bims-numges 2022-02-20 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–02–20
24 papers selected by
Sean Rudd, Karolinska Institutet

RB1 loss overrides PARP inhibitor sensitivity driven by RNASEH2B loss in prostate cancer.
Inosine Triphosphate Pyrophosphatase (ITPase): Functions, Mutations, Polymorphisms and Its Impact on Cancer Therapies.
Rev1 deficiency induces replication stress to cause metabolic dysfunction differently in males and females.
Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.
Metnase and EEPD1: DNA Repair Functions and Potential Targets in Cancer Therapy.
SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway.
R-Loop-Associated Genomic Instability and Implication of WRN and WRNIP1.
Therapeutic Targeting of DNA Damage Response in Cancer.
Immediate-Early, Early, and Late Responses to DNA Double Stranded Breaks.
RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity.
LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.
Etoposide-induced DNA damage is increased in p53 mutants: identification of ATR and other genes that influence effects of p53 mutations on Top2-induced cytotoxicity.
Disrupting the MAD2L2-Rev1 Complex Enhances Cell Death upon DNA Damage.
Sesquiterpene Lactones Potentiate Olaparib-Induced DNA Damage in p53 Wildtype Cancer Cells.
UBE2T-mediated Akt ubiquitination and Akt/β-catenin activation promotes hepatocellular carcinoma development by increasing pyrimidine metabolism.
The Role of Thymine DNA Glycosylase in Transcription, Active DNA Demethylation, and Cancer.
CNOT6: A Novel Regulator of DNA Mismatch Repair.
Characterization of Chromosomal Instability in Glioblastoma.
Chemotherapy Side-Effects: Not All DNA Damage Is Equal.
Recent advances of human dihydroorotate dehydrogenase inhibitors for cancer therapy: Current development and future perspectives.
Metabolic determination of cell fate through selective inheritance of mitochondria.
Modulation of DNA Damage Response by SAM and HD Domain Containing Deoxynucleoside Triphosphate Triphosphohydrolase (SAMHD1) Determines Prognosis and Treatment Efficacy in Different Solid Tumor Types.
Influence of Hypoxia on Radiosensitization of Cancer Cells by 5-Bromo-2'-deoxyuridine.
ABT199/venetoclax potentiates the cytotoxicity of alkylating agents and fludarabine in acute myeloid leukemia cells.

Sci Adv. 2022 Feb 18. 8(7): eabl9794

RB1 loss overrides PARP inhibitor sensitivity driven by RNASEH2B loss in prostate cancer.

Chenkui Miao, Takuya Tsujino, Tomoaki Takai, Fu Gui, Takeshi Tsutsumi, Zsofia Sztupinszki, Zengjun Wang, Haruhito Azuma, Zoltan Szallasi, Kent W Mouw, Lee Zou, Adam S Kibel, Li Jia.

Current targeted cancer therapies are largely guided by mutations of a single gene, which overlooks concurrent genomic alterations. Here, we show that RNASEH2B, RB1, and BRCA2, three closely located genes on chromosome 13q, are frequently deleted in prostate cancer individually or jointly. Loss of RNASEH2B confers cancer cells sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition due to impaired ribonucleotide excision repair and PARP trapping. When co-deleted with RB1, however, cells lose their sensitivity, in part, through E2F1-induced BRCA2 expression, thereby enhancing homologous recombination repair capacity. Nevertheless, loss of BRCA2 resensitizes RNASEH2B/RB1 co-deleted cells to PARP inhibition. Our results may explain some of the disparate clinical results from PARP inhibition due to interaction between multiple genomic alterations and support a comprehensive genomic test to determine who may benefit from PARP inhibition. Last, we show that ATR inhibition can disrupt E2F1-induced BRCA2 expression and overcome PARP inhibitor resistance caused by RB1 loss.

DOI: https://doi.org/10.1126/sciadv.abl9794
Cells. 2022 Jan 24. pii: 384. [Epub ahead of print]11(3):

Inosine Triphosphate Pyrophosphatase (ITPase): Functions, Mutations, Polymorphisms and Its Impact on Cancer Therapies.

Mazin A Zamzami.

  Inosine triphosphate pyrophosphatase (ITPase) is an enzyme encoded by the ITPA gene and functions to prevent the incorporation of noncanonical purine nucleotides into DNA and RNA. Specifically, the ITPase catalyzed the hydrolysis of (deoxy) nucleoside triphosphates ((d) NTPs) into the corresponding nucleoside monophosphate with the concomitant release of pyrophosphate. Recently, thiopurine drug metabolites such as azathioprine have been included in the lists of ITPase substrates. Interestingly, inosine or xanthosine triphosphate (ITP/XTP) and their deoxy analogs, deoxy inosine or xanthosine triphosphate (dITP/dXTP), are products of important biological reactions such as deamination that take place within the cellular compartments. However, the incorporation of ITP/XTP, dITP/dXTP, or the genetic deficiency or polymorphism of the ITPA gene have been implicated in many human diseases, including infantile epileptic encephalopathy, early onset of tuberculosis, and the responsiveness of patients to cancer therapy. This review provides an up-to-date report on the ITPase enzyme, including information regarding its discovery, analysis, and cellular localization, its implication in human diseases including cancer, and its therapeutic potential, amongst others.

Keywords:  cancer therapy; deamination; inosine triphosphate pyrophosphatase (ITPase); polymorphism; purine metabolism

DOI:  https://doi.org/10.3390/cells11030384
Am J Physiol Endocrinol Metab. 2022 Feb 14.

Rev1 deficiency induces replication stress to cause metabolic dysfunction differently in males and females.

Wietse In Het Panhuis, Anastasia Tsaalbi-Shtylik, Milena Schönke, Vanessa van Harmelen, Amanda C M Pronk, Trea C M Streefland, Hetty C M Sips, Salwa Afkir, Ko Willems van Dijk, Patrick C N Rensen, Niels de Wind, Sander Kooijman.

  DNA damage responses compete for cellular resources with metabolic pathways, but little is known about the metabolic consequences of impaired DNA replication, a process called replication stress. Here we characterized the metabolic consequences of DNA replication stress at endogenous DNA lesions by using mice with a disruption of Rev1, a translesion DNA polymerase specialized in the mutagenic replication of damaged DNA. Male and female Rev1 KO mice were compared to wild-type (WT) mice and followed over time to study the natural course of body weight gain and glucose tolerance. Follow-up measurements were performed in female mice for in-depth metabolic characterization. Body weight and fat mass were only increased in female KO mice versus WT mice, whereas glucose intolerance and a reduction in lean mass were observed in both sexes. Female KO mice showed reduced locomotor activity while male KO mice showed increased activity as compared with their WT littermates. Further characterization of female mice revealed that lipid handling was unaffected by Rev1 deletion. An increased respiratory exchange ratio, combined with elevated plasma lactate levels and increased hepatic gluconeogenesis indicated problems with aerobic oxidation and increased reliance of anaerobic glycolysis. Supplementation with the NAD+ precursor nicotinamide riboside to stimulate aerobic respiration failed to restore the metabolic phenotype. In conclusion, replication stress at endogenous DNA lesions induces a complex metabolic phenotype, most likely initiated by muscular metabolic dysfunction and increased dependence on anaerobic glycolysis. Nicotinamide riboside supplementation after the onset of the metabolic impairment did not rescue this phenotype.

Keywords:  adiposity; anaerobic glycolysis; hyperglycemia; skeletal muscle

DOI:  https://doi.org/10.1152/ajpendo.00357.2021
Elife. 2022 Feb 18. pii: e74175. [Epub ahead of print]11

Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.

Christl Gaubitz, Xingchen Liu, Joshua Pajak, Nicholas P Stone, Janelle A Hayes, Gabriel Demo, Brian A Kelch.

  Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the S. cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

Keywords:  S. cerevisiae; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.74175
Front Oncol. 2022 ;12 808757

Metnase and EEPD1: DNA Repair Functions and Potential Targets in Cancer Therapy.

Jac A Nickoloff, Neelam Sharma, Lynn Taylor, Sage J Allen, Suk-Hee Lee, Robert Hromas.

  Cells respond to DNA damage by activating signaling and DNA repair systems, described as the DNA damage response (DDR). Clarifying DDR pathways and their dysregulation in cancer are important for understanding cancer etiology, how cancer cells exploit the DDR to survive endogenous and treatment-related stress, and to identify DDR targets as therapeutic targets. Cancer is often treated with genotoxic chemicals and/or ionizing radiation. These agents are cytotoxic because they induce DNA double-strand breaks (DSBs) directly, or indirectly by inducing replication stress which causes replication fork collapse to DSBs. EEPD1 and Metnase are structure-specific nucleases, and Metnase is also a protein methyl transferase that methylates histone H3 and itself. EEPD1 and Metnase promote repair of frank, two-ended DSBs, and both promote the timely and accurate restart of replication forks that have collapsed to single-ended DSBs. In addition to its roles in HR, Metnase also promotes DSB repair by classical non-homologous recombination, and chromosome decatenation mediated by TopoIIα. Although mutations in Metnase and EEPD1 are not common in cancer, both proteins are frequently overexpressed, which may help tumor cells manage oncogenic stress or confer resistance to therapeutics. Here we focus on Metnase and EEPD1 DNA repair pathways, and discuss opportunities for targeting these pathways to enhance cancer therapy.

Keywords:  DNA damage; DNA double-strand breaks; DNA repair; chromosome decatenation; genome instability; homologous recombination; non-homologous end-joining

DOI:  https://doi.org/10.3389/fonc.2022.808757
EMBO Rep. 2022 Feb 14. e53639

SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway.

Lisa Schubert, Ivo A Hendriks, Emil P T Hertz, Wei Wu, Selene Sellés-Baiget, Saskia Hoffmann, Keerthana Stine Viswalingam, Irene Gallina, Satyakrishna Pentakota, Bente Benedict, Joachim Johansen, Katja Apelt, Martijn S Luijsterburg, Simon Rasmussen, Michael Lisby, Ying Liu, Michael L Nielsen, Niels Mailand, Julien P Duxin.

  DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)-mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error-free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error-free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR-mediated ICL repair is defective, and breaks are instead re-ligated by polymerase θ-dependent microhomology-mediated end-joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error-free ICL repair.

Keywords:  DNA interstrand crosslinks (ICLs); DNA repair; DNA replication; genome stability; translesion DNA synthesis (TLS)

DOI:  https://doi.org/10.15252/embr.202153639
Int J Mol Sci. 2022 Jan 28. pii: 1547. [Epub ahead of print]23(3):

R-Loop-Associated Genomic Instability and Implication of WRN and WRNIP1.

Veronica Marabitti, Pasquale Valenzisi, Giorgia Lillo, Eva Malacaria, Valentina Palermo, Pietro Pichierri, Annapaola Franchitto.

  Maintenance of genome stability is crucial for cell survival and relies on accurate DNA replication. However, replication fork progression is under constant attack from different exogenous and endogenous factors that can give rise to replication stress, a source of genomic instability and a notable hallmark of pre-cancerous and cancerous cells. Notably, one of the major natural threats for DNA replication is transcription. Encounters or conflicts between replication and transcription are unavoidable, as they compete for the same DNA template, so that collisions occur quite frequently. The main harmful transcription-associated structures are R-loops. These are DNA structures consisting of a DNA-RNA hybrid and a displaced single-stranded DNA, which play important physiological roles. However, if their homeostasis is altered, they become a potent source of replication stress and genome instability giving rise to several human diseases, including cancer. To combat the deleterious consequences of pathological R-loop persistence, cells have evolved multiple mechanisms, and an ever growing number of replication fork protection factors have been implicated in preventing/removing these harmful structures; however, many others are perhaps still unknown. In this review, we report the current knowledge on how aberrant R-loops affect genome integrity and how they are handled, and we discuss our recent findings on the role played by two fork protection factors, the Werner syndrome protein (WRN) and the Werner helicase-interacting protein 1 (WRNIP1) in response to R-loop-induced genome instability.

Keywords:  DNA repair; R-loops; RecQ helicases; genomic instability; replication stress

DOI:  https://doi.org/10.3390/ijms23031547
Int J Mol Sci. 2022 Feb 01. pii: 1701. [Epub ahead of print]23(3):

Therapeutic Targeting of DNA Damage Response in Cancer.

Wonyoung Choi, Eun Sook Lee.

  DNA damage response (DDR) is critical to ensure genome stability, and defects in this signaling pathway are highly associated with carcinogenesis and tumor progression. Nevertheless, this also provides therapeutic opportunities, as cells with defective DDR signaling are directed to rely on compensatory survival pathways, and these vulnerabilities have been exploited for anticancer treatments. Following the impressive success of PARP inhibitors in the treatment of BRCA-mutated breast and ovarian cancers, extensive research has been conducted toward the development of pharmacologic inhibitors of the key components of the DDR signaling pathway. In this review, we discuss the key elements of the DDR pathway and how these molecular components may serve as anticancer treatment targets. We also summarize the recent promising developments in the field of DDR pathway inhibitors, focusing on novel agents beyond PARP inhibitors. Furthermore, we discuss biomarker studies to identify target patients expected to derive maximal clinical benefits as well as combination strategies with other classes of anticancer agents to synergize and optimize the clinical benefits.

Keywords:  ATM; ATR; BRCA; CHK1; CHK2; DNA damage response; DNA-PK; PARP inhibitor; WEE1; homologous recombination

DOI:  https://doi.org/10.3390/ijms23031701
Front Genet. 2022 ;13 793884

Immediate-Early, Early, and Late Responses to DNA Double Stranded Breaks.

Shaylee R Kieffer, Noel F Lowndes.

  Loss or rearrangement of genetic information can result from incorrect responses to DNA double strand breaks (DSBs). The cellular responses to DSBs encompass a range of highly coordinated events designed to detect and respond appropriately to the damage, thereby preserving genomic integrity. In analogy with events occurring during viral infection, we appropriate the terms Immediate-Early, Early, and Late to describe the pre-repair responses to DSBs. A distinguishing feature of the Immediate-Early response is that the large protein condensates that form during the Early and Late response and are resolved upon repair, termed foci, are not visible. The Immediate-Early response encompasses initial lesion sensing, involving poly (ADP-ribose) polymerases (PARPs), KU70/80, and MRN, as well as rapid repair by so-called 'fast-kinetic' canonical non-homologous end joining (cNHEJ). Initial binding of PARPs and the KU70/80 complex to breaks appears to be mutually exclusive at easily ligatable DSBs that are repaired efficiently by fast-kinetic cNHEJ; a process that is PARP-, ATM-, 53BP1-, Artemis-, and resection-independent. However, at more complex breaks requiring processing, the Immediate-Early response involving PARPs and the ensuing highly dynamic PARylation (polyADP ribosylation) of many substrates may aid recruitment of both KU70/80 and MRN to DSBs. Complex DSBs rely upon the Early response, largely defined by ATM-dependent focal recruitment of many signalling molecules into large condensates, and regulated by complex chromatin dynamics. Finally, the Late response integrates information from cell cycle phase, chromatin context, and type of DSB to determine appropriate pathway choice. Critical to pathway choice is the recruitment of p53 binding protein 1 (53BP1) and breast cancer associated 1 (BRCA1). However, additional factors recruited throughout the DSB response also impact upon pathway choice, although these remain to be fully characterised. The Late response somehow channels DSBs into the appropriate high-fidelity repair pathway, typically either 'slow-kinetic' cNHEJ or homologous recombination (HR). Loss of specific components of the DSB repair machinery results in cells utilising remaining factors to effect repair, but often at the cost of increased mutagenesis. Here we discuss the complex regulation of the Immediate-Early, Early, and Late responses to DSBs proceeding repair itself.

Keywords:  DNA repair; Early response; Immediate-early response; Late response; double strand breaks (DSBs); homologous recombination (HR); non-homologous end joining (NHEJ); pre-repair responses

DOI:  https://doi.org/10.3389/fgene.2022.793884
Nat Commun. 2022 Feb 17. 13(1): 957

RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity.

Sumin Feng, Sai Ma, Kejiao Li, Shengxian Gao, Shaokai Ning, Jinfeng Shang, Ruiyuan Guo, Yingying Chen, Britny Blumenfeld, Itamar Simon, Qing Li, Rong Guo, Dongyi Xu.

The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identify a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

DOI: https://doi.org/10.1038/s41467-022-28588-y
Proc Natl Acad Sci U S A. 2022 Feb 22. pii: e2115999119. [Epub ahead of print]119(8):

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.

Wilson McKerrow, Xuya Wang, Carlos Mendez-Dorantes, Paolo Mita, Song Cao, Mark Grivainis, Li Ding, John LaCava, Kathleen H Burns, Jef D Boeke, David Fenyö.

  Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Keywords:  DNA damage response; LINE-1; cancer; copy number alteration; retrotransposon

DOI:  https://doi.org/10.1073/pnas.2115999119
Oncotarget. 2022 ;13 332-346

Etoposide-induced DNA damage is increased in p53 mutants: identification of ATR and other genes that influence effects of p53 mutations on Top2-induced cytotoxicity.

Daniel Menendez, Jay R Anand, Carri C Murphy, Whitney J Bell, Jiaqi Fu, Nadia Slepushkina, Eugen Buehler, Scott E Martin, Madhu Lal-Nag, John L Nitiss, Michael A Resnick.

  The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.

Keywords:  ICE assay; Top2 covalent complexes; siRNA screen; synthetic lethality; tumor suppressor

DOI:  https://doi.org/10.18632/oncotarget.28195
Molecules. 2022 Jan 19. pii: 636. [Epub ahead of print]27(3):

Disrupting the MAD2L2-Rev1 Complex Enhances Cell Death upon DNA Damage.

Nomi Pernicone, Maria Elias, Itay Onn, Dror Tobi, Tamar Listovsky.

  DNA-damaging chemotherapy agents such as cisplatin have been the first line of treatment for cancer for decades. While chemotherapy can be very effective, its long-term success is often reduced by intrinsic and acquired drug resistance, accompanied by chemotherapy-resistant secondary malignancies. Although the mechanisms causing drug resistance are quite distinct, they are directly connected to mutagenic translesion synthesis (TLS). The TLS pathway promotes DNA damage tolerance by supporting both replication opposite to a lesion and inaccurate single-strand gap filling. Interestingly, inhibiting TLS reduces both cisplatin resistance and secondary tumor formation. Therefore, TLS targeting is a promising strategy for improving chemotherapy. MAD2L2 (i.e., Rev7) is a central protein in TLS. It is an essential component of the TLS polymerase zeta (ζ), and it forms a regulatory complex with Rev1 polymerase. Here we present the discovery of two small molecules, c#2 and c#3, that directly bind both in vitro and in vivo to MAD2L2 and influence its activity. Both molecules sensitize lung cancer cell lines to cisplatin, disrupt the formation of the MAD2L2-Rev1 complex and increase DNA damage, hence underlining their potential as lead compounds for developing novel TLS inhibitors for improving chemotherapy treatments.

Keywords:  MAD2L2; TLS; small molecules

DOI:  https://doi.org/10.3390/molecules27030636
Int J Mol Sci. 2022 Jan 20. pii: 1116. [Epub ahead of print]23(3):

Sesquiterpene Lactones Potentiate Olaparib-Induced DNA Damage in p53 Wildtype Cancer Cells.

Hugh C Osborne, Igor Larrosa, Christine K Schmidt.

  Despite notable advances in utilising PARP inhibitor monotherapy, many cancers are not PARP inhibitor-sensitive or develop treatment resistance. In this work, we show that the two structurally-related sesquiterpene lactones, a 2-bromobenzyloxy derivative of dehydrosantonin (BdS) and alantolactone (ATL) sensitise p53 wildtype, homologous recombination-proficient cancer cells to low-dose treatment with the PARP inhibitor, olaparib. Exposure to combination treatments of olaparib with BdS or ATL induces cell-cycle changes, chromosomal instability, as well as considerable increases in nuclear area. Mechanistically, we uncover that mitotic errors likely depend on oxidative stress elicited by the electrophilic lactone warheads and olaparib-mediated PARP-trapping, culminating in replication stress. Combination treatments exhibit moderately synergistic effects on cell survival, probably attenuated by a p53-mediated, protective cell-cycle arrest in the G2 cell-cycle phase. Indeed, using a WEE1 inhibitor, AZD1775, to inhibit the G2/M cell-cycle checkpoint further decreased cell survival. Around half of all cancers diagnosed retain p53 functionality, and this proportion could be expected to increase with improved diagnostic approaches in the clinic. Utilising sublethal oxidative stress to sensitise p53 wildtype, homologous recombination-proficient cancer cells to low-dose PARP-trapping could therefore serve as the basis for future research into the treatment of cancers currently refractory to PARP inhibition.

Keywords:  2-bromobenzyloxy derivative of dehydrosantonin (BdS); DNA replication stress; PARP inhibitor (PARPi); alantolactone (ATL); cancer; olaparib; reactive oxygen species (ROS); sesquiterpene lactones

DOI:  https://doi.org/10.3390/ijms23031116
Cell Death Dis. 2022 02 15. 13(2): 154

UBE2T-mediated Akt ubiquitination and Akt/β-catenin activation promotes hepatocellular carcinoma development by increasing pyrimidine metabolism.

Zhenru Zhu, Chuanhui Cao, Dongyan Zhang, Zhihong Zhang, Li Liu, Dehua Wu, Jingyuan Sun.

The oncogene protein ubiquitin-conjugating enzyme E2T (UBE2T) is reported to be upregulated in hepatocellular carcinoma (HCC) and correlated with poor clinical outcomes of HCC patients. However, the underlying mechanism by which UBE2T exerts its oncogenic function in HCC remains largely unexplored. In this study, in vitro and in vivo experiments suggested that UBE2T promoted HCC development including proliferation and metastasis. GSEA analysis indicated that UBE2T was positively correlated with pyrimidine metabolism, and LC/MS-MS metabolomics profiling revealed that the key products of pyrimidine metabolism were significantly increased in UBE2T-overexpressing cells. UBE2T overexpression led to the upregulation of several key enzymes catalyzing de novo pyrimidine synthesis, including CAD, DHODH, and UMPS. Moreover, the utilization of leflunomide, a clinically approved DHODH inhibitor, blocked the effect of UBE2T in promoting HCC progression. Mechanistically, UBE2T increased Akt K63-mediated ubiquitination and Akt/β-catenin signaling pathway activation. The disruption of UBE2T-mediated ubiquitination on Akt, including E2-enzyme-deficient mutation (C86A) of UBE2T and ubiquitination-site-deficient mutation (K8/14 R) of Akt impaired UBE2T's effect in upregulating CAD, DHODH, and UMPS. Importantly, we demonstrated that UBE2T was positively correlated with p-Akt, β-catenin, CAD, DHODH, and UMPS in HCC tumor tissues. In summary, our study indicates that UBE2T increases pyrimidine metabolism by promoting Akt K63-linked ubiquitination, thus contributing to HCC development. This work provides a novel insight into HCC development and a potential therapeutic strategy for HCC patients.

DOI: https://doi.org/10.1038/s41419-022-04596-0
Cancers (Basel). 2022 Feb 01. pii: 765. [Epub ahead of print]14(3):

The Role of Thymine DNA Glycosylase in Transcription, Active DNA Demethylation, and Cancer.

Oladapo Onabote, Haider M Hassan, Majdina Isovic, Joseph Torchia.

  DNA methylation is an essential covalent modification that is required for growth and development. Once considered to be a relatively stable epigenetic mark, many studies have established that DNA methylation is dynamic. The 5-methylcytosine (5-mC) mark can be removed through active DNA demethylation in which 5-mC is converted to an unmodified cytosine through an oxidative pathway coupled to base excision repair (BER). The BER enzyme Thymine DNA Glycosylase (TDG) plays a key role in active DNA demethylation by excising intermediates of 5-mC generated by this process. TDG acts as a key player in transcriptional regulation through its interactions with various nuclear receptors and transcription factors, in addition to its involvement in classical BER and active DNA demethylation, which serve to protect the stability of the genome and epigenome, respectively. Recent animal studies have identified a connection between the loss of Tdg and the onset of tumorigenesis. In this review, we summarize the recent findings on TDG's function as a transcriptional regulator as well as the physiological relevance of TDG and active DNA demethylation in cancer.

Keywords:  Thymine DNA Glycosylase; active DNA demethylation; cancer; chromatin reorganization; coactivator; transcription; tumor suppressor

DOI:  https://doi.org/10.3390/cancers14030765
Cells. 2022 Feb 02. pii: 521. [Epub ahead of print]11(3):

CNOT6: A Novel Regulator of DNA Mismatch Repair.

Peng Song, Shaojun Liu, Dekang Liu, Guido Keijzers, Daniela Bakula, Shunlei Duan, Niels de Wind, Zilu Ye, Sergey Y Vakhrushev, Morten Scheibye-Knudsen, Lene Juel Rasmussen.

  DNA mismatch repair (MMR) is a highly conserved pathway that corrects both base-base mispairs and insertion-deletion loops (IDLs) generated during DNA replication. Defects in MMR have been linked to carcinogenesis and drug resistance. However, the regulation of MMR is poorly understood. Interestingly, CNOT6 is one of four deadenylase subunits in the conserved CCR4-NOT complex and it targets poly(A) tails of mRNAs for degradation. CNOT6 is overexpressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and androgen-independent prostate cancer cells, which suggests that an altered expression of CNOT6 may play a role in tumorigenesis. Here, we report that a depletion of CNOT6 sensitizes human U2OS cells to N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and leads to enhanced apoptosis. We also demonstrate that the depletion of CNOT6 upregulates MMR and decreases the mutation frequency in MMR-proficient cells. Furthermore, the depletion of CNOT6 increases the stability of mRNA transcripts from MMR genes, leading to the increased expression of MMR proteins. Our work provides insight into a novel CNOT6-dependent mechanism for regulating MMR.

Keywords:  cancer; gene regulation; genome stability; mRNA degradation; mammalian deadenylase; mismatch repair

DOI:  https://doi.org/10.3390/cells11030521
Front Genet. 2021 ;12 810793

Characterization of Chromosomal Instability in Glioblastoma.

Elisa Balzano, Elena Di Tommaso, Antonio Antoccia, Franca Pelliccia, Simona Giunta.

  Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system (CNS). The poor prognosis of GBM due to resistance to therapy has been associated with high chromosomal instability (CIN). Replication stress is a major cause of CIN that manifests as chromosome rearrangements, fragility, and breaks, including those cytologically expressed within specific chromosome regions named common fragile sites (CFSs). In this work, we characterized the expression of human CFSs in the glioblastoma U-251 MG cell line upon treatment with the inhibitor of DNA polymerase alpha aphidicolin (APH). We observed 52 gaps/breaks located within previously characterized CFSs. We found 17 to be CFSs in GBM cells upon treatment with APH, showing a frequency equal to at least 1% of the total gaps/breaks. We report that two CFSs localized to regions FRA2E (2p13/p12) and FRA2F (2q22) were only found in U-251 MG cells, but not lymphocytes or fibroblasts, after APH treatment. Notably, these glioblastoma-specific CFSs had a relatively high expression compared to the other CFSs with breakage frequency between ∼7 and 9%. Presence of long genes, incomplete replication, and delayed DNA synthesis during mitosis (MiDAS) after APH treatment suggest that an impaired replication process may contribute to this loci-specific fragility in U-251 MG cells. Altogether, our work offers a characterization of common fragile site expression in glioblastoma U-251 MG cells that may be further exploited for cytogenetic and clinical studies to advance our understanding of this incurable cancer.

Keywords:  cancer; chromosome instability; fragile sites; glioblastoma; replicative stress

DOI:  https://doi.org/10.3389/fgene.2021.810793
Cancers (Basel). 2022 Jan 26. pii: 627. [Epub ahead of print]14(3):

Chemotherapy Side-Effects: Not All DNA Damage Is Equal.

Winnie M C van den Boogaard, Daphne S J Komninos, Wilbert P Vermeij.

  Recent advances have increased survival rates of children and adults suffering from cancer thanks to effective anti-cancer therapy, such as chemotherapy. However, during treatment and later in life they are frequently confronted with the severe negative side-effects of their life-saving treatment. The occurrence of numerous features of accelerated aging, seriously affecting quality of life, has now become one of the most pressing problems associated with (pediatric) cancer treatment. Chemotherapies frequently target and damage the DNA, causing mutations or genome instability, a major hallmark of both cancer and aging. However, there are numerous types of chemotherapeutic drugs that are genotoxic and interfere with DNA metabolism in different ways, each with their own biodistribution, kinetics, and biological fate. Depending on the type of DNA lesion produced (e.g., interference with DNA replication or RNA transcription), the organ or cell type inflicted (e.g., cell cycle or differentiation status, metabolic state, activity of clearance and detoxification mechanisms, the cellular condition or micro-environment), and the degree of exposure, outcomes of cancer treatment can largely differ. These considerations provide a conceptual framework in which different classes of chemotherapeutics contribute to the development of toxicities and accelerated aging of different organ systems. Here, we summarize frequently observed side-effects in (pediatric) ex-cancer patients and discuss which types of DNA damage might be responsible.

Keywords:  DNA damage; cancer survivors; cancer treatment; chemotherapy; premature aging

DOI:  https://doi.org/10.3390/cancers14030627
Eur J Med Chem. 2022 Feb 03. pii: S0223-5234(22)00078-2. [Epub ahead of print]232 114176

Recent advances of human dihydroorotate dehydrogenase inhibitors for cancer therapy: Current development and future perspectives.

Lele Zhang, Jifa Zhang, Jiaxing Wang, Changyu Ren, Pan Tang, Liang Ouyang, Yuxi Wang.

  Human dihydroorotate dehydrogenase (hDHODH) is a flavin-dependent enzyme catalyzing the fourth step of pyrimidine de novo biosynthesis. Since aberrant pyrimidine metabolism is closely related abnormal cell proliferation, hDHODH is believed to have an intimate linkage with cancers. For instance, hDHODH induces the abrogation of β-catenin degradation and cell proliferation in esophageal squamous cell carcinoma (ESCC). Thus, small molecular inhibitors targeting hDHODH has been considered as a promising strategy for cancer treatment. In recent years, in exploiting novel structural hDHODH inhibitors (hDHODHi), a candidate drug PTC299 has entered clinical trials for treating acute myelocytic leukemia (AML) and other tumors. This review discusses tumor-related research of hDHODH and highlights the structure-activity relationships of hDHODHi, providing insights into new drugs targeting hDHODH for antitumor clinical practice.

Keywords:  Anticancer; Human dihydroorotate dehydrogenase (hDHODH); Small molecule inhibitors; Structure-activity relationships (SARs)

DOI:  https://doi.org/10.1016/j.ejmech.2022.114176
Nat Cell Biol. 2022 Feb;24(2): 148-154

Metabolic determination of cell fate through selective inheritance of mitochondria.

Julia Döhla, Emilia Kuuluvainen, Nadja Gebert, Ana Amaral, Johanna I Englund, Swetha Gopalakrishnan, Svetlana Konovalova, Anni I Nieminen, Ella S Salminen, Rubén Torregrosa Muñumer, Kati Ahlqvist, Yang Yang, Hien Bui, Timo Otonkoski, Reijo Käkelä, Ville Hietakangas, Henna Tyynismaa, Alessandro Ori, Pekka Katajisto.

Metabolic characteristics of adult stem cells are distinct from their differentiated progeny, and cellular metabolism is emerging as a potential driver of cell fate conversions1-4. How these metabolic features are established remains unclear. Here we identified inherited metabolism imposed by functionally distinct mitochondrial age-classes as a fate determinant in asymmetric division of epithelial stem-like cells. While chronologically old mitochondria support oxidative respiration, the electron transport chain of new organelles is proteomically immature and they respire less. After cell division, selectively segregated mitochondrial age-classes elicit a metabolic bias in progeny cells, with oxidative energy metabolism promoting differentiation in cells that inherit old mitochondria. Cells that inherit newly synthesized mitochondria with low levels of Rieske iron-sulfur polypeptide 1 have a higher pentose phosphate pathway activity, which promotes de novo purine biosynthesis and redox balance, and is required to maintain stemness during early fate determination after division. Our results demonstrate that fate decisions are susceptible to intrinsic metabolic bias imposed by selectively inherited mitochondria.

DOI: https://doi.org/10.1038/s41556-021-00837-0
Cancers (Basel). 2022 Jan 27. pii: 641. [Epub ahead of print]14(3):

Modulation of DNA Damage Response by SAM and HD Domain Containing Deoxynucleoside Triphosphate Triphosphohydrolase (SAMHD1) Determines Prognosis and Treatment Efficacy in Different Solid Tumor Types.

Eudald Felip, Lucía Gutiérrez-Chamorro, Maica Gómez, Edurne Garcia-Vidal, Margarita Romeo, Teresa Morán, Laura Layos, Laia Pérez-Roca, Eva Riveira-Muñoz, Bonaventura Clotet, Pedro Luis Fernandez, Ricard Mesía, Anna Martínez-Cardús, Ester Ballana, Mireia Margelí.

  SAMHD1 is a deoxynucleotide triphosphate (dNTP) triphosphohydrolase with important roles in the control of cell proliferation and apoptosis, either through the regulation of intracellular dNTPs levels or the modulation of the DNA damage response. However, SAMHD1's role in cancer evolution is still unknown. We performed the first in-depth study of SAMHD1's role in advanced solid tumors, by analyzing samples of 128 patients treated with chemotherapy agents based on platinum derivatives and/or antimetabolites, developing novel in vitro knock-out models to explore the mechanisms driving SAMHD1 function in cancer. Low (or no) expression of SAMHD1 was associated with a positive prognosis in breast, ovarian, and non-small cell lung cancer (NSCLC) cancer patients. A predictive value was associated with low-SAMHD1 expression in NSCLC and ovarian patients treated with antimetabolites in combination with platinum derivatives. In vitro, SAMHD1 knock-out cells showed increased γ-H2AX and apoptosis, suggesting that SAMHD1 depletion induces DNA damage leading to cell death. In vitro treatment with platinum-derived drugs significantly enhanced γ-H2AX and apoptotic markers expression in knock-out cells, indicating a synergic effect of SAMHD1 depletion and platinum-based treatment. SAMHD1 expression represents a new strong prognostic and predictive biomarker in solid tumors and, thus, modulation of the SAMHD1 function may constitute a promising target for the improvement of cancer therapy.

Keywords:  NSCLC; SAMHD1; breast cancer; ovarian cancer; solid tumors

DOI:  https://doi.org/10.3390/cancers14030641
Int J Mol Sci. 2022 Jan 27. pii: 1429. [Epub ahead of print]23(3):

Influence of Hypoxia on Radiosensitization of Cancer Cells by 5-Bromo-2'-deoxyuridine.

Magdalena Zdrowowicz, Paulina Spisz, Aleksandra Hać, Anna Herman-Antosiewicz, Janusz Rak.

  Radiotherapy is a crucial cancer treatment, but its outcome is still far from satisfactory. One of the reasons that cancer cells show resistance to ionizing radiation is hypoxia, defined as a low level of oxygenation, which is typical for solid tumors. In the hypoxic environment, cancer cells are 2-3 times more resistant to ionizing radiation than normoxic cells. To overcome this important impediment, radiosensitizers should be introduced to cancer therapy. When modified with an electrophilic substituent, nucleosides may undergo efficient dissociative electron attachment (DEA) that leaves behind nucleoside radicals, which, in secondary reactions, are able to induce DNA damage, leading to cancer cell death. We report the radiosensitizing effect of one of the best-known DEA-type radiosensitizers-5-bromo-2'-deoxyuridine (BrdU)-on breast (MCF-7) and prostate (PC3) cancer cells under both normoxia and hypoxia. MCF-7 and PC3 cells were treated with BrdU to investigate the effect of hypoxia on cell proliferation, incorporation into DNA and radiosensitivity. While the oxygen concentration did not significantly affect the efficiency of BrdU incorporation into DNA or the proliferation of tumor cells, the radiosensitizing effect of BrdU on hypoxic cells was more evident than on normoxic cells. Further mechanistic studies performed with the use of flow cytometry showed that under hypoxia, BrdU increased the level of histone H2A.X phosphorylation after X-ray exposure to a greater extent than under normal oxygenation conditions. These results confirm that the formation of double-strand breaks in hypoxic BrdU-treated cancer cells is more efficient. In addition, by performing stationary radiolysis of BrdU solution in the presence of an ●OH radical scavenger, we compared the degree of its electron-induced degradation under aerobic and anaerobic conditions. It was determined that radiodegradation under anaerobic conditions was almost twice as high as that under aerobic conditions.

Keywords:  DNA damage; X-ray; hypoxia; modified nucleosides; radiosensitizer; radiotherapy

DOI:  https://doi.org/10.3390/ijms23031429
Oncotarget. 2022 ;13 319-330

ABT199/venetoclax potentiates the cytotoxicity of alkylating agents and fludarabine in acute myeloid leukemia cells.

Benigno C Valdez, David Murray, Bin Yuan, Yago Nieto, Uday Popat, Borje S Andersson.

  The antineoplastic activity of pre-transplant regimens in hematopoietic stem cell transplantation (HSCT) is a critical factor for acute myeloid leukemia (AML) patients. There is an urgent need to identify novel approaches without jeopardizing patient safety. We hypothesized that combination of drugs with different mechanisms of action would provide better cytotoxicity. We, therefore, determined the synergistic cytotoxicity of various combinations of the alkylating agents busulfan (Bu) and 4-hydroperoxycyclophosphamide (4HC), the nucleoside analog fludarabine (Flu) and the BCL2 inhibitor ABT199/venetoclax in AML cells. [Bu+4HC] and [Bu+Flu] inhibited cell proliferation and activated apoptosis; addition of ABT199 to either combinations significantly increased these effects with combination indexes < 1. Apoptosis is suggested by cleavages of PARP1 and CASPASE 3, DNA fragmentation, increased reactive oxygen species, decreased mitochondrial membrane potential, and increased pro-apoptotic proteins in the cytoplasm. A similar enhancement of apoptosis was observed in patient-derived cell samples. ABT199/venetocalx upregulated anti-apoptotic MCL1 as a compensatory mechanism but addition of [Bu+4HC] or [Bu+Flu] negated this effect by CASPASE 3-mediated cleavage of MEK1/2 and its substrate MCL1. CASPASE 3 caused cleavage of pro-survival β-CATENIN, which likely contributed to the activation of stress signaling pathways involving SAPK/JNK and AMPK. The observed synergistic cytotoxicity was associated with an inhibition of pro-survival pathways involving STAT1, STAT5 and PI3K. These findings will be useful in designing clinical trials using these drug combinations as pre-transplant conditioning regimens for AML patients.

Keywords:  ABT199/venetoclax; acute myeloid leukemia; busulfan; cyclophosphamide; fludarabine

DOI:  https://doi.org/10.18632/oncotarget.28193