Communication between chromatin and homologous recombination.
  23. ADP-ribosylation of DNA and RNA.
  24. DNA-PK inhibitor peposertib enhances p53-dependent cytotoxicity of DNA double-strand break inducing therapy in acute leukemia.
  25. DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance.
  26. Structural Insights into the Mechanism of Base Excision by MBD4.
  27. DNA damage and regulation of protein homeostasis.
  28. Replication protein A plays multifaceted roles complementary to specialized helicases in processing G-quadruplex DNA.
  29. Diverse immune response of DNA damage repair-deficient tumors.
  30. Cryo-EM structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in nucleotide excision repair.
  31. Genetic variations in 3´UTRs of SMUG1 and NEIL2 genes modulate breast cancer risk, survival and therapy response.
  32. Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics.
  33. Clinical CDK4/6 inhibitors induce selective and immediate dissociation of p21 from cyclin D-CDK4 to inhibit CDK2.
  34. Preliminary Activity Seen with RAD51 Inhibitor.
  35. Activation of PARP2/ARTD2 by DNA damage induces conformational changes relieving enzyme autoinhibition.
  36. Tumor-specific delivery of 5FU-incorporated EGFR-targeted aptamers as an efficient treatment in pancreatic ductal adenocarcinoma models.
  37. Polθ reverse transcribes RNA and promotes RNA-templated DNA repair.
  38. AXL inhibition in macrophages stimulates host-versus-leukemia immunity and eradicates naive and treatment resistant leukemia.
  39. PELP1/SRC-3-dependent regulation of metabolic PFKFB kinases drives therapy resistant ER+ breast cancer.
  1. Mol Cell. 2021 May 29. pii: S1097-2765(21)00366-X. [Epub ahead of print]
    XRCC1 prevents toxic PARP1 trapping during DNA base excision repair.
    Annie A Demin, Kouji Hirota, Masataka Tsuda, Marek Adamowicz, Richard Hailstone, Jan Brazina, William Gittens, Ilona Kalasova, Zhengping Shao, Shan Zha, Hiroyuki Sasanuma, Hana Hanzlikova, Shunichi Takeda, Keith W Caldecott.
      Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.
    Keywords:  PARP inhibitors; PARP trapping; PARP1; XRCC1 protein complexes; base excision repair; single-strand breaks
    DOI:  https://doi.org/10.1016/j.molcel.2021.05.009
  2. Mol Cell. 2021 May 29. pii: S1097-2765(21)00390-7. [Epub ahead of print]
    RADX prevents genome instability by confining replication fork reversal to stalled forks.
    Archana Krishnamoorthy, Jessica Jackson, Taha Mohamed, Madison Adolph, Alessandro Vindigni, David Cortez.
      RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.
    Keywords:  BRCA2; DNA damage; DNA repair; DNA replication; electron microscopy; genome instability; telomere
    DOI:  https://doi.org/10.1016/j.molcel.2021.05.014
  3. Cancer Res. 2021 Jun 07. pii: canres.2114.2020. [Epub ahead of print]
    Dual covalent inhibition of PKM and IMPDH targets metabolism in cutaneous metastatic melanoma.
    Marwa Zerhouni, Anthony R Martin, Nathan Furstoss, Vincent S Gutierrez, Emilie Jaune, Nedra Tekaya, Guillaume E Beranger, Patricia Abbe, Claire Regazzetti, Hella Amdouni, Mohsine Driowya, Patrice Dubreuil, Frédéric Luciano, Arnaud Jacquel, Meri K Tulic, Thomas Cluzeau, Brendan P O'Hara, Issam Ben-Sahra, Thierry Passeron, Rachid Benhida, Guillaume Robert, Patrick Auberger, Stéphane Rocchi.
      Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of BRAFV600E cutaneous metastatic melanoma (CMM) patients eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming which profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafemib-sensitive and -resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro. Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-2114
  4. Curr Biol. 2021 Jun 07. pii: S0960-9822(21)00382-1. [Epub ahead of print]31(11): R710-R711
    Unchecked nick ligation can promote localized genome re-replication.
    Erik Johansson, John F X Diffley.
      Single-stranded DNA breaks, or nicks, are amongst the most common forms of DNA damage in cells. They can be repaired by ligation; however, if a nick occurs just ahead of an approaching replisome, the outcome is a collapsed replication fork comprising a single-ended double-strand break and a 'hybrid nick' with parental DNA on one side and nascent DNA on the other (Figure 1A). We realized that in eukaryotic cells, where replication initiates from multiple replication origins, a fork from an adjacent origin can promote localized re-replication if the hybrid nick is ligated. We have modelled this situation with purified proteins in vitro and have found that there is, indeed, an additional hazard that eukaryotic replisomes face. We discuss how this problem might be mitigated.
    DOI:  https://doi.org/10.1016/j.cub.2021.03.043
  5. Nat Cell Biol. 2021 Jun;23(6): 608-619
    Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability.
    Marit E Geijer, Di Zhou, Kathiresan Selvam, Barbara Steurer, Chirantani Mukherjee, Bastiaan Evers, Simona Cugusi, Marvin van Toorn, Melanie van der Woude, Roel C Janssens, Yannick P Kok, Wenzhi Gong, Anja Raams, Calvin S Y Lo, Joyce H G Lebbink, Bart Geverts, Dalton A Plummer, Karel Bezstarosti, Arjan F Theil, Richard Mitter, Adriaan B Houtsmuller, Wim Vermeulen, Jeroen A A Demmers, Shisheng Li, Marcel A T M van Vugt, Hannes Lans, René Bernards, Jesper Q Svejstrup, Arnab Ray Chaudhuri, John J Wyrick, Jurgen A Marteijn.
      Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.
    DOI:  https://doi.org/10.1038/s41556-021-00692-z
  6. Nat Cell Biol. 2021 Jun;23(6): 595-607
    ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation.
    Yana van der Weegen, Klaas de Lint, Diana van den Heuvel, Yuka Nakazawa, Tycho E T Mevissen, Janne J M van Schie, Marta San Martin Alonso, Daphne E C Boer, Román González-Prieto, Ishwarya V Narayanan, Noud H M Klaassen, Annelotte P Wondergem, Khashayar Roohollahi, Josephine C Dorsman, Yuichiro Hara, Alfred C O Vertegaal, Job de Lange, Johannes C Walter, Sylvie M Noordermeer, Mats Ljungman, Tomoo Ogi, Rob M F Wolthuis, Martijn S Luijsterburg.
      Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4CSA ubiquitin ligase. How CRL4CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.
    DOI:  https://doi.org/10.1038/s41556-021-00688-9
  7. Genome Biol. 2021 Jun 09. 22(1): 176
    Transcription shapes DNA replication initiation to preserve genome integrity.
    Yang Liu, Chen Ai, Tingting Gan, Jinchun Wu, Yongpeng Jiang, Xuhao Liu, Rusen Lu, Ning Gao, Qing Li, Xiong Ji, Jiazhi Hu.
      BACKGROUND: Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.RESULTS: We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage.
    CONCLUSION: RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.
    Keywords:  DNA damage; DNA replication initiation; Genome instability; MCM redistribution; Transcription; Transcription-replication initiation collision
    DOI:  https://doi.org/10.1186/s13059-021-02390-3
  8. DNA Repair (Amst). 2021 Jun 03. pii: S1568-7864(21)00098-7. [Epub ahead of print]104 103142
    mTOR pathway and DNA damage response: A therapeutic strategy in cancer therapy.
    Romina Danesh Pazhooh, Parnia Rahnamay Farnood, Zatollah Asemi, Liaosadat Mirsafaei, Bahman Yousefi, Hamed Mirzaei.
      The mammalian target of rapamycin (mTOR) is a conserved serine/threonine-protein kinase, comprising two subunit protein complexes: mTORC1 and mTORC2. In response to insult and cancer, the mTOR pathway plays a crucial role in regulating growth, metabolism, cell survival, and protein synthesis. Key subunits of mTORC1/2 catalyze the phosphorylation of various molecules, including eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), ribosomal protein S6 kinase β-1 (S6K1). The DNA damage response (DDR) maintains genomic stability and provides an opportunity for treating tumors with defects caused by DNA damaging agents. Many mTOR inhibitors are utilized for the treatment of cancers. However, several clinical trials are still assessing the efficacy of mTOR inhibitors. This paper discusses the role of the mTOR signaling pathway and its regulators in developing cancer. In the following, we will review the interaction between DDR and mTOR signaling and the innovative therapies applied in preclinical and clinical trials for treating cancers.
    Keywords:  Cancer; DNA damage; DNA repair; Therapy; mTOR
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103142
  9. Biochemistry. 2021 Jun 11.
    Rhodium Complexes Targeting DNA Mismatches as a Basis for New Therapeutics in Cancers Deficient in Mismatch Repair.
    Adela Nano, Joanne Dai, Julie M Bailis, Jacqueline K Barton.
      Cancers with microsatellite instability (MSI), which include ≤20% of solid tumors, are characterized by resistance to chemotherapy due to deficiency in the DNA mismatch repair (MMR) pathway. Rhodium metalloinsertors make up a class of compounds that bind DNA mismatches with high specificity and show selective cytotoxicity in MSI cancer cells. We determined that rhodium complexes with an N∧O coordination showed significantly increased cell potency compared with that of N∧N-coordinated compounds, and we identified [Rh(chrysi)(phen)(PPO)]2+ (RhPPO) as the most potent, selective compound in this class. Using matched cell lines that are MMR-deficient (HCT116O) and MMR-proficient (HCT116N), we demonstrated that RhPPO preferentially activates the DNA damage response and inhibits DNA replication and cell proliferation in HCT116O cells, leading to cell death by necrosis. Using a fluorescent conjugate of RhPPO, we established that the metalloinsertor localizes to DNA mismatches in the cell nucleus and causes DNA double-strand breaks at or near the mismatch sites. Evaluation of RhPPO across MMR-deficient and MMR-proficient cell lines confirmed the broad potential for RhPPO to target MSI cancers, with cell potency significantly higher than that of platinum complexes used broadly as chemotherapeutics. Moreover, in a mouse xenograft model of MSI cancer, RhPPO shows promising antitumor activity and increased survival. Thus, our studies indicate that RhPPO is a novel DNA-targeted therapy with improved potency and selectivity over standard-of-care platinum-based chemotherapy and, importantly, that DNA mismatches offer a critical new target in the design of chemotherapeutics for MSI cancers.
    DOI:  https://doi.org/10.1021/acs.biochem.1c00302
  10. BMC Genomics. 2021 Jun 09. 22(1): 430
    DNAscent v2: detecting replication forks in nanopore sequencing data with deep learning.
    Michael A Boemo.
      BACKGROUND: Measuring DNA replication dynamics with high throughput and single-molecule resolution is critical for understanding both the basic biology behind how cells replicate their DNA and how DNA replication can be used as a therapeutic target for diseases like cancer. In recent years, the detection of base analogues in Oxford Nanopore Technologies (ONT) sequencing reads has become a promising new method to supersede existing single-molecule methods such as DNA fibre analysis: ONT sequencing yields long reads with high throughput, and sequenced molecules can be mapped to the genome using standard sequence alignment software.RESULTS: This paper introduces DNAscent v2, software that uses a residual neural network to achieve fast, accurate detection of the thymidine analogue BrdU with single-nucleotide resolution. DNAscent v2 also comes equipped with an autoencoder that interprets the pattern of BrdU incorporation on each ONT-sequenced molecule into replication fork direction to call the location of replication origins termination sites. DNAscent v2 surpasses previous versions of DNAscent in BrdU calling accuracy, origin calling accuracy, speed, and versatility across different experimental protocols. Unlike NanoMod, DNAscent v2 positively identifies BrdU without the need for sequencing unmodified DNA. Unlike RepNano, DNAscent v2 calls BrdU with single-nucleotide resolution and detects more origins than RepNano from the same sequencing data. DNAscent v2 is open-source and available at https://github.com/MBoemo/DNAscent .
    CONCLUSIONS: This paper shows that DNAscent v2 is the new state-of-the-art in the high-throughput, single-molecule detection of replication fork dynamics. These improvements in DNAscent v2 mark an important step towards measuring DNA replication dynamics in large genomes with single-molecule resolution. Looking forward, the increase in accuracy in single-nucleotide resolution BrdU calls will also allow DNAscent v2 to branch out into other areas of genome stability research, particularly the detection of DNA repair.
    Keywords:  Budding yeast; DNA replication; DNAscent; Oxford nanopore; Replication forks; Replication origins; Residual neural networks
    DOI:  https://doi.org/10.1186/s12864-021-07736-6
  11. R Soc Open Sci. 2021 Jun 09. 8(6): 201932
    Mind the replication gap.
    Camelia Mocanu, Kok-Lung Chan.
      Unlike bacteria, mammalian cells need to complete DNA replication before segregating their chromosomes for the maintenance of genome integrity. Thus, cells have evolved efficient pathways to restore stalled and/or collapsed replication forks during S-phase, and when necessary, also to delay cell cycle progression to ensure replication completion. However, strong evidence shows that cells can proceed to mitosis with incompletely replicated DNA when under mild replication stress (RS) conditions. Consequently, the incompletely replicated genomic gaps form, predominantly at common fragile site regions, where the converging fork-like DNA structures accumulate. These branched structures pose a severe threat to the faithful disjunction of chromosomes as they physically interlink the partially duplicated sister chromatids. In this review, we provide an overview discussing how cells respond and deal with the under-replicated DNA structures that escape from the S/G2 surveillance system. We also focus on recent research of a mitotic break-induced replication pathway (also known as mitotic DNA repair synthesis), which has been proposed to operate during prophase in an attempt to finish DNA synthesis at the under-replicated genomic regions. Finally, we discuss recent data on how mild RS may cause chromosome instability and mutations that accelerate cancer genome evolution.
    Keywords:  break-induced replication; chromosome instability; common fragile sites; mitotic DNA repair synthesis; replication stress; ultrafine DNA bridges
    DOI:  https://doi.org/10.1098/rsos.201932
  12. J Biol Chem. 2021 Apr 29. pii: S0021-9258(21)00510-X. [Epub ahead of print] 100721
    Catalysis-dependent and redundant roles of Dma1 and Dma2 in maintenance of genome stability in Saccharomyces cerevisiae.
    Andrew R Yoblinski, Seoyoung Chung, Sophie B Robinson, Kaitlyn E Forester, Brian D Strahl, Raghuvar Dronamraju.
      DNA double strand breaks (DSBs) are among the deleterious lesions that are both endogenous and exogenous in origin, and are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). However, the molecular mechanisms responsible for maintaining genome stability remain incompletely understood. Here, we investigate the role of two E3 ligases, Dma1 and Dma2 (homologs of human RNF8) in the maintenance of genome stability in budding yeast. Using yeast spotting assays, Chromatin immunoprecipitation (ChIP) and plasmid and chromosomal repair assays, we establish that Dma1 and Dma2 act in a redundant and a catalysis-dependent manner in the maintenance of genome stability, as well as localize to transcribed regions of the genome and increase in abundance upon phleomycin treatment. Additionally, Dma1 and Dma2 are required for the normal kinetics of histone H4 acetylation under DNA damage conditions, genetically interact with RAD9 and SAE2, and are in a complex with Rad53 and histones. Taken together, our results demonstrate the requirement of Dma1 and Dma2 in regulating DNA repair pathway choice, preferentially affecting HR over NHEJ and open up the possibility of using these candidates in manipulating the repair pathways towards precision genome editing.
    Keywords:  DNA end resection; DNA repair; Dma1; Dma2; genome stability; histones; homologous recombination; yeast
    DOI:  https://doi.org/10.1016/j.jbc.2021.100721
  13. Mol Cell Biol. 2021 Jun 07. MCB0023421
    CDK-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity.
    Juan A Cantres-Velez, Justin L Blaize, David A Vierra, Rebecca A Boisvert, Jada L Garzon, Benjamin Piraino, Winnie Tan, Andrew J Deans, Niall G Howlett.
      Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA damaging agents and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.
    DOI:  https://doi.org/10.1128/MCB.00234-21
  14. Nucleic Acids Res. 2021 Jun 07. pii: gkab458. [Epub ahead of print]
    LEO1 is a partner for Cockayne syndrome protein B (CSB) in response to transcription-blocking DNA damage.
    Vinod Tiwari, Tomasz Kulikowicz, David M Wilson, Vilhelm A Bohr.
      Cockayne syndrome (CS) is an autosomal recessive genetic disorder characterized by photosensitivity, developmental defects, neurological abnormalities, and premature aging. Mutations in CSA (ERCC8), CSB (ERCC6), XPB, XPD, XPG, XPF (ERCC4) and ERCC1 can give rise to clinical phenotypes resembling classic CS. Using a yeast two-hybrid (Y2H) screening approach, we identified LEO1 (Phe381-Ser568 region) as an interacting protein partner of full-length and C-terminal (Pro1010-Cys1493) CSB in two independent screens. LEO1 is a member of the RNA polymerase associated factor 1 complex (PAF1C) with roles in transcription elongation and chromatin modification. Supportive of the Y2H results, purified, recombinant LEO1 and CSB directly interact in vitro, and the two proteins exist in a common complex within human cells. In addition, fluorescently tagged LEO1 and CSB are both recruited to localized DNA damage sites in human cells. Cell fractionation experiments revealed a transcription-dependent, coordinated association of LEO1 and CSB to chromatin following either UVC irradiation or cisplatin treatment of HEK293T cells, whereas the response to menadione was distinct, suggesting that this collaboration occurs mainly in the context of bulky transcription-blocking lesions. Consistent with a coordinated interaction in DNA repair, LEO1 knockdown or knockout resulted in reduced CSB recruitment to chromatin, increased sensitivity to UVC light and cisplatin damage, and reduced RNA synthesis recovery and slower excision of cyclobutane pyrimidine dimers following UVC irradiation; the absence of CSB resulted in diminished LEO1 recruitment. Our data indicate a reciprocal communication between CSB and LEO1 in the context of transcription-associated DNA repair and RNA transcription recovery.
    DOI:  https://doi.org/10.1093/nar/gkab458
  15. Proc Natl Acad Sci U S A. 2021 Jun 15. pii: e2019183118. [Epub ahead of print]118(24):
    The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes.
    Yang Liu, Lu Wang, Xin Xu, Yue Yuan, Bo Zhang, Zeyang Li, Yuchen Xie, Rui Yan, Zeqi Zheng, Jianguo Ji, Johanne M Murray, Antony M Carr, Daochun Kong.
      DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2 However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1 Chk2 -mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.
    Keywords:  CMG complex; checkpoint; replication fork stability
    DOI:  https://doi.org/10.1073/pnas.2019183118
  16. Nat Struct Mol Biol. 2021 Jun;28(6): 487-500
    Fanconi anemia proteins participate in a break-induced-replication-like pathway to counter replication stress.
    Xinlin Xu, Yixi Xu, Ruiyuan Guo, Ran Xu, Congcong Fu, Mengtan Xing, Hiroyuki Sasanuma, Qing Li, Minoru Takata, Shunichi Takeda, Rong Guo, Dongyi Xu.
      Fanconi anemia (FA) is a devastating hereditary disease characterized by bone marrow failure (BMF) and acute myeloid leukemia (AML). As FA-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), ICLs are widely assumed to be the lesions responsible for FA symptoms. Here, we show that FA-mutated cells are hypersensitive to persistent replication stress and that FA proteins play a role in the break-induced-replication (BIR)-like pathway for fork restart. Both the BIR-like pathway and ICL repair share almost identical molecular mechanisms of 53BP1-BRCA1-controlled signaling response, SLX4- and FAN1-mediated fork cleavage and POLD3-dependent DNA synthesis, suggesting that the FA pathway is intrinsically one of the BIR-like pathways. Replication stress not only triggers BMF in FA-deficient mice, but also specifically induces monosomy 7, which is associated with progression to AML in patients with FA, in FA-deficient cells.
    DOI:  https://doi.org/10.1038/s41594-021-00602-9
  17. Mol Cell. 2021 May 31. pii: S1097-2765(21)00367-1. [Epub ahead of print]
    Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin.
    Linchang Dai, Yaxin Dai, Jinhua Han, Yan Huang, Longge Wang, Jun Huang, Zheng Zhou.
      The BRCA1-BARD1 complex directs the DNA double-strand break (DSB) repair pathway choice to error-free homologous recombination (HR) during the S-G2 stages. Targeting BRCA1-BARD1 to DSB-proximal sites requires BARD1-mediated nucleosome interaction and histone mark recognition. Here, we report the cryo-EM structure of BARD1 bound to a ubiquitinated nucleosome core particle (NCPUb) at 3.1 Å resolution and illustrate how BARD1 simultaneously recognizes the DNA damage-induced mark H2AK15ub and DNA replication-associated mark H4K20me0 on the nucleosome. In vitro and in vivo analyses reveal that the BARD1-NCPUb complex is stabilized by BARD1-nucleosome interaction, BARD1-ubiquitin interaction, and BARD1 ARD domain-BARD1 BRCT domain interaction, and abrogating these interactions is detrimental to HR activity. We further identify multiple disease-causing BARD1 mutations that disrupt BARD1-NCPUb interactions and hence impair HR. Together, this study elucidates the mechanism of BRCA1-BARD1 complex recruitment and retention by DSB-flanking nucleosomes and sheds important light on cancer therapeutic avenues.
    Keywords:  BARD1; BRCA1; DNA double strand break repair; cryo-electron microscopy structure; homologous recombination; nucleosomes; structural biology; tumour-suppressor proteins
    DOI:  https://doi.org/10.1016/j.molcel.2021.05.010
  18. Anticancer Agents Med Chem. 2021 Jun 07.
    Recent Advances in Therapeutic Application of DNA Damage Response Inhibitors against Cancer.
    Stina George Fernandes, Prachi Shah, Ekta Khattar.
      DNA integrity is continuously challenged by intrinsic cellular processes and environmental agents. To overcome this genomic damage, cells have developed multiple signaling pathways collectively named as DNA damage response (DDR) and composed of three components: (i) sensor proteins, which detect DNA damage, (ii) mediators that relay the signal downstream and recruit the repair machinery, and (iii) the repair proteins, which restore the damaged DNA. A flawed DDR and failure to repair the damage lead to the accumulation of genetic lesions and increased genomic instability, which is recognized as a hallmark of cancer. Cancer cells tend to harbor increased mutations in DDR genes and often have fewer DDR pathways than normal cells. This makes cancer cells more dependent on particular DDR pathways and thus become more susceptible to compounds inhibiting those pathways compared to normal cells, which have all the DDR pathways intact. Understanding the roles of different DDR proteins in the DNA damage response and repair pathways and identification of their structures have paved the way for the development of their inhibitors as targeted cancer therapy. In this review, we describe the major participants of various DDR pathways, their significance in carcinogenesis, and focus on the inhibitors developed against several key DDR proteins.
    Keywords:  DNA damage response; checkpoint inhibitors; double strand breaks; response inhibitors; synthetic lethality; targeted cancer therapy
    DOI:  https://doi.org/10.2174/1871520621666210608105735
  19. DNA Repair (Amst). 2021 May 11. pii: S1568-7864(21)00090-2. [Epub ahead of print]105 103134
    Perspectives on formaldehyde dysregulation: Mitochondrial DNA damage and repair in mammalian cells.
    Cristina A Nadalutti, Rajendra Prasad, Samuel H Wilson.
      Maintaining genome stability involves coordination between different subcellular compartments providing cells with DNA repair systems that safeguard against environmental and endogenous stresses. Organisms produce the chemically reactive molecule formaldehyde as a component of one-carbon metabolism, and cells maintain systems to regulate endogenous levels of formaldehyde under physiological conditions, preventing genotoxicity, among other adverse effects. Dysregulation of formaldehyde is associated with several diseases, including cancer and neurodegenerative disorders. In the present review, we discuss the complex topic of endogenous formaldehyde metabolism and summarize advances in research on fo dysregulation, along with future research perspectives.
    Keywords:  DNA damage; Formaldehyde; Mitochondrial DNA; One carbon metabolism
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103134
  20. Nat Commun. 2021 06 08. 12(1): 3448
    Dynamics of replication origin over-activation.
    Haiqing Fu, Christophe E Redon, Bhushan L Thakur, Koichi Utani, Robin Sebastian, Sang-Min Jang, Jacob M Gross, Sara Mosavarpour, Anna B Marks, Sophie Z Zhuang, Sarah B Lazar, Mishal Rao, Shira T Mencer, Adrian M Baris, Lorinc S Pongor, Mirit I Aladjem.
      Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.
    DOI:  https://doi.org/10.1038/s41467-021-23835-0
  21. Mol Cancer Res. 2021 Jun 07. pii: molcanres.0780.2020. [Epub ahead of print]
    RAD51AP1 loss attenuates colorectal cancer stem cell renewal and sensitizes to chemotherapy.
    Allison E Bridges, Sabarish Ramachandran, Kavin Tamizhmani, Utkarsh Parwal, Adrienne Lester, Pragya Rajpurohit, Daley S Morera, Sarah L Hasanali, Pachiappan Arjunan, Ravirajsingh N Jedeja, Nikhil Patel, Pamela M Marin, Hasan Korkaya, Nagendra Singh, Santhakumar Manicassamy, Puttur D Prasad, Vinata B Lokeshwar, Bal L Lokeshwar, Vadivel Ganapathy, Muthusamy Thangaraju.
      DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer (CRC). DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in CRC is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced CRC growth, we unraveled the role of RAD51AP1 (RAD51-Associated Protein 1), a protein involved in HR, in genotoxic carcinogen (Azoxymethane, AOM)-induced CRC. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and Dextran Sulfate Sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human CRC tissues, including BRAF/KRAS mutant CRC, and associated with reduced treatment response and poor prognosis. Rad51ap1 deficient mice were protected against AOM/DSS-induced CRC. These observations were recapitulated in a genetically engineered mouse model of CRC (ApcMin/+). Further, chemotherapy-resistant CRC is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and CRC stem cell (CRCSC) self-renewal. Implications: This study provides first in vivo evidence that RAD51AP1 plays a critical role in CRC growth and drug resistance by regulating CRCSC self-renewal.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0780
  22. Curr Opin Genet Dev. 2021 Jun 04. pii: S0959-437X(21)00060-5. [Epub ahead of print]71 1-9
    Communication between chromatin and homologous recombination.
    Priyanka Verma, Roger A Greenberg.
      Higher-order chromatin packing serves as a structural barrier to the recognition and repair of genomic lesions. The initiation and outcome of the repair response is dictated by a highly coordinated yet complex interplay between chromatin modifying enzymes and their cognate readers, damage induced chemical modifications, nucleosome density, transcriptional state, and cell cycle-dependent availability of DNA repair machinery. The physical and chemical properties of the DNA lesions themselves further regulate the nature of ensuing chromatin responses. Here we review recent discoveries across these various contexts, where chromatin regulates the homology-guided double-strand break repair mechanism, homologous recombination, and also highlight the key knowledge gaps vital to generate a holistic understanding of this process and its contributions to genome integrity.
    DOI:  https://doi.org/10.1016/j.gde.2021.05.006
  23. DNA Repair (Amst). 2021 Jun 03. pii: S1568-7864(21)00100-2. [Epub ahead of print]105 103144
    ADP-ribosylation of DNA and RNA.
    Joséphine Groslambert, Evgeniia Prokhorova, Ivan Ahel.
      ADP-ribosylation is a chemical modification of macromolecules found across all domains of life and known to regulate a variety of cellular processes. Notably, it has a well-established role in the DNA damage response. While it was historically known as a post-translational modification of proteins, recent studies have shown that nucleic acids can also serve as substrates of reversible ADP-ribosylation. More precisely, ADP-ribosylation of DNA bases, phosphorylated DNA ends and phosphorylated RNA ends have been reported. We will discuss these three types of modification in details. In a variety of bacterial species, including Mycobacterium tuberculosis, ADP-ribosylation of thymidine has emerged as the mode of action of a toxin-antitoxin system named DarTG, with the resultant products perceived as DNA damage by the cell. On the other hand, mammalian DNA damage sensors PARP1, PARP2 and PARP3 were shown to ADP-ribosylate phosphorylated ends of double-stranded DNA in vitro. Additionally, TRPT1 and several PARP enzymes, including PARP10, can add ADP-ribose to the 5'-phosphorylated end of single-stranded RNA in vitro, representing a novel RNA capping mechanism. Together, these discoveries have led to the emergence of a new and exciting research area, namely DNA and RNA ADP-ribosylation, that is likely to have far-reaching implications for the fields of DNA repair, replication and epigenetics.
    Keywords:  ADP-ribosylation; DNA damage response; DNA modification; PARP; RNA modification
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103144
  24. Sci Rep. 2021 Jun 09. 11(1): 12148
    DNA-PK inhibitor peposertib enhances p53-dependent cytotoxicity of DNA double-strand break inducing therapy in acute leukemia.
    Eric Haines, Yuki Nishida, Michael I Carr, Rafael Heinz Montoya, Lauren B Ostermann, Weiguo Zhang, Frank T Zenke, Andree Blaukat, Michael Andreeff, Lyubomir T Vassilev.
      Peposertib (M3814) is a potent and selective DNA-PK inhibitor in early clinical development. It effectively blocks non-homologous end-joining repair of DNA double-strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By suppressing DNA-PK catalytic activity in the presence of DNA DSB, M3814 potentiates ATM/p53 signaling leading to enhanced p53-dependent antitumor activity in tumor cells. Here, we investigated the therapeutic potential of M3814 in combination with DSB-inducing agents in leukemia cells and a patient-derived tumor. We show that in the presence of IR or topoisomerase II inhibitors, M3814 boosts the ATM/p53 response in acute leukemia cells leading to the elevation of p53 protein levels as well as its transcriptional activity. M3814 synergistically sensitized p53 wild-type, but not p53-deficient, AML cells to killing by DSB-inducing agents via p53-dependent apoptosis involving both intrinsic and extrinsic effector pathways. The antileukemic effect was further potentiated by enhancing daunorubicin-induced myeloid cell differentiation. Further, combined with the fixed-ratio liposomal formulation of daunorubicin and cytarabine, CPX-351, M3814 enhanced the efficacy against leukemia cells in vitro and in vivo without increasing hematopoietic toxicity, suggesting that DNA-PK inhibition could offer a novel clinical strategy for harnessing the anticancer potential of p53 in AML therapy.
    DOI:  https://doi.org/10.1038/s41598-021-90500-3
  25. iScience. 2021 May 21. 24(5): 102494
    DHODH inhibition modulates glucose metabolism and circulating GDF15, and improves metabolic balance.
    Juan Zhang, Graciela Terán, Mihaela Popa, Harsha Madapura, Marcus James Graeme Watson Ladds, Danai Lianoudaki, Jacob Grünler, Marie Arsenian-Henriksson, Emmet McCormack, Martin Enrique Rottenberg, Sergiu-Bogdan Catrina, Sonia Laín, Suhas Darekar.
      Dihydroorotate dehydrogenase (DHODH) is essential for the de novo synthesis of pyrimidine ribonucleotides, and as such, its inhibitors have been long used to treat autoimmune diseases and are in clinical trials for cancer and viral infections. Interestingly, DHODH is located in the inner mitochondrial membrane and contributes to provide ubiquinol to the respiratory chain. Thus, DHODH provides the link between nucleotide metabolism and mitochondrial function. Here we show that pharmacological inhibition of DHODH reduces mitochondrial respiration, promotes glycolysis, and enhances GLUT4 translocation to the cytoplasmic membrane and that by activating tumor suppressor p53, increases the expression of GDF15, a cytokine that reduces appetite and prolongs lifespan. In addition, similar to the antidiabetic drug metformin, we observed that in db/db mice, DHODH inhibitors elevate levels of circulating GDF15 and reduce food intake. Further analysis using this model for obesity-induced diabetes revealed that DHODH inhibitors delay pancreatic β cell death and improve metabolic balance.
    Keywords:  Biological sciences; Cellular physiology; Diabetology; Endocrinology; Physiology
    DOI:  https://doi.org/10.1016/j.isci.2021.102494
  26. J Mol Biol. 2021 Jun 06. pii: S0022-2836(21)00321-1. [Epub ahead of print] 167097
    Structural Insights into the Mechanism of Base Excision by MBD4.
    Lakshmi S Pidugu, Hilary Bright, Wen-Jen Lin, Chandrima Majumdar, Robert P Van Ostrand, Sheila S David, Edwin Pozharski, Alexander C Drohat.
      DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2'-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.
    Keywords:  5-methylcytosine; DNA glycosylase; G/T mismatch; base excision repair; helix-hairpin-helix motif
    DOI:  https://doi.org/10.1016/j.jmb.2021.167097
  27. DNA Repair (Amst). 2021 Jun 08. pii: S1568-7864(21)00111-7. [Epub ahead of print]105 103155
    DNA damage and regulation of protein homeostasis.
    Tanya T Paull.
      The accumulation of unrepaired DNA lesions is associated with many pathological outcomes in humans, particularly in neurodegenerative diseases and in normal aging. Evidence supporting a causal role for DNA damage in the onset and progression of neurodegenerative disease has come from rare human patients with mutations in DNA damage response genes as well as from model organisms; however, the generality of this relationship in the normal population is unclear. In addition, the relevance of DNA damage in the context of proteotoxic stress-the widely accepted paradigm for pathology during neurodegeneration-is not well understood. Here, observations supporting intertwined roles of DNA damage and proteotoxicity in aging-related neurological outcomes are reviewed, with particular emphasis on recent insights into the relationships between DNA repair and autophagy, the ubiquitin proteasome system, formation of protein aggregates, poly-ADP-ribose polymerization, and transcription-driven DNA lesions.
    Keywords:  DNA repair; Neurodegeneration; PARP; Protein aggregation; Protein homeostasis
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103155
  28. iScience. 2021 May 21. 24(5): 102493
    Replication protein A plays multifaceted roles complementary to specialized helicases in processing G-quadruplex DNA.
    Yi-Ran Wang, Ting-Ting Guo, Ya-Ting Zheng, Chang-Wei Lai, Bo Sun, Xu-Guang Xi, Xi-Miao Hou.
      G-quadruplexes (G4s) are non-canonical DNA structures with critical roles in DNA metabolisms. To resolve those structures that can cause replication fork stalling and genomic instability, single-stranded DNA-binding proteins and helicases are required. Here, we characterized the interplay between RPA and helicases on G4s using single-molecule FRET. We first discovered that human RPA efficiently prevents G4 formation by preempting ssDNA before its folding. RPA also differentially interacts with the folded G4s. However, helicases such as human BLM and yeast Pif1 have different G4 preferences from RPA mainly based on loop lengths. More importantly, both RPA and these helicases are required for the stable G4 unfolding, as RPA promotes helicase-mediated repetitive unfolding into durative linear state. Furthermore, BLM can traverse G4 obstacles temporarily disrupted by RPA and continue to unwind downstream duplex. We finally proposed the mechanisms underlying above functions of RPA in preventing, resolving, and assisting helicases to eliminate G4s.
    Keywords:  Molecular biology; Molecular structure
    DOI:  https://doi.org/10.1016/j.isci.2021.102493
  29. Cell Rep Med. 2021 May 18. 2(5): 100276
    Diverse immune response of DNA damage repair-deficient tumors.
    Tao Qing, Tomi Jun, Katherine E Lindblad, Amaia Lujambio, Michal Marczyk, Lajos Pusztai, Kuan-Lin Huang.
      Tumors with DNA damage repair (DDR) deficiency accumulate genomic alterations that may serve as neoantigens and increase sensitivity to immune checkpoint inhibitor. However, over half of DDR-deficient tumors are refractory to immunotherapy, and it remains unclear which mutations may promote immunogenicity in which cancer types. We integrate deleterious somatic and germline mutations and methylation data of DDR genes in 10,080 cancers representing 32 cancer types and evaluate the associations of these alterations with tumor neoantigens and immune infiltrates. Our analyses identify DDR pathway mutations that are associated with higher neoantigen loads, adaptive immune markers, and survival outcomes of immune checkpoint inhibitor-treated animal models and patients. Different immune phenotypes are associated with distinct types of DDR deficiency, depending on the cancer type context. The comprehensive catalog of immune response-associated DDR deficiency may explain variations in immunotherapy outcomes across DDR-deficient cancers and facilitate the development of genomic biomarkers for immunotherapy.
    Keywords:  DNA damage repair deficiency; germline variant; immune checkpoint inhibitor; immune infiltrate; immunogenicity; methylation; mismatch repair; neoantigen; somatic mutations; tumor mutation burden
    DOI:  https://doi.org/10.1016/j.xcrm.2021.100276
  30. Nat Commun. 2021 06 07. 12(1): 3338
    Cryo-EM structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in nucleotide excision repair.
    Trevor van Eeuwen, Yoonjung Shim, Hee Jong Kim, Tingting Zhao, Shrabani Basu, Benjamin A Garcia, Craig D Kaplan, Jung-Hyun Min, Kenji Murakami.
      The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.
    DOI:  https://doi.org/10.1038/s41467-021-23684-x
  31. Mutagenesis. 2021 Jun 07. pii: geab017. [Epub ahead of print]
    Genetic variations in 3´UTRs of SMUG1 and NEIL2 genes modulate breast cancer risk, survival and therapy response.
    Andrea Cumova, Veronika Vymetalkova, Alena Opattova, Veronika Bouskova, Barbara Pardini, Katerina Kopeckova, Renata Kozevnikovova, Katerina Lickova, Miloslav Ambrus, Ludmila Vodickova, Alessio Naccarati, Pavel Soucek, Pavel Vodicka.
      Breast cancer (BC) is the most frequent malignancy in women accounting for approximately 2 million new cases worldwide annually. Several genetic, epigenetic and environmental factors are known to be involved in BC development and progression, including alterations in post-transcriptional gene regulation mediated by microRNAs (miRNAs). Single nucleotide polymorphisms (SNPs) located in miRNA binding sites (miRSNPs) in 3'-untranslated (UTR) regions of target genes may affect miRNA-binding affinity and consequently modulate gene expression. We have previously reported a significant association of miRSNPs in the SMUG1 and NEIL2 genes with overall survival in colorectal cancer patients. SMUG1 and NEIL2 are DNA glycosylases involved in base excision DNA repair (BER). Assuming that certain genetic traits are common for solid tumours, we have investigated wherever variations in SMUG1 and NEIL2 genes display an association with BC risk, prognosis, and therapy response in a group of 673 BC patients and 675 healthy female controls. Patients with TC genotype of NEIL2 rs6997097 and receiving only hormonal therapy displayed markedly shorter overall survival (OS) (HR=4.15, 95% CI=1.7-10.16, P= 0.002) and disease-free survival (DFS) (HR=2.56, 95% CI=1.5-5.7, P= 0.02). Our results suggest that regulation of base excision repair glycosylases operated by miRNAs may modulate the prognosis of hormonally treated BC.
    Keywords:  NEIL2; SMUG1; base excision repair; breast cancer; miRSNPs
    DOI:  https://doi.org/10.1093/mutage/geab017
  32. Sci Signal. 2021 Jun 08. pii: eabc7405. [Epub ahead of print]14(686):
    Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics.
    Veerle W Daniels, Jason J Zoeller, Nick van Gastel, Kelley E McQueeney, Salma Parvin, Danielle S Potter, Geoffrey G Fell, Vinícius G Ferreira, Binyam Yilma, Rajat Gupta, Johan Spetz, Patrick D Bhola, Jennifer E Endress, Isaac S Harris, Emanuel Carrilho, Kristopher A Sarosiek, David T Scadden, Joan S Brugge, Anthony Letai.
      Cancer cells have differential metabolic dependencies compared to their nonmalignant counterparts. However, few metabolism-targeting compounds have been successful in clinical trials. Here, we investigated the metabolic vulnerabilities of triple-negative breast cancer (TNBC), particularly those metabolic perturbations that increased mitochondrial apoptotic priming and sensitivity to BH3 mimetics (drugs that antagonize antiapoptotic proteins). We used high-throughput dynamic BH3 profiling (HT-DBP) to screen a library of metabolism-perturbing small molecules, which revealed inhibitors of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) as top candidates. In some TNBC cells but not in nonmalignant cells, NAMPT inhibitors increased overall apoptotic priming and induced dependencies on specific antiapoptotic BCL-2 family members. Treatment of TNBC cells with NAMPT inhibitors sensitized them to subsequent treatment with BH3 mimetics. The combination of a NAMPT inhibitor (FK866) and an MCL-1 antagonist (S63845) reduced tumor growth in a TNBC patient-derived xenograft model in vivo. We found that NAMPT inhibition reduced NAD+ concentrations below a critical threshold that resulted in depletion of adenine, which was the metabolic trigger that primed TNBC cells for apoptosis. These findings demonstrate a close interaction between metabolic and mitochondrial apoptotic signaling pathways and reveal that exploitation of a tumor-specific metabolic vulnerability can sensitize some TNBC to BH3 mimetics.
    DOI:  https://doi.org/10.1126/scisignal.abc7405
  33. Nat Commun. 2021 06 07. 12(1): 3356
    Clinical CDK4/6 inhibitors induce selective and immediate dissociation of p21 from cyclin D-CDK4 to inhibit CDK2.
    Lindsey R Pack, Leighton H Daigh, Mingyu Chung, Tobias Meyer.
      Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors-palbociclib, ribociclib, or abemaciclib-immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms.
    DOI:  https://doi.org/10.1038/s41467-021-23612-z
  34. Cancer Discov. 2021 Jun 09.
    Preliminary Activity Seen with RAD51 Inhibitor.
      An investigational agent that inhibits RAD51-mediated homologous recombination DNA repair has shown preliminary signs of activity. In a phase I trial, CYT-0851 elicited three responses in patients with relapsed/refractory hematologic malignancies and advanced solid tumors and was associated with only mild side effects.
    DOI:  https://doi.org/10.1158/2159-8290.CD-NB2021-0355
  35. Nat Commun. 2021 06 09. 12(1): 3479
    Activation of PARP2/ARTD2 by DNA damage induces conformational changes relieving enzyme autoinhibition.
    Ezeogo Obaji, Mirko M Maksimainen, Albert Galera-Prat, Lari Lehtiö.
      Human PARP2/ARTD2 is an ADP-ribosyltransferase which, when activated by 5'-phosphorylated DNA ends, catalyses poly-ADP-ribosylation of itself, other proteins and DNA. In this study, a crystal structure of PARP2 in complex with an activating 5'-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, including reorganization of helical fragments, in the PARP2 regulatory domain. A comparison of PARP1 and PARP2 crystal structures reveals how binding to a DNA damage site leads to formation of a catalytically competent conformation. In this conformation, PARP2 is capable of binding substrate NAD+ and histone PARylation factor 1 that changes PARP2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.
    DOI:  https://doi.org/10.1038/s41467-021-23800-x
  36. Gastroenterology. 2021 Jun 04. pii: S0016-5085(21)03081-X. [Epub ahead of print]
    Tumor-specific delivery of 5FU-incorporated EGFR-targeted aptamers as an efficient treatment in pancreatic ductal adenocarcinoma models.
    Ujjwal M Mahajan, Qi Li, Ahmed Alnatsha, Jessica Maas, Michael Orth, Sebastian Hubertus Maier, Julian Peterhansl, Ivonne Regel, Matthias Sendler, Preshit R Wagh, Neha Mishra, Yonggan Xue, Prince Allawadhi, Georg Beyer, Jens-Peter Kühn, Thomas Marshall, Bettina Appel, Felix Lämmerhirt, Claus Belka, Sabine Müller, Frank-Ulrich Weiss, Kirsten Lauber, Markus M Lerch, Julia Mayerle.
      BACKGROUNDS AND AIMS: Fluoropyrimidine 5-fluorouracil (5FU) increasingly represents the chemotherapeutic backbone for neoadjuvant, adjuvant and palliative treatment of pancreatic ductal adenocarcinoma (PDAC). Even in combination with other agents, 5FU efficacy remains transient and limited. One explanation for the inadequate response is insufficient and nonspecific delivery of 5FU to the tumor.METHODS: We designed, generated and characterized 5FU-incorporated SELEX-selected EGFR-targeted aptamers for tumor-specific delivery of 5FU to PDAC cells and tested their therapeutic efficacy in vitro and in vivo.
    RESULTS: 5FU-EGFR-aptamers reduced proliferation in a concentration-dependent manner in mouse and human pancreatic cancer cell lines. Time-lapsed live imaging revealed EGFR-specific uptake of aptamers via clathrin-dependent endocytosis. 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell-lines. Biweekly treatment with 5FU-EGFR-aptamers reduced tumor burden in a syngeneic orthotopic transplantation model of PDAC, in an autochthonously growing genetically engineered PDAC model (LSL-KrasG12D/+, LSL-Trp53flox/+, Ptf1a-Cre (KPC)), in an orthotopic cell-line derived xenograft model using human PDAC cells in athymic mice (CDX; Crl:NU-Foxn1nu) as well as in patient-derived organoids (PDOs). Tumor growth was significantly attenuated during 5FU-EGFR-aptamer treatment in the course of follow-up.
    CONCLUSION: Tumor-specific targeted delivery of 5FU using EGFR-aptamers as carrier achieved high target specificity, overcame 5FU resistance, proved to be effective in a syngeneic orthotopic transplantation model, in KPC mice, in a CDX model as well as PDOs and therefore represents a promising backbone for pancreatic cancer chemotherapy in patients. Furthermore, our approach has the potential to target virtually any cancer entity sensitive to 5FU treatment by incorporating 5FU into a cancer-cell-targeting aptamers as delivery platform.
    Keywords:  5FU; aptamers; pancreatic cancer; targeted delivery
    DOI:  https://doi.org/10.1053/j.gastro.2021.05.055
  37. Sci Adv. 2021 Jun;pii: eabf1771. [Epub ahead of print]7(24):
    Polθ reverse transcribes RNA and promotes RNA-templated DNA repair.
    Gurushankar Chandramouly, Jiemin Zhao, Shane McDevitt, Timur Rusanov, Trung Hoang, Nikita Borisonnik, Taylor Treddinick, Felicia Wednesday Lopezcolorado, Tatiana Kent, Labiba A Siddique, Joseph Mallon, Jacklyn Huhn, Zainab Shoda, Ekaterina Kashkina, Alessandra Brambati, Jeremy M Stark, Xiaojiang S Chen, Richard T Pomerantz.
      Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Å crystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2'-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair.
    DOI:  https://doi.org/10.1126/sciadv.abf1771
  38. Cancer Discov. 2021 Jun 08. pii: candisc.1378.2020. [Epub ahead of print]
    AXL inhibition in macrophages stimulates host-versus-leukemia immunity and eradicates naive and treatment resistant leukemia.
    Irene Tirado-Gonzalez, Arnaud Descot, Devona Soetopo, Aleksandra Nevmerzhitskaya, Alexander Schaffer, Ivan-Maximiliano Kur, Ewelina Czlonka, Carolin Wachtel, Ioanna Tsoukala, Luise Muller, Anna-Lena Schafer, Maresa Weitmann, Petra Dinse, Emily Alberto, Michele C Buck, Jonathan J M Landry, Bianka Baying, Julia Slotta-Huspenina, Jenny Roesler, Patrick N Harter, Anne-Sophie Kubasch, Jörn Meinel, Eiman Elwakeel, Elisabeth Strack, Christine Tran Quang, Omar Abdel-Wahab, Marc Schmitz, Andreas Weigert, Tobias Schmid, Uwe Platzbecker, Vladimir Benes, Jacques Ghysdael, Halvard Bonig, Katharina S Gotze, Carla V Rothlin, Sourav Ghosh, Hind Medyouf.
      Acute leukemias are systemic malignancies associated with a dire outcome. Due to low immunogenicity, leukemias display a remarkable ability to evade immune control and are often resistant to checkpoint blockade. Here, we discover that leukemia cells actively establish a suppressive environment to prevent immune attacks by co-opting a signaling axis that skews macrophages towards a tumor promoting tissue repair phenotype, namely the GAS6/AXL axis. Using aggressive leukemia models, we demonstrate that ablation of the AXL receptor specifically in macrophages, or its ligand GAS6 in the environment, stimulates anti-leukemic immunity and elicits effective and lasting NK- and T-cell dependent immune response against naive and treatment resistant leukemia. Remarkably, AXL deficiency in macrophages also enables PD1 checkpoint blockade in PD1-refractory leukemias. Lastly, we provide proof-of-concept that a clinical grade AXL inhibitor can be used in combination with standard of care therapy to cure established leukemia, regardless on AXL expression in malignant cells.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-1378
  39. Oncogene. 2021 Jun 08.
    PELP1/SRC-3-dependent regulation of metabolic PFKFB kinases drives therapy resistant ER+ breast cancer.
    Thu H Truong, Elizabeth A Benner, Kyla M Hagen, Nuri A Temiz, Carlos Perez Kerkvliet, Ying Wang, Emilio Cortes-Sanchez, Chieh-Hsiang Yang, Marygrace C Trousdell, Thomas Pengo, Katrin P Guillen, Bryan E Welm, Camila O Dos Santos, Sucheta Telang, Carol A Lange, Julie H Ostrander.
      Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance.
    DOI:  https://doi.org/10.1038/s41388-021-01871-w
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