bims-numges 2022-01-02 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–01–02
twelve papers selected by
Sean Rudd, Karolinska Institutet

The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase.
Target Actionability Review: a systematic evaluation of replication stress as a therapeutic target for paediatric solid malignancies.
p16 Represses DNA Damage Repair via a Novel Ubiquitin-Dependent Signaling Cascade.
Chromatin and Nuclear Dynamics in the Maintenance of Replication Fork Integrity.
Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy.
Mitotic and DNA Damage Response Proteins: Maintaining the Genome Stability and Working for the Common Good.
Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.
DNA-PKcs kinase activity orchestrates both end-processing and end-ligation.
Transcription-Replication Collisions and Chromosome Fragility.
Assessing kinetics and recruitment of DNA repair factors using high content screens.
FDI-6 and olaparib synergistically inhibit the growth of pancreatic cancer by repressing BUB1, BRCA1 and CDC25A signaling pathways.
Decoding Cancer Variants of Unknown Significance for Helicase-Nuclease-RPA Complexes Orchestrating DNA Repair During Transcription and Replication.

J Biol Chem. 2021 Dec 22. pii: S0021-9258(21)01341-7. [Epub ahead of print] 101531

The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase.

Lisa N Heppler, Sanaz Attarha, Rosanne Persaud, Jennifer I Brown, Peng Wang, Boryana Petrova, Isidora Tošić, Foster B Burton, Yael Flamand, Sarah R Walker, Jennifer E Yeh, Roman A Zubarev, Massimiliano Gaetani, Naama Kanarek, Brent D G Page, David A Frank.

  Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many pro-tumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased, high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration (PISA) coupled with a metabolomic analysis. We identified human dihydrofolate reductase (DHFR) as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of DHFR inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.

Keywords:  Cancer therapy; STAT3; dihydrofolate reductase; folate metabolism; inhibition mechanism; pyrimethamine; small molecule; transcription regulation

DOI:  https://doi.org/10.1016/j.jbc.2021.101531
Eur J Cancer. 2021 Dec 25. pii: S0959-8049(21)01254-5. [Epub ahead of print]162 107-117

Target Actionability Review: a systematic evaluation of replication stress as a therapeutic target for paediatric solid malignancies.

Kaylee M Keller, Sonja Krausert, Apurva Gopisetty, Dan Luedtke, Jan Koster, Nil A Schubert, Ana Rodríguez, Sander R van Hooff, Damian Stichel, M Emmy M Dolman, Gilles Vassal, Stefan M Pfister, Hubert N Caron, Louis F Stancato, Jan J Molenaar, Natalie Jäger, Marcel Kool.

   BACKGROUND: Owing to the high numbers of paediatric cancer-related deaths, advances in therapeutic options for childhood cancer is a heavily studied field, especially over the past decade. Classical chemotherapy offers some therapeutic benefit but has proven long-term complications in survivors, and there is an urgent need to identify novel target-driven therapies. Replication stress is a major cause of genomic instability in cancer, triggering the stalling of the replication fork. Failure of molecular response by DNA damage checkpoints, DNA repair mechanisms and restarting the replication forks can exacerbate replication stress and initiate cell death pathways, thus presenting as a novel therapeutic target. To bridge the gap between preclinical evidence and clinical utility thereof, we apply the literature-driven systematic target actionability review methodology to published proof-of-concept (PoC) data related to the process of replication stress.
METHODS: A meticulous PubMed literature search was performed to gather replication stress-related articles (published between 2014 and 2021) across 16 different paediatric solid tumour types. Articles that fulfilled inclusion criteria were uploaded into the R2 informatics platform [r2.amc.nl] and assessed by critical appraisal. Key evidence based on nine pre-established PoC modules was summarised, and scores based on the quality and outcome of each study were assigned by two separate reviewers. Articles with discordant modules/scores were re-scored by a third independent reviewer, and a final consensus score was agreed upon by adjudication between all three reviewers. To visualise the final scores, an interactive heatmap summarising the evidence and scores associated with each PoC module across all, including paediatric tumour types, were generated.
RESULTS AND CONCLUSIONS: 145 publications related to targeting replication stress in paediatric tumours were systematically reviewed with an emphasis on DNA repair pathways and cell cycle checkpoint control. Although various targets in these pathways have been studied in these diseases to different extents, the results of this extensive literature search show that ATR, CHK1, PARP or WEE1 are the most promising targets using either single agents or in combination with chemotherapy or radiotherapy in neuroblastoma, osteosarcoma, high-grade glioma or medulloblastoma. Targeting these pathways in other paediatric malignancies may work as well, but here, the evidence was more limited. The evidence for other targets (such as ATM and DNA-PK) was also limited but showed promising results in some malignancies and requires more studies in other tumour types. Overall, we have created an extensive overview of targeting replication stress across 16 paediatric tumour types, which can be explored using the interactive heatmap on the R2 target actionability review platform [https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2_targetmap_v1].

Keywords:  Cell cycle checkpoints; DNA repair; Paediatric oncology; Preclinical research; Replication stress; Systematic review; Targeted drugs

DOI:  https://doi.org/10.1016/j.ejca.2021.11.030
Cancer Res. 2021 Dec 29. pii: canres.2101.2021. [Epub ahead of print]

p16 Represses DNA Damage Repair via a Novel Ubiquitin-Dependent Signaling Cascade.

David P Molkentine, Jessica M Molkentine, Kathleen A Bridges, David R Valdecanas, Annika Dhawan, Reshub Bahri, Andrew J Hefner, Manish Kumar, Liangpeng Yang, Mohamed Abdelhakiem, Phillip M Pifer, Vlad Sandulache, Aakash Sheth, Beth M Beadle, Howard D Thames, Kathryn A Mason, Curtis R Pickering, Raymond E Meyn, Heath D Skinner.

Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies than its HPV-negative counterpart. Here we show that p16, the clinically utilized surrogate for HPV positivity, renders cells more sensitive to radiation via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription, and degradation of ubiquitin-specific protease 7 (USP7) and TRIP12. Activation of this pathway in HPV-positive disease led to decreased homologous recombination (HR) and improved response to radiation, a phenomenon that can be recapitulated in HPV-negative disease using USP7 inhibitors in clinical development. This p16-driven axis induced sensitivity to PARP inhibition and potentially leads to "BRCAness" in head and neck squamous cell carcinoma (HNSCC) cells. Thus, these findings support a functional role for p16 in HPV-positive tumors in driving response to DNA damage, which can be exploited to improve outcomes in both HPV-positive and HPV-negative HNSCC patients.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2101
Front Genet. 2021 ;12 773426

Chromatin and Nuclear Dynamics in the Maintenance of Replication Fork Integrity.

Jack Wootton, Evi Soutoglou.

  Replication of the eukaryotic genome is a highly regulated process and stringent control is required to maintain genome integrity. In this review, we will discuss the many aspects of the chromatin and nuclear environment that play key roles in the regulation of both unperturbed and stressed replication. Firstly, the higher order organisation of the genome into A and B compartments, topologically associated domains (TADs) and sub-nuclear compartments has major implications in the control of replication timing. In addition, the local chromatin environment defined by non-canonical histone variants, histone post-translational modifications (PTMs) and enrichment of factors such as heterochromatin protein 1 (HP1) plays multiple roles in normal S phase progression and during the repair of replicative damage. Lastly, we will cover how the spatial organisation of stalled replication forks facilitates the resolution of replication stress.

Keywords:  DNA damage; DNA repair; DNA replication; chromatin; nucleus

DOI:  https://doi.org/10.3389/fgene.2021.773426
Front Immunol. 2021 ;12 797880

Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy.

Zu Ye, Yin Shi, Susan P Lees-Miller, John A Tainer.

  The DNA damage response (DDR) is an organized network of multiple interwoven components evolved to repair damaged DNA and maintain genome fidelity. Conceptually the DDR includes damage sensors, transducer kinases, and effectors to maintain genomic stability and accurate transmission of genetic information. We have recently gained a substantially improved molecular and mechanistic understanding of how DDR components are interconnected to inflammatory and immune responses to stress. DDR shapes both innate and adaptive immune pathways: (i) in the context of innate immunity, DDR components mainly enhance cytosolic DNA sensing and its downstream STimulator of INterferon Genes (STING)-dependent signaling; (ii) in the context of adaptive immunity, the DDR is needed for the assembly and diversification of antigen receptor genes that is requisite for T and B lymphocyte development. Imbalances between DNA damage and repair impair tissue homeostasis and lead to replication and transcription stress, mutation accumulation, and even cell death. These impacts from DDR defects can then drive tumorigenesis, secretion of inflammatory cytokines, and aberrant immune responses. Yet, DDR deficiency or inhibition can also directly enhance innate immune responses. Furthermore, DDR defects plus the higher mutation load in tumor cells synergistically produce primarily tumor-specific neoantigens, which are powerfully targeted in cancer immunotherapy by employing immune checkpoint inhibitors to amplify immune responses. Thus, elucidating DDR-immune response interplay may provide critical connections for harnessing immunomodulatory effects plus targeted inhibition to improve efficacy of radiation and chemotherapies, of immune checkpoint blockade, and of combined therapeutic strategies.

Keywords:  DNA damage; DNA repair; adaptive immunity; cGAS-STING; cancer therapy; immune response; immunomodulatory; innate immunity

DOI:  https://doi.org/10.3389/fimmu.2021.797880
Front Cell Dev Biol. 2021 ;9 700162

Mitotic and DNA Damage Response Proteins: Maintaining the Genome Stability and Working for the Common Good.

Fernando Luna-Maldonado, Marco A Andonegui-Elguera, José Díaz-Chávez, Luis A Herrera.

  Cellular function is highly dependent on genomic stability, which is mainly ensured by two cellular mechanisms: the DNA damage response (DDR) and the Spindle Assembly Checkpoint (SAC). The former provides the repair of damaged DNA, and the latter ensures correct chromosome segregation. This review focuses on recently emerging data indicating that the SAC and the DDR proteins function together throughout the cell cycle, suggesting crosstalk between both checkpoints to maintain genome stability.

Keywords:  DNA damage response (DDR); DNA repair; cancer; genomic stability; mitosis; spindle assembly checkpoint (SAC)

DOI:  https://doi.org/10.3389/fcell.2021.700162
Nat Struct Mol Biol. 2021 Dec 28.

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.

Julia F Greiwe, Thomas C R Miller, Julia Locke, Fabrizio Martino, Steven Howell, Anne Schreiber, Andrea Nans, John F X Diffley, Alessandro Costa.

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

DOI: https://doi.org/10.1038/s41594-021-00698-z
Trends Cell Biol. 2021 Dec 18. pii: S0962-8924(21)00248-8. [Epub ahead of print]

DNA-PKcs kinase activity orchestrates both end-processing and end-ligation.

Demis Menolfi, Shan Zha.

  Recent structural studies have revealed the atomic details of how the DNA-PKcs kinase protects DNA ends, recruits and activates Artemis endonuclease for end-processing, and phosphorylates itself for end-ligation, revealing the molecular details from end-processing to end-ligation, two critical phases of the non-homologous end-joining (NHEJ) DNA-double strand break repair pathway.

Keywords:  Artemis; DNA end-ligation; DNA end-processing; DNA-PK holoenzyme; non-homologous end-joining

DOI:  https://doi.org/10.1016/j.tcb.2021.12.002
Front Genet. 2021 ;12 804547

Transcription-Replication Collisions and Chromosome Fragility.

Wei Wu, Jing Na He, Mengjiao Lan, Pumin Zhang, Wai Kit Chu.

  Accurate replication of the entire genome is critical for cell division and propagation. Certain regions in the genome, such as fragile sites (common fragile sites, rare fragile sites, early replicating fragile sites), rDNA and telomeres, are intrinsically difficult to replicate, especially in the presence of replication stress caused by, for example, oncogene activation during tumor development. Therefore, these regions are particularly prone to deletions and chromosome rearrangements during tumorigenesis, rendering chromosome fragility. Although, the mechanism underlying their "difficult-to-replicate" nature and genomic instability is still not fully understood, accumulating evidence suggests transcription might be a major source of endogenous replication stress (RS) leading to chromosome fragility. Here, we provide an updated overview of how transcription affects chromosome fragility. Furthermore, we will use the well characterized common fragile sites (CFSs) as a model to discuss pathways involved in offsetting transcription-induced RS at these loci with a focus on the recently discovered atypical DNA synthesis repair pathway Mitotic DNA Synthesis (MiDAS).

Keywords:  fragile sites; mitotic DNA synthesis (MiDAS); replication; replication stress; transcription

DOI:  https://doi.org/10.3389/fgene.2021.804547
Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01676-4. [Epub ahead of print]37(13): 110176

Assessing kinetics and recruitment of DNA repair factors using high content screens.

Barbara Martinez-Pastor, Giorgia G Silveira, Thomas L Clarke, Dudley Chung, Yuchao Gu, Claudia Cosentino, Lance S Davidow, Gadea Mata, Sylvana Hassanieh, Jayme Salsman, Alberto Ciccia, Narkhyun Bae, Mark T Bedford, Diego Megias, Lee L Rubin, Alejo Efeyan, Graham Dellaire, Raul Mostoslavsky.

  Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

Keywords:  DNA damage foci; DNA repair; GOF; PHF20; chromatin accessibility; high throughput; laser microirradiation

DOI:  https://doi.org/10.1016/j.celrep.2021.110176
Pharmacol Res. 2021 Dec 22. pii: S1043-6618(21)00624-1. [Epub ahead of print]175 106040

FDI-6 and olaparib synergistically inhibit the growth of pancreatic cancer by repressing BUB1, BRCA1 and CDC25A signaling pathways.

Shi-Qi Wu, Shi-Hui Huang, Qian-Wen Lin, Yi-Xuan Tang, Lei Huang, Yun-Gen Xu, Shu-Ping Wang.

  Inducing homologous recombination (HR) deficiency is a promising strategy to broaden the indication of PARP1/2 inhibitors in pancreatic cancer treatment. In addition to inhibition kinases, repression of the transcriptional function of FOXM1 has been reported to inhibit HR-mediated DNA repair. We found that FOXM1 inhibitor FDI-6 and PARP1/2 inhibitor Olaparib synergistically inhibited the malignant growth of pancreatic cancer cells in vitro and in vivo. The results of bioinformatic analysis and mechanistic study showed that FOXM1 directly interacted with PARP1. Olaparib induced the feedback overexpression of PARP1/2, FOXM1, CDC25A, CCND1, CDK1, CCNA2, CCNB1, CDC25B, BRCA1/2 and Rad51 to promote the acceleration of cell mitosis and recovery of DNA repair, which caused the generation of adaptive resistance. FDI-6 reversed Olaparib-induced adaptive resistance and inhibited cell cycle progression and DNA damage repair by repressing the expression of FOXM1, PARP1/2, BUB1, CDC25A, BRCA1 and other genes-involved in cell cycle control and DNA damage repair. We believe that targeting FOXM1 and PARP1/2 is a promising combination therapy for pancreatic cancer without HR deficiency.

Keywords:  Cell cycle; DNA repair; FDI-6 (PubChem CID: 5175738); FOXM1; Olaparib (PubChem CID: 23725625); PARP1; Pancreatic cancer; Propidium bromide (PubChem CID: 16043037)

DOI:  https://doi.org/10.1016/j.phrs.2021.106040
Front Mol Biosci. 2021 ;8 791792

Decoding Cancer Variants of Unknown Significance for Helicase-Nuclease-RPA Complexes Orchestrating DNA Repair During Transcription and Replication.

Susan E Tsutakawa, Albino Bacolla, Panagiotis Katsonis, Amer Bralić, Samir M Hamdan, Olivier Lichtarge, John A Tainer, Chi-Lin Tsai.

  All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase-nuclease-RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary "driver" and "passenger" mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

Keywords:  VUS; cancer mutations; evolutionary action; helicase-nuclease; nucleotide excision repair; protein structure; replication forks; transcription

DOI:  https://doi.org/10.3389/fmolb.2021.791792