bims-numges 2022-06-26 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022‒06‒26
thirty papers selected by
Sean Rudd
Karolinska Institutet

WRN rescues replication forks compromised by a BRCA2 deficiency: Predictions for how inhibition of a helicase that suppresses premature aging tilts the balance to fork demise and chromosomal instability in cancer.
Post-Translational Modifications of PCNA: Guiding for the Best DNA Damage Tolerance Choice.
The TIP60-ATM axis regulates replication fork stability in BRCA-deficient cells.
Mechanisms used by cancer cells to tolerate drug-induced replication stress.
Rrp1, Rrp2 and Uls1 - Yeast SWI2/SNF2 DNA dependent translocases in genome stability maintenance.
Cockayne syndrome group B protein uses its DNA translocase activity to promote mitotic DNA synthesis.
DDK: The Outsourced Kinase of Chromosome Maintenance.
Clickable Cisplatin Derivatives as Versatile Tools to Probe the DNA Damage Response to Chemotherapy.
Essential Roles of Ribonucleotide Reductases under DNA Damage and Replication Stresses in Cryptococcus neoformans.
Migrating bubble synthesis promotes mutagenesis through lesions in its template.
DNA damage-induced transcription stress triggers the genome-wide degradation of promoter-bound Pol II.
Imaging the Response to DNA Damage in Heterochromatin Domains.
Synthetic Lethality Targeting Polθ.
Effective Radiosensitization of Bladder Cancer Cells by Pharmacological Inhibition of DNA-PK and ATR.
The Influence of PARP, ATR, CHK1 Inhibitors on Premature Mitotic Entry and Genomic Instability in High-Grade Serous BRCAMUT and BRCAWT Ovarian Cancer Cells.
WASHC1 interacts with MCM2-7 complex to promote cell survival under replication stress.
Structural and Molecular Kinetic Features of Activities of DNA Polymerases.
Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function.
Comprehensive analysis of cis- and trans-acting factors affecting ectopic Break-Induced Replication.
Monitoring DNA polymerase β mitochondrial localization and dynamics.
Inhibitors of the Cancer Target Ribonucleotide Reductase, Past and Present.
A Lack of Effectiveness in the ATM-Orchestrated DNA Damage Response Contributes to the DNA Repair Defect of HPV-Positive Head and Neck Cancer Cells.
Lethal and Non-Lethal Functions of Caspases in the DNA Damage Response.
RNAPII response to transcription-blocking DNA lesions in mammalian cells.
A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA.
PBRM1 Deficiency Sensitizes Renal Cancer Cells to DNMT Inhibitor 5-Fluoro-2'-Deoxycytidine.
Targeting therapeutic resistance and multinucleate giant cells in CCNE1-amplified HR-proficient ovarian cancer.
A CDK activity buffer ensures mitotic completion.
Identification of Human Dihydroorotate Dehydrogenase Inhibitor by a Pharmacophore-Based Virtual Screening Study.
Temozolomide-induced guanine mutations create exploitable vulnerabilities of guanine-rich DNA and RNA regions in drug-resistant gliomas.

Bioessays. 2022 Jun 25. e2200057

WRN rescues replication forks compromised by a BRCA2 deficiency: Predictions for how inhibition of a helicase that suppresses premature aging tilts the balance to fork demise and chromosomal instability in cancer.

Arindam Datta, Robert M Brosh.

  Hereditary breast and ovarian cancers are frequently attributed to germline mutations in the tumor suppressor genes BRCA1 and BRCA2. BRCA1/2 act to repair double-strand breaks (DSBs) and suppress the demise of unstable replication forks. Our work elucidated a dynamic interplay between BRCA2 and the WRN DNA helicase/exonuclease defective in the premature aging disorder Werner syndrome. WRN and BRCA2 participate in complementary pathways to stabilize replication forks in cancer cells, allowing them to proliferate. Whether the functional overlap of WRN and BRCA2 is relevant to replication at gaps between newly synthesized DNA fragments, protection of telomeres, and/or metabolism of secondary DNA structures remain to be determined. Advances in understanding the mechanisms elicited during replication stress have prompted the community to reconsider avenues for cancer therapy. Insights from studies of PARP or topoisomerase inhibitors provide working models for the investigation of WRN's mechanism of action. We discuss these topics, focusing on the implications of the WRN-BRCA2 genetic interaction under conditions of replication stress.

Keywords:  BRCA2; DNA repair; WRN; aging; cancer; genetic disease; genomic instability; replication stress; synthetic lethality

DOI:  https://doi.org/10.1002/bies.202200057
J Fungi (Basel). 2022 Jun 10. pii: 621. [Epub ahead of print]8(6):

Post-Translational Modifications of PCNA: Guiding for the Best DNA Damage Tolerance Choice.

Gemma Bellí, Neus Colomina, Laia Castells-Roca, Neus P Lorite.

  The sliding clamp PCNA is a multifunctional homotrimer mainly linked to DNA replication. During this process, cells must ensure an accurate and complete genome replication when constantly challenged by the presence of DNA lesions. Post-translational modifications of PCNA play a crucial role in channeling DNA damage tolerance (DDT) and repair mechanisms to bypass unrepaired lesions and promote optimal fork replication restart. PCNA ubiquitination processes trigger the following two main DDT sub-pathways: Rad6/Rad18-dependent PCNA monoubiquitination and Ubc13-Mms2/Rad5-mediated PCNA polyubiquitination, promoting error-prone translation synthesis (TLS) or error-free template switch (TS) pathways, respectively. However, the fork protection mechanism leading to TS during fork reversal is still poorly understood. In contrast, PCNA sumoylation impedes the homologous recombination (HR)-mediated salvage recombination (SR) repair pathway. Focusing on Saccharomyces cerevisiae budding yeast, we summarized PCNA related-DDT and repair mechanisms that coordinately sustain genome stability and cell survival. In addition, we compared PCNA sequences from various fungal pathogens, considering recent advances in structural features. Importantly, the identification of PCNA epitopes may lead to potential fungal targets for antifungal drug development.

Keywords:  DNA damage tolerance; DNA replication forks; DNA replication stress; PCNA; fungal genome stability; post-translational modifications; salvage recombination; template switch; translesion synthesis

DOI:  https://doi.org/10.3390/jof8060621
Oncogenesis. 2022 Jun 18. 11(1): 33

The TIP60-ATM axis regulates replication fork stability in BRCA-deficient cells.

Emily M Schleicher, Ashna Dhoonmoon, Lindsey M Jackson, Jude B Khatib, Claudia M Nicolae, George-Lucian Moldovan.

Maintenance of replication fork stability is essential for genome preservation. Stalled replication forks can be reversed by translocases such as SMARCAL1, and unless protected through the activity of the BRCA pathway, are subsequently subjected to nucleolytic degradation. The ATM and ATR kinases are master regulators of the DNA damage response. ATM activation upon DNA damage is mediated by the acetyltransferase TIP60. Here, we show that the TIP60-ATM pathway promotes replication fork reversal by recruiting SMARCAL1 to stalled forks. This enables fork degradation in BRCA-deficient cells. We also show that this ATM activity is not shared by ATR. Moreover, we performed a series of genome-wide CRISPR knockout genetic screens to identify genetic determinants of the cellular sensitivity to ATM inhibition in wildtype and BRCA2-knockout cells, and validated the top hits from multiple screens. We provide a valuable list of common genes which regulate the response to multiple ATM inhibitors. Importantly, we identify a differential response of wildtype and BRCA2-deficient cells to these inhibitors. In BRCA2-knockout cells, DNA repair genes (including RAD17, MDC1, and USP28) were essential for survival upon ATM inhibitor treatment, which was not the case in wild-type cells. These findings may eventually help guide the way for rational deployment of ATM inhibitors in the clinic.

DOI: https://doi.org/10.1038/s41389-022-00410-w
Cancer Lett. 2022 Jun 21. pii: S0304-3835(22)00288-9. [Epub ahead of print] 215804

Mechanisms used by cancer cells to tolerate drug-induced replication stress.

Hendrika A Segeren, Bart Westendorp.

  Activation of oncogenes in cancer cells forces cell proliferation, leading to DNA replication stress (RS). As a consequence, cancer cells heavily rely on the intra S-phase checkpoint for survival. This fundamental principle formed the basis for the development of inhibitors against key players of the intra S-phase checkpoint, ATR and CHK1. These drugs are often combined with chemotherapeutic drugs that interfere with DNA replication to exacerbate RS and exhaust the intra S-phase checkpoint in cancer cells. However, drug resistance impedes efficient clinical use, suggesting that some cancer cells tolerate severe RS. In this review, we describe how an increased nucleotide pool, boosted stabilization and repair of stalled forks and firing of dormant origins fortify the RS response in cancer cells. Notably, the vast majority of the genes that confer RS tolerance are regulated by the E2F and NRF2 transcription factors. These transcriptional programs are frequently activated in cancer cells, allowing simultaneous activation of multiple tolerance avenues. We propose that the E2F and NRF2 transcriptional programs can be used as biomarker to select patients for treatment with RS-inducing drugs and as novel targets to kill RS-tolerant cancer cells. Together, this review aims to provide a framework to maximally exploit RS as an Achilles' heel of cancer cells.

Keywords:  Chemoresistance; E2F; Intra S-Phase checkpoint; NRF2

DOI:  https://doi.org/10.1016/j.canlet.2022.215804
DNA Repair (Amst). 2022 Jun 10. pii: S1568-7864(22)00089-1. [Epub ahead of print]116 103356

Rrp1, Rrp2 and Uls1 - Yeast SWI2/SNF2 DNA dependent translocases in genome stability maintenance.

Karol Kramarz, Dorota Dziadkowiec.

  Multiple eukaryotic SWI2/SNF2 DNA translocases safeguard genome integrity, mostly by remodelling nucleosomes, but also by fine-tuning mechanisms of DNA repair, such as homologous recombination. Among this large family there is a unique class of Rad5/16-like enzymes, including Saccharomyces cerevisiae Uls1 and its Schizosaccharomyces pombe orthologues Rrp1 and Rrp2, that have both translocase and E3 ubiquitin ligase activities, and are often directed towards their substrates by SUMOylation. Here we summarize recent advances in understanding how different activities of these yeast proteins jointly contribute to their important roles in replication stress response particularly at centromeres and telomeres. This extends the possible range of functions performed by this class of SNF2 enzymes in human cells involving both their translocase and ubiquitin ligase activities and related to SUMOylation pathways within the nucleus.

Keywords:  DNA repair; Replication stress; SUMOylation; Translocase; Ubiquitin ligase; Yeast

DOI:  https://doi.org/10.1016/j.dnarep.2022.103356
DNA Repair (Amst). 2022 Jun 17. pii: S1568-7864(22)00087-8. [Epub ahead of print]116 103354

Cockayne syndrome group B protein uses its DNA translocase activity to promote mitotic DNA synthesis.

Shixin Cui, John R Walker, Nicole L Batenburg, Xu-Dong Zhu.

  Mitotic DNA synthesis, also known as MiDAS, has been suggested to be a form of RAD52-dependent break-induced replication (BIR) that repairs under-replicated DNA regions of the genome in mitosis prior to chromosome segregation. Cockayne syndrome group B (CSB) protein, a chromatin remodeler of the SNF2 family, has been implicated in RAD52-dependent BIR repair of stalled replication forks. However, whether CSB plays a role in MiDAS has not been characterized. Here, we report that CSB functions epistatically with RAD52 to promote MiDAS at common fragile sites in response to replication stress, and prevents genomic instability associated with defects in MiDAS. We show that CSB is dependent upon the conserved phenylalanine at position 796 (F796), which lies in the recently-reported pulling pin that is required for CSB's translocase activity, to mediate MiDAS, suggesting that CSB uses its DNA translocase activity to promote MiDAS. Structural analysis reveals that CSB shares with a subset of SNF2 family proteins a translocase regulatory region (TRR), which is important for CSB's function in MiDAS. We further demonstrate that phosphorylation of S1013 in the TRR regulates the function of CSB in MiDAS and restart of stalled forks but not in fork degradation in BRCA2-deficient cells and UV repair. Taken together, these results suggest that the DNA translocase activity of CSB in vivo is likely to be highly regulated by post-translational modification in a context-specific manner.

Keywords:  Cockayne syndrome group B (CSB) protein; DNA replication stress; DNA translocase activity; Mitotic DNA synthesis; SNF2 chromatin remodelers

DOI:  https://doi.org/10.1016/j.dnarep.2022.103354
Biology (Basel). 2022 Jun 07. pii: 877. [Epub ahead of print]11(6):

DDK: The Outsourced Kinase of Chromosome Maintenance.

Peter J Gillespie, J Julian Blow.

  The maintenance of genomic stability during the mitotic cell-cycle not only demands that the DNA is duplicated and repaired with high fidelity, but that following DNA replication the chromatin composition is perpetuated and that the duplicated chromatids remain tethered until their anaphase segregation. The coordination of these processes during S phase is achieved by both cyclin-dependent kinase, CDK, and Dbf4-dependent kinase, DDK. CDK orchestrates the activation of DDK at the G1-to-S transition, acting as the 'global' regulator of S phase and cell-cycle progression, whilst 'local' control of the initiation of DNA replication and repair and their coordination with the re-formation of local chromatin environments and the establishment of chromatid cohesion are delegated to DDK. Here, we discuss the regulation and the multiple roles of DDK in ensuring chromosome maintenance. Regulation of replication initiation by DDK has long been known to involve phosphorylation of MCM2-7 subunits, but more recent results have indicated that Treslin:MTBP might also be important substrates. Molecular mechanisms by which DDK regulates replisome stability and replicated chromatid cohesion are less well understood, though important new insights have been reported recently. We discuss how the 'outsourcing' of activities required for chromosome maintenance to DDK allows CDK to maintain outright control of S phase progression and the cell-cycle phase transitions whilst permitting ongoing chromatin replication and cohesion establishment to be completed and achieved faithfully.

Keywords:  CDK; DDK; DNA repair; DNA replication; chromatid cohesion; epigenetic inheritance; replication fork stability

DOI:  https://doi.org/10.3390/biology11060877
Front Oncol. 2022 ;12 874201

Clickable Cisplatin Derivatives as Versatile Tools to Probe the DNA Damage Response to Chemotherapy.

Amandine Moretton, Jana Slyskova, Marwan E Simaan, Emili A Arasa-Verge, Mathilde Meyenberg, D Alonso Cerrón-Infantes, Miriam M Unterlass, Joanna I Loizou.

  Cisplatin induces DNA crosslinks that are highly cytotoxic. Hence, platinum complexes are frequently used in the treatment of a broad range of cancers. Efficiency of cisplatin treatment is limited by the tumor-specific DNA damage response to the generated lesions. We reasoned that better tools to investigate the repair of DNA crosslinks induced by cisplatin would therefore be highly useful in addressing drug limitations. Here, we synthesized a series of cisplatin derivatives that are compatible with click chemistry, thus allowing visualization and isolation of DNA-platinum crosslinks from cells to study cellular responses. We prioritized one alkyne and one azide Pt(II) derivative, Pt-alkyne-53 and Pt-azide-64, for further biological characterization. We demonstrate that both compounds bind DNA and generate DNA lesions and that the viability of treated cells depends on the active DNA repair machinery. We also show that the compounds are clickable with both a fluorescent probe as well as biotin, thus they can be visualized in cells, and their ability to induce crosslinks in genomic DNA can be quantified. Finally, we show that Pt-alkyne-53 can be used to identify DNA repair proteins that bind within its proximity to facilitate its removal from DNA. The compounds we report here can be used as valuable experimental tools to investigate the DNA damage response to platinum complexes and hence might shed light on mechanisms of chemoresistance.

Keywords:  DNA Damage; DNA crosslinks; DNA repair; chemoresistance; chemotherapy; cisplatin; click chemistry

DOI:  https://doi.org/10.3389/fonc.2022.874201
Microbiol Spectr. 2022 Jun 23. e0104422

Essential Roles of Ribonucleotide Reductases under DNA Damage and Replication Stresses in Cryptococcus neoformans.

Kwang-Woo Jung, Sunhak Kwon, Jong-Hyun Jung, Yong-Sun Bahn.

  A balance in the deoxyribonucleotide (dNTPs) intracellular concentration is critical for the DNA replication and repair processes. In the model yeast Saccharomyces cerevisiae, the Mec1-Rad53-Dun1 kinase cascade mainly regulates the ribonucleotide reductase (RNR) gene expression during DNA replication and DNA damage stress. However, the RNR regulatory mechanisms in basidiomycete fungi during DNA replication and damage stress remain elusive. Here, we observed that in C. neoformans, RNR1 (large RNR subunit) and RNR21 (one small RNR subunit) were required for cell viability, but not RNR22 (another small RNR subunit). RNR22 overexpression compensated for the lethality of RNR21 suppression. In contrast to the regulatory mechanisms of RNRs in S. cerevisiae, Rad53 and Chk1 kinases cooperatively or divergently controlled RNR1 and RNR21 expression under DNA damage and DNA replication stress. In particular, this study revealed that Chk1 mainly regulated RNR1 expression during DNA replication stress, whereas Rad53, rather than Chk1, played a significant role in controlling the expression of RNR21 during DNA damage stress. Furthermore, the expression of RNR22, not but RNR1 and RNR21, was suppressed by the Ssn6-Tup1 complex during DNA replication stress. Notably, we observed that RNR1 expression was mainly regulated by Mbs1, whereas RNR21 expression was cooperatively controlled by Mbs1 and Bdr1 as downstream factors of Rad53 and Chk1 during DNA replication and damage stress. Collectively, the regulation of RNRs in C. neoformans has both evolutionarily conserved and divergent features in DNA replication and DNA damage stress, compared with other yeasts. IMPORTANCE Upon DNA replication or damage stresses, it is critical to provide proper levels of deoxynucleotide triphosphates (dNTPs) and activate DNA repair machinery. Ribonucleotide reductases (RNRs), which are composed of large and small subunits, are required for synthesizing dNTP. An imbalance in the intracellular concentration of dNTPs caused by the perturbation of RNR results in a reduction in DNA repair fidelity. Despite the importance of their roles, functions and regulations of RNR have not been elucidated in the basidiomycete fungi. In this study, we found that the roles of RNR1, RNR21, and RNR22 genes encoding RNR subunits in the viability of C. neoformans. Furthermore, their expression levels are divergently regulated by the Rad53-Chk1 pathway and the Ssn6-Tup1 complex in response to DNA replication and damage stresses. Therefore, this study provides insight into the regulatory mechanisms of RNR genes to DNA replication and damage stresses in basidiomycete fungi.

Keywords:  Cryptococcus neoformans; DNA damage stress; DNA replication stress; ribonucleotide reductase

DOI:  https://doi.org/10.1128/spectrum.01044-22
Nucleic Acids Res. 2022 Jun 24. pii: gkac520. [Epub ahead of print]

Migrating bubble synthesis promotes mutagenesis through lesions in its template.

Beth Osia, Jerzy Twarowski, Tyler Jackson, Kirill Lobachev, Liping Liu, Anna Malkova.

Break-induced replication (BIR) proceeds via a migrating D-loop for hundreds of kilobases and is highly mutagenic. Previous studies identified long single-stranded (ss) nascent DNA that accumulates during leading strand synthesis to be a target for DNA damage and a primary source of BIR-induced mutagenesis. Here, we describe a new important source of mutagenic ssDNA formed during BIR: the ssDNA template for leading strand BIR synthesis formed during D-loop migration. Specifically, we demonstrate that this D-loop bottom template strand (D-BTS) is susceptible to APOBEC3A (A3A)-induced DNA lesions leading to mutations associated with BIR. Also, we demonstrate that BIR-associated ssDNA promotes an additional type of genetic instability: replication slippage between microhomologies stimulated by inverted DNA repeats. Based on our results we propose that these events are stimulated by both known sources of ssDNA formed during BIR, nascent DNA formed by leading strand synthesis, and the D-BTS that we describe here. Together we report a new source of mutagenesis during BIR that may also be shared by other homologous recombination pathways driven by D-loop repair synthesis.

DOI: https://doi.org/10.1093/nar/gkac520
Nat Commun. 2022 Jun 24. 13(1): 3624

DNA damage-induced transcription stress triggers the genome-wide degradation of promoter-bound Pol II.

Barbara Steurer, Roel C Janssens, Marit E Geijer, Fernando Aprile-Garcia, Bart Geverts, Arjan F Theil, Barbara Hummel, Martin E van Royen, Bastiaan Evers, René Bernards, Adriaan B Houtsmuller, Ritwick Sawarkar, Jurgen Marteijn.

The precise regulation of RNA Polymerase II (Pol II) transcription after genotoxic stress is crucial for proper execution of the DNA damage-induced stress response. While stalling of Pol II on transcription-blocking lesions (TBLs) blocks transcript elongation and initiates DNA repair in cis, TBLs additionally elicit a response in trans that regulates transcription genome-wide. Here we uncover that, after an initial elongation block in cis, TBLs trigger the genome-wide VCP-mediated proteasomal degradation of promoter-bound, P-Ser5-modified Pol II in trans. This degradation is mechanistically distinct from processing of TBL-stalled Pol II, is signaled via GSK3, and contributes to the TBL-induced transcription block, even in transcription-coupled repair-deficient cells. Thus, our data reveal the targeted degradation of promoter-bound Pol II as a critical pathway that allows cells to cope with DNA damage-induced transcription stress and enables the genome-wide adaptation of transcription to genotoxic stress.

DOI: https://doi.org/10.1038/s41467-022-31329-w
Front Cell Dev Biol. 2022 ;10 920267

Imaging the Response to DNA Damage in Heterochromatin Domains.

Audrey Chansard, Enrico Pobega, Pierre Caron, Sophie E Polo.

  The eukaryotic genome is assembled in a nucleoprotein complex called chromatin, whose organization markedly influences the repair of DNA lesions. For instance, compact chromatin states, broadly categorized as heterochromatin, present a challenging environment for DNA damage repair. Through transcriptional silencing, heterochromatin also plays a vital role in the maintenance of genomic integrity and cellular homeostasis. It is thus of critical importance to decipher whether and how heterochromatin affects the DNA damage response (DDR) to understand how this chromatin state is preserved after DNA damage. Here, we present two laser micro-irradiation-based methods for imaging the DDR in heterochromatin domains in mammalian cells. These methods allow DNA damage targeting to specific subnuclear compartments, direct visualization of the DDR and image-based quantification of the repair response. We apply them to study DNA double-strand break repair pathways in facultative heterochromatin and the repair of UV photoproducts in constitutive heterochromatin. We discuss the advantages and limitations of these methods compared to other targeted approaches for DNA damage induction.

Keywords:  DNA damage; DNA repair; UV; confocal microscopy; heterochromatin; laser micro-irradiation

DOI:  https://doi.org/10.3389/fcell.2022.920267
Genes (Basel). 2022 Jun 20. pii: 1101. [Epub ahead of print]13(6):

Synthetic Lethality Targeting Polθ.

Małgorzata Drzewiecka, Gabriela Barszczewska-Pietraszek, Piotr Czarny, Tomasz Skorski, Tomasz Śliwiński.

  Research studies regarding synthetic lethality (SL) in human cells are primarily motivated by the potential of this phenomenon to be an effective, but at the same time, safe to the patient's anti-cancer chemotherapy. Among the factors that are targets for the induction of the synthetic lethality effect, those involved in DNA repair seem to be the most relevant. Specifically, when mutation in one of the canonical DNA double-strand break (DSB) repair pathways occurs, which is a frequent event in cancer cells, the alternative pathways may be a promising target for the elimination of abnormal cells. Currently, inhibiting RAD52 and/or PARP1 in the tumor cells that are deficient in the canonical repair pathways has been the potential target for inducing the effect of synthetic lethality. Unfortunately, the development of resistance to commonly used PARP1 inhibitors (PARPi) represents the greatest obstacle to working out a successful treatment protocol. DNA polymerase theta (Polθ), encoded by the POLQ gene, plays a key role in an alternative DSB repair pathway-theta-mediated end joining (TMEJ). Thus, it is a promising target in the treatment of tumors harboring deficiencies in homologous recombination repair (HRR), where its inhibition can induce SL. In this review, the authors discuss the current state of knowledge on Polθ as a potential target for synthetic lethality-based anticancer therapies.

Keywords:  DNA damage; DNA repair; personalized medicine; polymerase theta; synthetic lethality

DOI:  https://doi.org/10.3390/genes13061101
Biomedicines. 2022 May 30. pii: 1277. [Epub ahead of print]10(6):

Effective Radiosensitization of Bladder Cancer Cells by Pharmacological Inhibition of DNA-PK and ATR.

Ahmed Ali Chughtai, Julia Pannhausen, Pia Dinger, Julia Wirtz, Ruth Knüchel, Nadine T Gaisa, Michael J Eble, Michael Rose.

  This study aims at analyzing the impact of the pharmacological inhibition of DNA damage response (DDR) targets (DNA-PK and ATR) on radiosensitization of bladder cancer cell lines of different molecular/histological subtypes. Applying DNA-PK (AZD7648) and ATR (Ceralasertib) inhibitors on SCaBER, J82 and VMCUB-1 bladder cancer cell lines, we revealed sensitization upon ionizing radiation (IR), i.e., the IC50 for each drug shifted to a lower drug concentration with increased IR doses. In line with this, drug exposure retarded DNA repair after IR-induced DNA damage visualized by a neutral comet assay. Western blot analyses confirmed specific inhibition of targeted DDR pathways in the analyzed bladder cancer cell lines, i.e., drugs blocked DNA-PK phosphorylation at Ser2056 and the ATR downstream mediator CHK1 at Ser317. Interestingly, clonogenic survival assays indicated a cell-line-dependent synergism of combined DDR inhibition upon IR. Calculating combined index (CI) values, with and without IR, according to the Chou-Talalay method, confirmed drug- and IR-dose-specific synergistic CI values. Thus, we provide functional evidence that DNA-PK and ATR inhibitors specifically target corresponding DDR pathways retarding the DNA repair process at nano-molar concentrations. This, in turn, leads to a strong radiosensitizing effect and impairs the survival of bladder cancer cells.

Keywords:  ATR; AZD7648; Ceralasertib; DNA-PK; bladder cancer; ionizing radiation (IR); targeted therapy

DOI:  https://doi.org/10.3390/biomedicines10061277
Cells. 2022 Jun 10. pii: 1889. [Epub ahead of print]11(12):

The Influence of PARP, ATR, CHK1 Inhibitors on Premature Mitotic Entry and Genomic Instability in High-Grade Serous BRCAMUT and BRCAWT Ovarian Cancer Cells.

Patrycja Gralewska, Arkadiusz Gajek, Dorota Rybaczek, Agnieszka Marczak, Aneta Rogalska.

  Olaparib is a poly (ADP-ribose) polymerase inhibitor (PARPi) that inhibits PARP1/2, leading to replication-induced DNA damage that requires homologous recombination repair. Olaparib is often insufficient to treat BRCA-mutated (BRCAMUT) and BRCA wild-type (BRCAWT) high-grade serous ovarian carcinomas (HGSOCs). We examined the short-term (up to 48 h) efficacy of PARPi treatment on a DNA damage response pathway mediated by ATR and CHK1 kinases in BRCAMUT (PEO-1) and BRCAWT (SKOV-3 and OV-90) cells. The combination of ATRi/CHK1i with PARPi was not more cytotoxic than ATR and CHK1 monotherapy. The combination of olaparib with inhibitors of the ATR/CHK1 pathway generated chromosomal abnormalities, independent on BRCAMUT status of cells and formed of micronuclei (MN). However, the beneficial effect of the PARPi:ATRi combination on MN was seen only in the PEO1 BRCAMUT line. Monotherapy with ATR/CHK1 inhibitors reduced BrdU incorporation due to a slower rate of DNA synthesis, which resulted from elevated levels of replication stress, while simultaneous blockade of PARP and ATR caused beneficial effects only in OV-90 cells. Inhibition of ATR/CHK1 increased the formation of double-strand breaks as measured by increased γH2AX expression at collapsed replication forks, resulting in increased levels of apoptosis. Our findings indicate that ATR and CHK1 inhibitors provoke premature mitotic entry, leading to genomic instability and ultimately cell death.

Keywords:  ATR inhibitor; CHK1 inhibitor; PARP inhibitor; ovarian cancer; replication stress; targeted therapy

DOI:  https://doi.org/10.3390/cells11121889
Mol Biol Rep. 2022 Jun 22.

WASHC1 interacts with MCM2-7 complex to promote cell survival under replication stress.

Yu Hong, He Sun, Xian Hong, Cai-Ping Yang, Daniel D Billadeau, Tao Wang, Zhi-Hui Deng.

  BACKGROUND: WASHC1 is a member of the Wiskott-Aldrich syndrome protein (WASP) family and is involved in endosomal protein sorting and trafficking through the generation of filamentous actin (F-actin) via activation of the Arp2/3 complex. There is increasing evidence that WASHC1 is present in the nucleus and nuclear WASHC1 plays important roles in regulating gene transcription, DNA repair as well as maintaining nuclear organization. However, the multi-faceted functions of nuclear WASHC1 still need to be clarified.METHODS AND RESULTS: We show here that WASHC1 interacts with several components of the minichromosome maintenance (MCM) 2-7 complex by using co-immunoprecipitation and in situ proximity ligation assay. WASHC1-depleted cells display normal DNA replication and S-phase progression. However, loss of WASHC1 sensitizes HeLa cells to DNA replication inhibitor hydroxyurea (HU) and increases chromosome instability of HeLa and 3T3 cells under condition of HU-induced replication stress. Re-expression of nuclear WASHC1 in WASHC1KO 3T3 cells rescues the deficiency of WASHC1KO cells in the chromosomal stability after HU treatment. Moreover, chromatin immunoprecipitation assay indicates that WASHC1 associates with DNA replication origins, and knockdown of WASHC1 inhibits MCM protein loading at origins.
CONCLUSIONS: Since efficient loading of excess MCM2-7 complexes is required for cells to survive replicative stress, these results demonstrate that WASHC1 promotes cell survival and maintain chromosomal stability under replication stress through recruitment of excess MCM complex to origins.

Keywords:  Cell survival; Genomic stability; MCM2-7; Replication stress; WASHC1

DOI:  https://doi.org/10.1007/s11033-022-07650-4
Int J Mol Sci. 2022 Jun 07. pii: 6373. [Epub ahead of print]23(12):

Structural and Molecular Kinetic Features of Activities of DNA Polymerases.

Aleksandra A Kuznetsova, Olga S Fedorova, Nikita A Kuznetsov.

  DNA polymerases catalyze DNA synthesis during the replication, repair, and recombination of DNA. Based on phylogenetic analysis and primary protein sequences, DNA polymerases have been categorized into seven families: A, B, C, D, X, Y, and RT. This review presents generalized data on the catalytic mechanism of action of DNA polymerases. The structural features of different DNA polymerase families are described in detail. The discussion highlights the kinetics and conformational dynamics of DNA polymerases from all known polymerase families during DNA synthesis.

Keywords:  DNA polymerase; catalytic mechanism; kinetics; protein–DNA interaction; structural family

DOI:  https://doi.org/10.3390/ijms23126373
Science. 2022 Jun 24. 376(6600): 1471-1476

Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function.

Maurice Michel, Carlos Benítez-Buelga, Patricia A Calvo, Bishoy M F Hanna, Oliver Mortusewicz, Geoffrey Masuyer, Jonathan Davies, Olov Wallner, Kumar Sanjiv, Julian J Albers, Sergio Castañeda-Zegarra, Ann-Sofie Jemth, Torkild Visnes, Ana Sastre-Perona, Akhilesh N Danda, Evert J Homan, Karthick Marimuthu, Zhao Zhenjun, Celestine N Chi, Antonio Sarno, Elisée Wiita, Catharina von Nicolai, Anna J Komor, Varshni Rajagopal, Sarah Müller, Emily C Hank, Marek Varga, Emma R Scaletti, Monica Pandey, Stella Karsten, Hanne Haslene-Hox, Simon Loevenich, Petra Marttila, Azita Rasti, Kirill Mamonov, Florian Ortis, Fritz Schömberg, Olga Loseva, Josephine Stewart, Nicholas D'Arcy-Evans, Tobias Koolmeister, Martin Henriksson, Dana Michel, Ana de Ory, Lucia Acero, Oriol Calvete, Martin Scobie, Christian Hertweck, Ivan Vilotijevic, Christina Kalderén, Ana Osorio, Rosario Perona, Alexandra Stolz, Pål Stenmark, Ulrika Warpman Berglund, Miguel de Vega, Thomas Helleday.

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

DOI: https://doi.org/10.1126/science.abf8980
PLoS Genet. 2022 Jun 21. 18(6): e1010124

Comprehensive analysis of cis- and trans-acting factors affecting ectopic Break-Induced Replication.

Tannia Uribe-Calvillo, Laetitia Maestroni, Marie-Claude Marsolier, Basheer Khadaroo, Christine Arbiol, Jonathan Schott, Bertrand Llorente.

Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating ectopic BIR efficiency, we found that ectopic BIR efficiency is the highest close to chromosome ends. The variations of ectopic BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of ectopic BIR depends on the length of DNA already synthesized. Ectopic BIR is more susceptible to disruption during the synthesis of the first ~35-40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes ectopic BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway.

DOI: https://doi.org/10.1371/journal.pgen.1010124
DNA Repair (Amst). 2022 Jun 11. pii: S1568-7864(22)00090-8. [Epub ahead of print]116 103357

Monitoring DNA polymerase β mitochondrial localization and dynamics.

Julie K Horton, Agnes K Janoshazi, Cristina A Nadalutti, Ming-Lang Zhao, Donna F Stefanick, Samuel H Wilson.

  Mouse fibroblasts lacking (null) DNA polymerase β (pol β) were transfected with fluorescently tagged pol β and stained with biomarkers to allow visualization within living cells by confocal microscopy. Transient transfection resulted in varying pol β expression levels. Separating cells into three groups based on pol β fluorescence intensity and morphological distribution, permitted analysis of the concentration dependence and spatial distribution of cytoplasmic pol β. Colocalization between pol β and mitochondria was pol β concentration dependent. A decrease in overlap with nucleoids containing mitochondrial DNA (mtDNA) was observed at the highest pol β intensity where pol β exhibits a tubular appearance, suggesting the ability to load elevated levels of pol β into mitochondria readily available for relocation to damaged mtDNA. The dynamics of pol β and mitochondrial nucleoids were followed by confocal recording of time series images. Two populations of mitochondrial nucleoids were observed, with and without pol β. Micro-irradiation, known to form DNA single-strand breaks, in a line across nucleus and cytoplasm of pol β stably transfected cells enhanced apparent localization of pol β with mitochondria in the perinuclear region of the cytoplasm near the nuclear membrane. Exposure of pol β expressing cells to H2O2 resulted in a time-dependent increase in cytoplasmic pol β observed by immunofluorescence analysis of fixed cells. Further screening revealed increased levels of colocalization of pol β with a mitochondrial probe and an increase in oxidative DNA damage in the cytoplasm. ELISA quantification confirmed an increase of an oxidative mitochondrial base lesion, 7,8-dihydro-8-oxoguanine, after H2O2 treatment. Taken together, the results suggest that pol β is recruited to mitochondria in response to oxidatively-induced mtDNA damage to participate in mtDNA repair.

Keywords:  DNA damage; DNA polymerase β; DNA repair; Laser micro-irradiation; Mitochondria; Mitochondrial DNA; Mitochondrial nucleoids

DOI:  https://doi.org/10.1016/j.dnarep.2022.103357
Biomolecules. 2022 Jun 10. pii: 815. [Epub ahead of print]12(6):

Inhibitors of the Cancer Target Ribonucleotide Reductase, Past and Present.

Sarah E Huff, Jordan M Winter, Chris G Dealwis.

  Ribonucleotide reductase (RR) is an essential multi-subunit enzyme found in all living organisms; it catalyzes the rate-limiting step in dNTP synthesis, namely, the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates. As expression levels of human RR (hRR) are high during cell replication, hRR has long been considered an attractive drug target for a range of proliferative diseases, including cancer. While there are many excellent reviews regarding the structure, function, and clinical importance of hRR, recent years have seen an increase in novel approaches to inhibiting hRR that merit an updated discussion of the existing inhibitors and strategies to target this enzyme. In this review, we discuss the mechanisms and clinical applications of classic nucleoside analog inhibitors of hRRM1 (large catalytic subunit), including gemcitabine and clofarabine, as well as inhibitors of the hRRM2 (free radical housing small subunit), including triapine and hydroxyurea. Additionally, we discuss novel approaches to targeting RR and the discovery of new classes of hRR inhibitors.

Keywords:  COH 29; acyl hydrazone; breast cancer; cancer chemotherapy; cladribine; clofarabine; drug discovery; fludarabine; gemcitabine; hydrazone; pancreatic cancer; ribonucleotide reductase; ribonucleotide reductase inhibitors; small cell lung cancer; small molecule; triapine

DOI:  https://doi.org/10.3390/biom12060815
Front Oncol. 2022 ;12 765968

A Lack of Effectiveness in the ATM-Orchestrated DNA Damage Response Contributes to the DNA Repair Defect of HPV-Positive Head and Neck Cancer Cells.

Sabrina Köcher, Henrike Barbara Zech, Leonie Krug, Fruzsina Gatzemeier, Sabrina Christiansen, Felix Meyer, Ruth Rietow, Nina Struve, Wael Yassin Mansour, Malte Kriegs, Cordula Petersen, Christian Betz, Kai Rothkamm, Thorsten Rieckmann.

  Patients with human papillomavirus-positive squamous cell carcinoma of the head and neck (HPV+ HNSCC) have a favorable prognosis compared to those with HPV-negative (HPV-) ones. We have shown previously that HPV+ HNSCC cell lines are characterized by enhanced radiation sensitivity and impaired DNA double-strand break (DSB) repair. Since then, various publications have suggested a defect in homologous recombination (HR) and dysregulated expression of DSB repair proteins as underlying mechanisms, but conclusions were often based on very few cell lines. When comparing the expression levels of suggested proteins and other key repair factors in 6 HPV+ vs. 5 HPV- HNSCC strains, we could not confirm most of the published differences. Furthermore, HPV+ HNSCC strains did not demonstrate enhanced sensitivity towards PARP inhibition, questioning a general HR defect. Interestingly, our expression screen revealed minimal levels of the central DNA damage response kinase ATM in the two most radiosensitive HPV+ strains. We therefore tested whether insufficient ATM activity may contribute to the enhanced cellular radiosensitivity. Irrespective of their ATM expression level, radiosensitive HPV+ HNSCC cells displayed DSB repair kinetics similar to ATM-deficient cells. Upon ATM inhibition, HPV+ cell lines showed only a marginal increase in residual radiation-induced γH2AX foci and induction of G2 cell cycle arrest as compared to HPV- ones. In line with these observations, ATM inhibition sensitized HPV+ HNSCC strains less towards radiation than HPV- strains, resulting in similar levels of sensitivity. Unexpectedly, assessment of the phosphorylation kinetics of the ATM targets KAP-1 and Chk2 as well as ATM autophosphorylation after radiation did not indicate directly compromised ATM activity in HPV-positive cells. Furthermore, ATM inhibition delayed radiation induced DNA end resection in both HPV+ and HPV- cells to a similar extent, further suggesting comparable functionality. In conclusion, DNA repair kinetics and a reduced effectiveness of ATM inhibition clearly point to an impaired ATM-orchestrated DNA damage response in HPV+ HNSCC cells, but since ATM itself is apparently functional, the molecular mechanisms need to be further explored.

Keywords:  DNA damage response (DDR); DNA double-strand break repair; ataxia telangiectasia mutated (ATM); head and neck cancer; human papillomavirus (HPV); radiation sensitivity

DOI:  https://doi.org/10.3389/fonc.2022.765968
Cells. 2022 Jun 10. pii: 1887. [Epub ahead of print]11(12):

Lethal and Non-Lethal Functions of Caspases in the DNA Damage Response.

Karla E Lopez, Lisa Bouchier-Hayes.

  Members of the caspase family are well known for their roles in the initiation and execution of cell death. Due to their function in the removal of damaged cells that could otherwise become malignant, caspases are important players in the DNA damage response (DDR), a network of pathways that prevent genomic instability. However, emerging evidence of caspases positively or negatively impacting the accumulation of DNA damage in the absence of cell death demonstrates that caspases play a role in the DDR that is independent of their role in apoptosis. This review highlights the apoptotic and non-apoptotic roles of caspases in the DDR and how they can impact genomic stability and cancer treatment.

Keywords:  DNA damage; apoptosis; caspases; cell cycle; genomic instability

DOI:  https://doi.org/10.3390/cells11121887
FEBS J. 2022 Jun 22.

RNAPII response to transcription-blocking DNA lesions in mammalian cells.

Jianming Wang, Martina Muste Sadurni, Marco Saponaro.

  RNA polymerase II moves along genes to decode genetic information stored in the mammalian genome into messenger RNA and different forms of non-coding RNA. However, the transcription process is frequently challenged by DNA lesions caused by exogenous and endogenous insults, among which helix-distorting DNA lesions and double-stranded DNA breaks are particularly harmful for cell survival. In response to these DNA damages, RNA polymerase II transcription is regulated both locally and globally by multi-layer mechanisms, while transcription-blocking lesions are repaired before transcription can recover. Failure in the DNA damage repair will cause genome instability and cell death. Although recent studies have expanded our understanding of RNA polymerase II regulation confronting DNA lesions, it is still not always clear what the direct contribution of RNA polymerase II is in the DNA damage repair processes. In this review, we will focus on how RNA polymerase II and transcription are both repressed by transcription stalling lesions like DNA-adducts and double strand breaks, but also how they are actively regulated to support the cellular response to DNA damage and favour the repair of lesions.

Keywords:  DNA damage; R-loop; RNA Polymerase II; RNA transcription; double strand break; homologous recombination; nucleotide excision repair

DOI:  https://doi.org/10.1111/febs.16561
Elife. 2022 Jun 22. pii: e77483. [Epub ahead of print]11

A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA.

Xingchen Liu, Christl Gaubitz, Joshua Pajak, Brian A Kelch.

  Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC's external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.

Keywords:  S. cerevisiae; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.77483
Front Oncol. 2022 ;12 870229

PBRM1 Deficiency Sensitizes Renal Cancer Cells to DNMT Inhibitor 5-Fluoro-2'-Deoxycytidine.

Di Gu, Kai Dong, Aimin Jiang, Shaoqin Jiang, Zhibin Fu, Yewei Bao, Fuzhao Huang, Chenghua Yang, Linhui Wang.

  PBRM1 is a tumor suppressor frequently mutated in clear cell renal cell carcinoma. However, no effective targeted therapies exist for ccRCC with PBRM1 loss. To identify novel therapeutic approaches to targeting PBRM1-deficient renal cancers, we employed a synthetic lethality compound screening in isogenic PBRM1+/+ and PBRM1-/- 786-O renal tumor cells and found that a DNMT inhibitor 5-Fluoro-2'-deoxycytidine (Fdcyd) selectively inhibit PBRM1-deficient tumor growth. RCC cells lacking PBRM1 show enhanced DNA damage response, which leads to sensitivity to DNA toxic drugs. Fdcyd treatment not only induces DNA damage, but also re-activated a pro-apoptotic factor XAF1 and further promotes the genotoxic stress-induced PBRM1-deficient cell death. This study shows a novel synthetic lethality interaction between PBRM1 loss and Fdcyd treatment and indicates that DNMT inhibitor represents a novel strategy for treating ccRCC with PBRM1 loss-of-function mutations.

Keywords:  DNA methyltransferase inhibitor; FdCyd; PBRM1 gene mutation; renal cell carcinoma; synthetic lethality

DOI:  https://doi.org/10.3389/fonc.2022.870229
Mol Cancer Ther. 2022 Jun 23. pii: molcanther.MCT-21-0873-A.2021. [Epub ahead of print]

Targeting therapeutic resistance and multinucleate giant cells in CCNE1-amplified HR-proficient ovarian cancer.

Shoumei Bai, Sarah E Taylor, Mohd Azrin Jamalruddin, Stacy McGonigal, Edward Grimley, Dongli Yang, Kara A Bernstein, Ronald J Buckanovich.

Approximately 20% of high-grade serous ovarian cancers (HGSOC) have CCNE1 amplification. CCNE1-amplified tumors are homologous recombination (HR) proficient and resistant to standard therapies. Therapy resistance is associated with increased numbers of polyploid giant cancer cells (PGCCs). We sought to identify new therapeutic approaches for patients with CCNE1-amplified tumors. Using TCGA data, we find that the mTOR, HR, and DNA checkpoint pathways are enriched in CCNE1-amplifed ovarian cancers. Furthermore, Interactome Mapping Analysis linked the mTOR activity with upregulation of HR and DNA checkpoint pathways. Indeed, we find that mTOR inhibitors (mTORi) downregulate HR/checkpoint genes in CCNE1-amplifed tumors. As CCNE1-amplified tumors are dependent on the HR pathway for viability, mTORi proved selectively effective in CCNE1-amplified tumors. Similarly, via downregulation of HR genes, mTORi increased CCNE1-amplifed HGSOC response to PARPi. In contrast, overexpression of HR/checkpoint proteins (RAD51 or ATR), induced resistance to mTORi. In vivo, mTORi alone potently reduced CCNE1-amplified tumor growth and the combination of mTORi and PARPi increased response and tumor eradication. Tumors treated with mTORi demonstrated a significant reduction in ALDH+ PGCCs. Finally, as a proof of principle, we identified three patients with CCNE1 amplified tumors who were treated with an mTORi. All three obtained clinical benefit from the therapy. Our studies and clinical experience indicate mTOR inhibitors are a potential therapeutic approach for patients with CCNE1-amplified tumors.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0873
J Cell Sci. 2022 Jun 15. pii: jcs259626. [Epub ahead of print]135(12):

A CDK activity buffer ensures mitotic completion.

Souradeep Basu, James O Patterson, Theresa U Zeisner, Paul Nurse.

  The eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases (CDKs). CDK activity rises over 50-fold during the cell cycle, from a low level in G1 to a high level in mitosis. However, it is not known whether the entire range of CDK activity is necessary for cell cycle progression, or whether cells can tolerate a reduction in CDK activity level. Here, in fission yeast, we show that sublethal CDK inhibition lengthens the time cells spend in mitosis but does not cause misordering of mitotic events. Maximum attainable CDK activity exceeds the amount necessary for mitosis, and thus forms a CDK activity buffer between sufficient and maximal possible CDK activities. This CDK activity buffer is needed for mitotic completion when CDK activity is compromised, and CDK inhibition only becomes lethal to cells when this buffer is exhausted. Finally, we explore what factors influence this CDK activity buffer, and find that it is influenced by CDK-counteracting phosphatases. Therefore, maximum attainable CDK activity is not necessary for mitosis but provides robustness to CDK activity reduction to ensure mitotic completion.

Keywords:   Schizosaccharomyces pombe ; CDK inhibitor; Cdk1; Cyclin-dependent kinase; Kinase activity; Mitosis

DOI:  https://doi.org/10.1242/jcs.259626
Molecules. 2022 Jun 07. pii: 3660. [Epub ahead of print]27(12):

Identification of Human Dihydroorotate Dehydrogenase Inhibitor by a Pharmacophore-Based Virtual Screening Study.

Salvatore Galati, Stefano Sainas, Marta Giorgis, Donatella Boschi, Marco L Lolli, Gabriella Ortore, Giulio Poli, Tiziano Tuccinardi.

  Human dihydroorotate dehydrogenase (hDHODH) is an enzyme belonging to a flavin mononucleotide (FMN)-dependent family involved in de novo pyrimidine biosynthesis, a key biological pathway for highly proliferating cancer cells and pathogens. In fact, hDHODH proved to be a promising therapeutic target for the treatment of acute myelogenous leukemia, multiple myeloma, and viral and bacterial infections; therefore, the identification of novel hDHODH ligands represents a hot topic in medicinal chemistry. In this work, we reported a virtual screening study for the identification of new promising hDHODH inhibitors. A pharmacophore-based approach combined with a consensus docking analysis and molecular dynamics simulations was applied to screen a large database of commercial compounds. The whole virtual screening protocol allowed for the identification of a novel compound that is endowed with promising inhibitory activity against hDHODH and is structurally different from known ligands. These results validated the reliability of the in silico workflow and provided a valuable starting point for hit-to-lead and future lead optimization studies aimed at the development of new potent hDHODH inhibitors.

Keywords:  human dihydroorotate dehydrogenase; pharmacophore model; virtual screening

DOI:  https://doi.org/10.3390/molecules27123660
Sci Adv. 2022 Jun 24. 8(25): eabn3471

Temozolomide-induced guanine mutations create exploitable vulnerabilities of guanine-rich DNA and RNA regions in drug-resistant gliomas.

Deanna M Tiek, Beril Erdogdu, Roham Razaghi, Lu Jin, Norah Sadowski, Carla Alamillo-Ferrer, J Robert Hogg, Bassem R Haddad, David H Drewry, Carrow I Wells, Julie E Pickett, Xiao Song, Anshika Goenka, Bo Hu, Samuel A Goldlust, William J Zuercher, Mihaela Pertea, Winston Timp, Shi-Yuan Cheng, Rebecca B Riggins.

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Although initially beneficial, TMZ resistance is universal and second-line interventions are an unmet clinical need. Here, we took advantage of the known mechanism of action of TMZ to target guanines (G) and investigated G-rich G-quadruplex (G4) and splice site changes that occur upon TMZ resistance. We report that TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4-stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2-like kinase. Last, we show that the G4 and RNA binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker.

DOI: https://doi.org/10.1126/sciadv.abn3471