bims-numges 2021-07-25 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–07–25
39 papers selected by
Sean Rudd, Karolinska Institutet

Motif WFYY of human PrimPol is crucial to stabilize the incoming 3'-nucleotide during replication fork restart.
Harmful R-loops are prevented via different cell cycle-specific mechanisms.
PFKFB3 Inhibition Sensitizes DNA Crosslinking Chemotherapies by Suppressing Fanconi Anemia Repair.
Roles of Homologous Recombination in Response to Ionizing Radiation-Induced DNA Damage.
Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage.
ATR/ATM-mediated phosphorylation of BRCA1 T1394 promotes homologous recombinational repair and G2/M checkpoint maintenance.
Opportunities for Utilization of DNA Repair Inhibitors in Homologous Recombination Repair-Deficient and Proficient Pancreatic Adenocarcinoma.
Discovery of novel inhibitors of human phosphoglycerate dehydrogenase by activity-directed combinatorial chemical synthesis strategy.
Reconsidering pathway choice: a sequential model of mammalian DNA double-strand break pathway decisions.
OB-Folds and Genome Maintenance: Targeting Protein-DNA Interactions for Cancer Therapy.
A wake-up call for cancer DNA damage: the role of Schlafen 11 (SLFN11) across multiple cancers.
Intersection of Two Checkpoints: Could Inhibiting the DNA Damage Response Checkpoint Rescue Immune Checkpoint-Refractory Cancer?
BRCA mutations in pancreatic cancer and progress in their targeting.
MTA2 sensitizes gastric cancer cells to PARP inhibition by induction of DNA replication stress.
DNA-PKc inhibition overcomes taxane resistance by promoting taxane-induced DNA damage in prostate cancer cells.
Epstein-Barr Virus Induced Cytidine Metabolism Roles in Transformed B-Cell Growth and Survival.
CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.
Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition.
Exploiting DNA Damage Repair in Precision Cancer Therapy: BRCA1 as a Prime Therapeutic Target.
Crossover or non-crossover outcomes: tailored processing of homologous recombination intermediates.
Pyrophosphate release acts as a kinetic checkpoint during high-fidelity DNA replication by the Staphylococcus aureus replicative polymerase PolC.
Nucleotide stress responses in neural crest cell fate and melanoma.
Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity.
Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.
Deciphering CAD: Structure and function of a mega-enzymatic pyrimidine factory in health and disease.
Understanding and overcoming resistance to PARP inhibitors in cancer therapy.
The BET Inhibitor JQ1 Augments the Antitumor Efficacy of Gemcitabine in Preclinical Models of Pancreatic Cancer.
Differential Inhibition of Equilibrative Nucleoside Transporter 1 (ENT1) Activity by Tyrosine Kinase Inhibitors.
KSHV-encoded vCyclin can modulate HIF1α levels to promote DNA replication in hypoxia.
Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma.
A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.
Structural basis for ligand binding modes of CTP synthase.
Knockdown of DNA polymerase ζ relieved the chemoresistance of glioma via inhibiting the PI3K/AKT signaling pathway.
The WRN helicase: resolving a new target in microsatellite unstable cancers.
One carbon metabolism in human lung cancer.
A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition.
Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.
A DNA-repair pathway can affect transcriptional noise to promote cell fate transitions.
Comparable radiation sensitivity in p53 wild-type and p53 deficient tumor cells associated with different cell death modalities.

Nucleic Acids Res. 2021 Jul 24. pii: gkab634. [Epub ahead of print]

Motif WFYY of human PrimPol is crucial to stabilize the incoming 3'-nucleotide during replication fork restart.

Patricia A Calvo, María I Martínez-Jiménez, Marcos Díaz, Gorazd Stojkovic, Kazutoshi Kasho, Susana Guerra, Sjoerd Wanrooij, Juan Méndez, Luis Blanco.

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3'-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.

DOI: https://doi.org/10.1093/nar/gkab634
Nat Commun. 2021 07 22. 12(1): 4451

Harmful R-loops are prevented via different cell cycle-specific mechanisms.

Marta San Martin-Alonso, María E Soler-Oliva, María García-Rubio, Tatiana García-Muse, Andrés Aguilera.

Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.

DOI: https://doi.org/10.1038/s41467-021-24737-x
Cancers (Basel). 2021 Jul 18. pii: 3604. [Epub ahead of print]13(14):

PFKFB3 Inhibition Sensitizes DNA Crosslinking Chemotherapies by Suppressing Fanconi Anemia Repair.

Anna Huguet Ninou, Jemina Lehto, Dimitrios Chioureas, Hannah Stigsdotter, Korbinian Schelzig, Emma Åkerlund, Greta Gudoityte, Ulrika Joneborg, Joseph Carlson, Jos Jonkers, Brinton Seashore-Ludlow, Nina Marie Susanne Gustafsson.

  Replicative repair of interstrand crosslinks (ICL) generated by platinum chemotherapeutics is orchestrated by the Fanconi anemia (FA) repair pathway to ensure resolution of stalled replication forks and the maintenance of genomic integrity. Here, we identify novel regulation of FA repair by the cancer-associated glycolytic enzyme PFKFB3 that has functional consequences for replication-associated ICL repair and cancer cell survival. Inhibition of PFKFB3 displays a cancer-specific synergy with platinum compounds in blocking cell viability and restores sensitivity in treatment-resistant models. Notably, the synergies are associated with DNA-damage-induced chromatin association of PFKFB3 upon cancer transformation, which further increases upon platinum resistance. FA pathway activation triggers the PFKFB3 assembly into nuclear foci in an ATR- and FANCM-dependent manner. Blocking PFKFB3 activity disrupts the assembly of key FA repair factors and consequently prevents fork restart. This results in an incapacity to replicate cells to progress through S-phase, an accumulation of DNA damage in replicating cells, and fork collapse. We further validate PFKFB3-dependent regulation of FA repair in ex vivo cultures from cancer patients. Collectively, targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.

Keywords:  DNA repair; FANCD2; Fanconi anemia pathway; KAN0438757; PFKFB3

DOI:  https://doi.org/10.3390/cancers13143604
Int J Radiat Biol. 2021 Jul 20. 1-33

Roles of Homologous Recombination in Response to Ionizing Radiation-Induced DNA Damage.

Jac A Nickoloff, Neelam Sharma, Christopher P Allen, Lynn Taylor, Sage J Allen, Aruna S Jaiswal, Robert Hromas.

   PURPOSE: Ionizing radiation induces a vast array of DNA lesions including base damage, and single- and double-strand breaks (SSB, DSB). DSBs are among the most cytotoxic lesions, and mis-repair causes small- and large-scale genome alterations that can contribute to carcinogenesis. Indeed, ionizing radiation is a 'complete' carcinogen. DSBs arise immediately after irradiation, termed 'frank DSBs,' as well as several hours later in a replication-dependent manner, termed 'secondary' or 'replication-dependent DSBs. DSBs resulting from replication fork collapse are single-ended and thus pose a distinct problem from two-ended, frank DSBs. DSBs are repaired by error-prone non-homologous end-joining (NHEJ), or generally error-free homologous recombination (HR), each with sub-pathways. Clarifying how these pathways operate in normal and tumor cells is critical to increasing tumor control and minimizing side effects during radiotherapy.
CONCLUSIONS: The choice between NHEJ and HR is regulated during the cell cycle and by other factors. DSB repair pathways are major contributors to cell survival after ionizing radiation, including tumor-resistance to radiotherapy. Several nucleases are important for HR-mediated repair of replication-dependent DSBs and thus replication fork restart. These include three structure-specific nucleases, the 3' MUS81 nuclease, and two 5' nucleases, EEPD1 and Metnase, as well as three end-resection nucleases, MRE11, EXO1, and DNA2. The three structure-specific nucleases evolved at very different times, suggesting incremental acceleration of replication fork restart to limit toxic HR intermediates and genome instability as genomes increased in size during evolution, including the gain of large numbers of HR-prone repetitive elements. Ionizing radiation also induces delayed effects, observed days to weeks after exposure, including delayed cell death and delayed HR. In this review we highlight the roles of HR in cellular responses to ionizing radiation, and discuss the importance of HR as an exploitable target for cancer radiotherapy.

Keywords:  DNA double-strand breaks; DNA repair; cancer radiotherapy; homologous recombination; ionizing radiation; replication stress

DOI:  https://doi.org/10.1080/09553002.2021.1956001
Front Mol Biosci. 2021 ;8 712971

Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage.

Thomas A Guilliam.

  The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template.

Keywords:  DNA damage tolerance; DNA replication; recoupling; replication fork; replisome; repriming; translesion synthesis

DOI:  https://doi.org/10.3389/fmolb.2021.712971
Cancer Res. 2021 Jul 23. pii: canres.2723.2020. [Epub ahead of print]

ATR/ATM-mediated phosphorylation of BRCA1 T1394 promotes homologous recombinational repair and G2/M checkpoint maintenance.

Tzeh K Foo, Gabrele Vincelli, Eric Huselid, Joonyoung Her, Haiyan Zheng, Srilatha Simhadri, Meiling Wang, Yanying Huo, Tao Li, Xiaochun Yu, Hong Li, Weixing Zhao, Samuel F Bunting, Bing Xia.

BRCA1 maintains genome integrity and suppresses tumorigenesis by promoting homologous recombination (HR)-mediated repair of DNA double strand breaks (DSB) and DNA damage-induced cell cycle checkpoints. Phosphorylation of BRCA1 by ATM, ATR, CHK2, CDK, and PLK1 kinases has been reported to regulate its functions. Here we show that ATR and ATM-mediated phosphorylation of BRCA1 on T1394, a highly conserved but functionally uncharacterized site, is a key modification for its function in the DNA damage response. Following DNA damage, T1394 phosphorylation ensured faithful repair of DSBs by promoting HR and preventing single strand annealing, a deletion-generating repair process. BRCA1 T1394 phosphorylation further safeguarded chromosomal integrity by maintaining the G2/M checkpoint. Moreover, multiple patient-derived BRCA1 variants of unknown significance were shown to affect T1394 phosphorylation. These results establish an important regulatory mechanism of BRCA1 function in the DNA damage response and may have implications in the development or prognosis of BRCA1-associated cancers.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-2723
Clin Cancer Res. 2021 Jul 20. pii: clincanres.1367.2021. [Epub ahead of print]

Opportunities for Utilization of DNA Repair Inhibitors in Homologous Recombination Repair-Deficient and Proficient Pancreatic Adenocarcinoma.

James M Cleary, Brian M Wolpin, Stephanie K Dougan, Srivatsan Raghavan, Harshabad Singh, Brandon Huffman, Nilay S Sethi, Jonathan A Nowak, Geoffrey I Shapiro, Andrew J Aguirre, Alan D D'Andrea.

Pancreatic cancer is rapidly progressive and notoriously difficult to treat with cytotoxic chemotherapy and targeted agents. Recent demonstration of the efficacy of maintenance PARP inhibition in germline BRCA mutated pancreatic cancer has raised hopes that increased understanding of the DNA damage response pathway will lead to new therapies in both homologous recombination (HR) repair-deficient and proficient pancreatic cancer. Here, we review the potential mechanisms of exploiting HR deficiency, replicative stress, and DNA damage-mediated immune activation through targeted inhibition of DNA repair regulatory proteins.

DOI: https://doi.org/10.1158/1078-0432.CCR-21-1367
Bioorg Chem. 2021 Jul 08. pii: S0045-2068(21)00536-8. [Epub ahead of print]115 105159

Discovery of novel inhibitors of human phosphoglycerate dehydrogenase by activity-directed combinatorial chemical synthesis strategy.

Xia Zhou, Yuping Tan, Kun Gou, Lei Tao, Yuan Luo, Yue Zhou, Zeping Zuo, Qingxiang Sun, Youfu Luo, Yinglan Zhao.

  Serine, the source of the one-carbon units essential for de novo purine and deoxythymidine synthesis plays a crucial role in the growth of cancer cells. Phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first, rate-limiting step in de novo serine biosynthesis has become a promising target for the cancer treatment. Here we identified H-G6 as a potential PHGDH inhibitor from the screening of an in-house small molecule library based on the enzymatic assay. We adopted activity-directed combinatorial chemical synthesis strategy to optimize this hit compound. Compound b36 was found to be the noncompetitive and the most promising one with IC50 values of 5.96 ± 0.61 μM against PHGDH. Compound b36 inhibited the proliferation of human breast cancer and ovarian cancer cells, reduced intracellular serine synthesis, damaged DNA synthesis, and induced cell cycle arrest. Collectively, our results suggest that b36 is a novel PHGDH inhibitor, which could be a promising modulator to reprogram the serine synthesis pathway and might be a potential anticancer lead worth further exploration.

Keywords:  3-phosphoglycerate dehydrogenase; Combinatorial chemical synthesis; Inhibitor; Serine synthesis

DOI:  https://doi.org/10.1016/j.bioorg.2021.105159
Curr Opin Genet Dev. 2021 Jul 19. pii: S0959-437X(21)00081-2. [Epub ahead of print]71 55-62

Reconsidering pathway choice: a sequential model of mammalian DNA double-strand break pathway decisions.

Tanya T Paull.

DNA double-strand breaks can be repaired through ligation-based pathways (non-homologous end-joining) or replication-based pathways (homologous recombination) in eukaryotic cells. The decisions that govern these outcomes are widely viewed as a competition between factors that recognize DNA ends and physically promote association of factors specific to each pathway, commonly known as 'pathway choice'. Here I review recent results in the literature and propose that this decision is better described as a sequential set of binding and end processing events, with non-homologous end joining as the first decision point. Physical association and co-localization of end resection factors with non-homologous end-joining factors suggests that ends are transferred between these complexes, thus the ultimate outcome is not the result of a competition but is more akin to a relay race that is determined by the efficiency of the initial end-joining event and the availability of activated DNA end-processing enzymes.

DOI: https://doi.org/10.1016/j.gde.2021.06.011
Cancers (Basel). 2021 Jul 03. pii: 3346. [Epub ahead of print]13(13):

OB-Folds and Genome Maintenance: Targeting Protein-DNA Interactions for Cancer Therapy.

Sui Par, Sofia Vaides, Pamela S VanderVere-Carozza, Katherine S Pawelczak, Jason Stewart, John J Turchi.

  Genome stability and maintenance pathways along with their requisite proteins are critical for the accurate duplication of genetic material, mutation avoidance, and suppression of human diseases including cancer. Many of these proteins participate in these pathways by binding directly to DNA, and a subset employ oligonucleotide/oligosaccharide binding folds (OB-fold) to facilitate the protein-DNA interactions. OB-fold motifs allow for sequence independent binding to single-stranded DNA (ssDNA) and can serve to position specific proteins at specific DNA structures and then, via protein-protein interaction motifs, assemble the machinery to catalyze the replication, repair, or recombination of DNA. This review provides an overview of the OB-fold structural organization of some of the most relevant OB-fold containing proteins for oncology and drug discovery. We discuss their individual roles in DNA metabolism, progress toward drugging these motifs and their utility as potential cancer therapeutics. While protein-DNA interactions were initially thought to be undruggable, recent reports of success with molecules targeting OB-fold containing proteins suggest otherwise. The potential for the development of agents targeting OB-folds is in its infancy, but if successful, would expand the opportunities to impinge on genome stability and maintenance pathways for more effective cancer treatment.

Keywords:  DNA binding; DNA damage response; DNA repair; DNA replication; OB-fold; cancer therapy; drug development; genome stability; single-stranded DNA

DOI:  https://doi.org/10.3390/cancers13133346
Br J Cancer. 2021 Jul 22.

A wake-up call for cancer DNA damage: the role of Schlafen 11 (SLFN11) across multiple cancers.

Bingnan Zhang, Kavya Ramkumar, Robert John Cardnell, Carl Michael Gay, C Allison Stewart, Wei-Lien Wang, Junya Fujimoto, Ignacio I Wistuba, Lauren Averett Byers.

DNA-damaging agents exploit increased genomic instability, a hallmark of cancer. Recently, inhibitors targeting the DNA damage response (DDR) pathways, such as PARP inhibitors, have also shown promising therapeutic potential. However, not all tumors respond well to these treatments, suggesting additional determinants of response are required. Schlafen 11 (SLFN11), a putative DNA/RNA helicase that induces irreversible replication block, is emerging as an important regulator of cellular response to DNA damage. Preclinical and emerging clinical trial data suggest that SLFN11 is a predictive biomarker of response to a wide range of therapeutics that cause DNA damage including platinum salts and topoisomerase I/II inhibitors, as well as PARP inhibitors, which has raised exciting possibilities for its clinical application. In this article, we review the function, prevalence, and clinical testing of SLFN11 in tumor biopsy samples and circulating tumor cells. We discuss mounting evidence of SLFN11 as a key predictive biomarker for a wide range of cancer therapeutics and as a prognostic marker across several cancer types. Furthermore, we discuss emerging areas of investigation such as epigenetic reactivation of SLFN11 and its role in activating immune response. We then provide perspectives on open questions and future directions in studying this important biomarker.

DOI: https://doi.org/10.1038/s41416-021-01476-w
Cancers (Basel). 2021 Jul 08. pii: 3415. [Epub ahead of print]13(14):

Intersection of Two Checkpoints: Could Inhibiting the DNA Damage Response Checkpoint Rescue Immune Checkpoint-Refractory Cancer?

Peter H Goff, Rashmi Bhakuni, Thomas Pulliam, Jung Hyun Lee, Evan T Hall, Paul Nghiem.

  Metastatic cancers resistant to immunotherapy require novel management strategies. DNA damage response (DDR) proteins, including ATR (ataxia telangiectasia and Rad3-related), ATM (ataxia telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase), have been promising therapeutic targets for decades. Specific, potent DDR inhibitors (DDRi) recently entered clinical trials. Surprisingly, preclinical studies have now indicated that DDRi may stimulate anti-tumor immunity to augment immunotherapy. The mechanisms governing how DDRi could promote anti-tumor immunity are not well understood; however, early evidence suggests that they can potentiate immunogenic cell death to recruit and activate antigen-presenting cells to prime an adaptive immune response. Merkel cell carcinoma (MCC) is well suited to test these concepts. It is inherently immunogenic as ~50% of patients with advanced MCC persistently benefit from immunotherapy, making MCC one of the most responsive solid tumors. As is typical of neuroendocrine cancers, dysfunction of p53 and Rb with upregulation of Myc leads to the very rapid growth of MCC. This suggests high replication stress and susceptibility to DDRi and DNA-damaging agents. Indeed, MCC tumors are particularly radiosensitive. Given its inherent immunogenicity, cell cycle checkpoint deficiencies and sensitivity to DNA damage, MCC may be ideal for testing whether targeting the intersection of the DDR checkpoint and the immune checkpoint could help patients with immunotherapy-refractory cancers.

Keywords:  ATM; ATR; DNA damage response inhibitors; DNA-PK; Merkel cell carcinoma; PD-1 pathway; cell cycle checkpoint; immune checkpoint inhibitors; immunogenic cell death

DOI:  https://doi.org/10.3390/cancers13143415
Expert Opin Ther Targets. 2021 Jul 22.

BRCA mutations in pancreatic cancer and progress in their targeting.

Samer Alkassis, Omid Yazdanpanah, Philip Agob Philip.

   INTRODUCTION: Genomic instability resulting from DNA damage repair (DDR) deficiencies is hallmark of cancer and offers treatment opportunities. Homologous recombination DDR defect is result of multiple critical gene mutations including BRCA1/2. Targeting DNA DDR defects in pancreatic cancer (PC) is emerging as potential treatment strategy with current focus on BRCA mutations.
AREAS COVERED: Challenges in treating patients with PC are explained. We review DDR defects as a treatment target in PC. Specifically, germline BRCA mutation and sensitivity to platinum compounds and exploiting the strategy of synthetic lethality using poly (ADP-ribose) polymerase (PARP) inhibition. Literature review was undertaken through PubMed, Google Scholar, and Clinicaltrials.gov website.
EXPERT OPINION: DDR defects are promising targets for novel therapies in PC. Early application of such strategy is in patient subgroup with BRCA germline mutation is seen in only 5-7% of the PC population. The oral PARP inhibitor olaparib in the maintenance setting represents the first targeted therapy in metastatic PC based on a phase 3 study. There is a very modest benefit for patients with PC using PARP inhibitors. Future work must improve our understanding of mechanisms of sensitivity and resistance to PARP inhibitors in PC and to enhance molecular selection of patients for such therapy.

Keywords:  BRCA mutation; DNA damage repair; PARP inhibitor; PARP resistance; homologous recombination; pancreatic cancer; synthetic lethality

DOI:  https://doi.org/10.1080/14728222.2021.1957462
Transl Oncol. 2021 Jul 17. pii: S1936-5233(21)00159-5. [Epub ahead of print]14(10): 101167

MTA2 sensitizes gastric cancer cells to PARP inhibition by induction of DNA replication stress.

Jinwen Shi, Xiaofeng Zhang, Jin'e Li, Wenwen Huang, Yini Wang, Yi Wang, Jun Qin.

  Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib selectively kills cancer cells with BRCA-deficiency and is approved for BRCA-mutated breast, ovarian and pancreatic cancers by FDA. However, phase III study of olaparib failed to show a significant improvement in overall survival in patients with gastric cancer (GC). To discover an effective biomarker for GC patient-selection in olaparib treatment, we analyzed proteomic profiling of 12 GC cell lines. MTA2 was identified to confer sensitivity to olaparib by aggravating olaparib-induced replication stress in cancer cells. Mechanistically, we applied Cleavage Under Targets and Tagmentation assay to find that MTA2 proteins preferentially bind regions of replication origin-associated DNA sequences, which could be enhanced by olaparib treatment. Furthermore, MTA2 was validated here to render cancer cells susceptible to combination of olaparib with ATR inhibitor AZD6738. In general, our study identified MTA2 as a potential biomarker for olaparib sensitivity by aggravating olaparib-induced replication stress.

Keywords:  Gastric cancer; MTA2; Olaparib; Replication origins; Replication stress

DOI:  https://doi.org/10.1016/j.tranon.2021.101167
Prostate. 2021 Jul 23.

DNA-PKc inhibition overcomes taxane resistance by promoting taxane-induced DNA damage in prostate cancer cells.

Olivia S Chao, Oscar B Goodman.

   BACKGROUND: Overcoming taxane resistance remains a major clinical challenge in metastatic castrate-resistant prostate cancer (mCRPC). Loss of DNA repair proteins is associated with resistance to anti-microtubule agents. We propose that alterations in DNA damage response (DDR) pathway contribute to taxane resistance, and identification of these alterations may provide a potential therapeutic target to resensitize docetaxel-refractory mCRPC to taxane-based therapy.
METHODS: Alterations in DDR gene expression in our prostate cancer cell line model of docetaxel-resistance (DU145-DxR) derived from DU-145 cells were determined by DDR pathway-specific polymerase chain reaction array and immunoblotting. The PRKDC gene encoding DNA-PKc (DNA-dependent protein kinase catalytic unit), was noted to be overexpressed and evaluated for its role in docetaxel resistance. Cell viability and clonogenic survival of docetaxel-treated DU145-DxR cells were assessed after pharmacologic inhibition of DNA-PKc with three different inhibitors-NU7441, LTURM34, and M3814. Response to second-line cytotoxic agents, cabazitaxel and etoposide upon DNA-PKc inhibition was also tested. The impact of DNA-PKc upregulation on DNA damage repair was evaluated by comet assay and analysis of double-strand breaks marker, γH2AX and Rad51. Lastly, DNA-PKc inhibitor's effect on MDR1 activity was assessed by rhodamine 123 efflux assay.
RESULTS: DDR pathway-specific gene profiling revealed significant upregulation of PRKDC and CDK7, and downregulation of MSH3 in DU145-DxR cells. Compared to parental DU145, DU145-DxR cells sustained significantly less DNA damage when exposed to etoposide and docetaxel. Pharmacologic inhibition of DNA-PKc, a component of NHEJ repair machinery, with all three inhibitors, significantly resensitized DU145-DxR cells to docetaxel. Furthermore, DNA-PKc inhibition also resensitized DU145-DxR to cabazitaxel and etoposide, which demonstrated cross-resistance. Inhibition of DNA-PKc led to increased DNA damage in etoposide- and docetaxel-treated DU145-DxR cells. Finally, DNA-PKc inhibition did not affect MDR1 activity, indicating that DNA-PKc inhibitors resensitized taxane-resistant cells via an MDR1-independent mechanism.
CONCLUSION: This study supports a role of DDR genes, particularly, DNA-PKc in promoting resistance to taxanes in mCRPC. Targeting prostatic DNA-PKc may provide a novel strategy to restore taxane sensitivity in taxane-refractory mCRPC.

Keywords:  DNA damage response; PRKDC; cabazitaxel; cross resistance; docetaxel; multidrug resistance

DOI:  https://doi.org/10.1002/pros.24200
mBio. 2021 Jul 20. e0153021

Epstein-Barr Virus Induced Cytidine Metabolism Roles in Transformed B-Cell Growth and Survival.

Jin Hua Liang, Chong Wang, Stephanie Pei Tung Yiu, Bo Zhao, Rui Guo, Benjamin E Gewurz.

  Epstein-Barr virus (EBV) is associated with 200,000 cancers annually, including B-cell lymphomas in immunosuppressed hosts. Hypomorphic mutations of the de novo pyrimidine synthesis pathway enzyme cytidine 5' triphosphate synthase 1 (CTPS1) suppress cell-mediated immunity, resulting in fulminant EBV infection and EBV+ central nervous system (CNS) lymphomas. Since CTP is a critical precursor for DNA, RNA, and phospholipid synthesis, this observation raises the question of whether the isozyme CTPS2 or cytidine salvage pathways help meet CTP demand in EBV-infected B cells. Here, we found that EBV upregulated CTPS1 and CTPS2 with distinct kinetics in newly infected B cells. While CRISPR CTPS1 knockout caused DNA damage and proliferation defects in lymphoblastoid cell lines (LCLs), which express the EBV latency III program observed in CNS lymphomas, double CTPS1/2 knockout caused stronger phenotypes. EBNA2, MYC, and noncanonical NF-κB positively regulated CTPS1 expression. CTPS1 depletion impaired EBV lytic DNA synthesis, suggesting that latent EBV may drive pathogenesis with CTPS1 deficiency. Cytidine rescued CTPS1/2 deficiency phenotypes in EBV-transformed LCLs and Burkitt B cells, highlighting CTPS1/2 as a potential therapeutic target for EBV-driven lymphoproliferative disorders. Collectively, our results suggest that CTPS1 and CTPS2 have partially redundant roles in EBV-transformed B cells and provide insights into EBV pathogenesis with CTPS1 deficiency.

Keywords:  B-cell deficiency; chronic active EBV; gammaherpesvirus; lymphoproliferative disease; mononucleosis; nucleotide metabolism; primary immunodeficiency; pyrimidine metabolism; tumor virus

DOI:  https://doi.org/10.1128/mBio.01530-21
J Cell Sci. 2021 Jul 19. pii: jcs.258601. [Epub ahead of print]

CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.

Swati Priya, Ekjot Kaur, Swati Kulshrestha, Awadhesh Pandit, Isabelle Gross, Nitin Kumar, Himanshi Agarwal, Aamir Khan, Radhey Shyam, Prakash Bhagat, Jyothi S Prabhu, Perumal Nagarajan, S V S Deo, Avinash Bajaj, Jean-Noël Freund, Arnab Mukhopadhyay, Sagar Sengupta.

  Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2 containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 and RNF8. CDX2 directly regulated the DDSMs which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival, and thereby could be used as a prognostic biomarker.

Keywords:  ATM; BLM; BRCA1; Chk1; Colon cancer; DNA damage response; DNA repair; MicroRNA; RNF8

DOI:  https://doi.org/10.1242/jcs.258601
Cancers (Basel). 2021 Jul 10. pii: 3464. [Epub ahead of print]13(14):

Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition.

Miriam Pons, Yanira Zeyn, Stella Zahn, Nisintha Mahendrarajah, Brent D G Page, Patrick T Gunning, Richard Moriggl, Walburgis Brenner, Falk Butter, Oliver H Krämer.

  The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.

Keywords:  AML; BCL-XL; BCR-ABL1; CML; FLT3; RAF1; apoptosis; hydroxyurea; replication stress

DOI:  https://doi.org/10.3390/cancers13143464
Cancers (Basel). 2021 Jul 09. pii: 3438. [Epub ahead of print]13(14):

Exploiting DNA Damage Repair in Precision Cancer Therapy: BRCA1 as a Prime Therapeutic Target.

Liliana Raimundo, Juliana Calheiros, Lucília Saraiva.

  Precision medicine aims to identify specific molecular alterations, such as driver mutations, allowing tailored and effective anticancer therapies. Poly(ADP)-ribose polymerase inhibitors (PARPi) are the prototypical example of targeted therapy, exploiting the inability of cancer cells to repair DNA damage. Following the concept of synthetic lethality, PARPi have gained great relevance, particularly in BRCA1 dysfunctional cancer cells. In fact, BRCA1 mutations culminate in DNA repair defects that can render cancer cells more vulnerable to therapy. However, the efficacy of these drugs has been greatly affected by the occurrence of resistance due to multi-connected DNA repair pathways that may compensate for each other. Hence, the search for additional effective agents targeting DNA damage repair (DDR) is of crucial importance. In this context, BRCA1 has assumed a central role in developing drugs aimed at inhibiting DNA repair activity. Collectively, this review provides an in-depth understanding of the biology and regulatory mechanisms of DDR pathways, highlighting the potential of DDR-associated molecules, particularly BRCA1 and its interconnected partners, in precision cancer medicine. It also affords an overview about what we have achieved and a reflection on how much remains to be done in this field, further addressing encouraging clues for the advance of DDR targeted therapy.

Keywords:  BRCA1; DNA damage repair; synthetic lethality; targeted anticancer therapy

DOI:  https://doi.org/10.3390/cancers13143438
Curr Opin Genet Dev. 2021 Jul 19. pii: S0959-437X(21)00082-4. [Epub ahead of print]71 39-47

Crossover or non-crossover outcomes: tailored processing of homologous recombination intermediates.

Aurore Sanchez, Giordano Reginato, Petr Cejka.

DNA breaks may arise accidentally in vegetative cells or in a programmed manner in meiosis. The usage of a DNA template makes homologous recombination potentially error-free, however, recombination is not always accurate. Cells possess a remarkable capacity to tailor processing of recombination intermediates to fulfill a particular need. Vegetatively growing cells aim to maintain genome stability and therefore repair accidental breaks largely accurately, using sister chromatids as templates, into mostly non-crossovers products. Recombination in meiotic cells is instead more likely to employ homologous chromosomes as templates and result in crossovers to allow proper chromosome segregation and promote genetic diversity. Here we review models explaining the processing of recombination intermediates in vegetative and meiotic cells and its regulation, with a focus on MLH1-MLH3-dependent crossing-over during meiotic recombination.

DOI: https://doi.org/10.1016/j.gde.2021.06.012
Nucleic Acids Res. 2021 Jul 24. pii: gkab613. [Epub ahead of print]

Pyrophosphate release acts as a kinetic checkpoint during high-fidelity DNA replication by the Staphylococcus aureus replicative polymerase PolC.

Sean P Fagan, Purba Mukherjee, William J Jaremko, Rachel Nelson-Rigg, Ryan C Wilson, Tyler L Dangerfield, Kenneth A Johnson, Indrajit Lahiri, Janice D Pata.

Bacterial replication is a fast and accurate process, with the bulk of genome duplication being catalyzed by the α subunit of DNA polymerase III within the bacterial replisome. Structural and biochemical studies have elucidated the overall properties of these polymerases, including how they interact with other components of the replisome, but have only begun to define the enzymatic mechanism of nucleotide incorporation. Using transient-state methods, we have determined the kinetic mechanism of accurate replication by PolC, the replicative polymerase from the Gram-positive pathogen Staphylococcus aureus. Remarkably, PolC can recognize the presence of the next correct nucleotide prior to completing the addition of the current nucleotide. By modulating the rate of pyrophosphate byproduct release, PolC can tune the speed of DNA synthesis in response to the concentration of the next incoming nucleotide. The kinetic mechanism described here would allow PolC to perform high fidelity replication in response to diverse cellular environments.

DOI: https://doi.org/10.1093/nar/gkab613
Cell Cycle. 2021 Jul 19. 1-13

Nucleotide stress responses in neural crest cell fate and melanoma.

Audrey Sporrij, Leonard I Zon.

  Melanoma is the deadliest form of skin cancer. While clinical developments have significantly improved patient prognosis, effective treatment is often obstructed by limited response rates, intrinsic or acquired resistance to therapy, and adverse events. Melanoma initiation and progression are associated with transcriptional reprogramming of melanocytes to a cell state that resembles the lineage from which the cells are specified during development, that is the neural crest. Convergence to a neural crest cell (NCC)-like state revealed the therapeutic potential of targeting developmental pathways for the treatment of melanoma. Neural crest cells have a unique sensitivity to metabolic dysregulation, especially nucleotide depletion. Mutations in the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) particularly affect neural crest-derived tissues and cause Miller syndrome, a genetic disorder characterized by craniofacial malformations in patients. The developmental susceptibility of the neural crest to nucleotide deficiency is conserved in melanoma and provides a metabolic vulnerability that can be exploited for therapeutic purposes. We review the current knowledge on nucleotide stress responses in neural crest and melanoma and discuss how the recent scientific advances that have improved our understanding of transcriptional regulation during nucleotide depletion can impact melanoma treatment.

Keywords:  DHODH; HEXIM; Neural crest; RNA helicase DDX21; melanoma; nucleotide stress

DOI:  https://doi.org/10.1080/15384101.2021.1947567
PLoS One. 2021 ;16(7): e0247227

Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity.

Randall C Mazzarino, Veronika Baresova, Marie Zikánová, Nathan Duval, Terry G Wilkinson, David Patterson, Guido N Vacano.

In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We selected the Cluster of Differentiation (CD36) gene for initial analysis based on its elevated expression in crGART versus HeLa as well as its high basal expression, high log2 value, and minimal P-value.

DOI: https://doi.org/10.1371/journal.pone.0247227
Pharmacol Res Perspect. 2021 Aug;9(4): e00831

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Siennah R Miller, Joseph L Jilek, Meghan E McGrath, Raymond K Hau, Erin Q Jennings, James J Galligan, Stephen H Wright, Nathan J Cherrington.

  Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

Keywords:  Sertoli cells; blood-testis barrier; clofarabine; nucleoside transporter; nucleosides

DOI:  https://doi.org/10.1002/prp2.831
Protein Sci. 2021 Jul 19.

Deciphering CAD: Structure and function of a mega-enzymatic pyrimidine factory in health and disease.

Francisco Del Caño-Ochoa, Santiago Ramón-Maiques.

  CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein divided into different enzymatic domains, each catalyzing one of the initial reactions for de novo biosynthesis of pyrimidine nucleotides: glutaminase-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase. The pathway for de novo pyrimidine synthesis is essential for cell proliferation and is conserved in all living organisms, but the covalent linkage of the first enzymatic activities into a multienzymatic CAD particle is unique to animals. In other organisms, these enzymatic activities are encoded as monofunctional proteins for which there is abundant structural and biochemical information. However, the knowledge about CAD is scarce and fragmented. Understanding CAD requires not only to determine the three-dimensional structures and define the catalytic and regulatory mechanisms of the different enzymatic domains, but also to comprehend how these domains entangle and work in a coordinated and regulated manner. This review summarizes significant progress over the past 10 years toward the characterization of CAD's architecture, function, regulatory mechanisms, and cellular compartmentalization, as well as the recent finding of a new and rare neurometabolic disorder caused by defects in CAD activities.

Keywords:  aspartate transcarbamoylase; carbamoyl phosphate synthetase; de novo pyrimidine biosynthesis; dihydroorotase; multienzymatic protein; nucleotide metabolism; rare diseases

DOI:  https://doi.org/10.1002/pro.4158
Nat Rev Clin Oncol. 2021 Jul 20.

Understanding and overcoming resistance to PARP inhibitors in cancer therapy.

Mariana Paes Dias, Sarah C Moser, Shridar Ganesan, Jos Jonkers.

Developing novel targeted anticancer therapies is a major goal of current research. The use of poly(ADP-ribose) polymerase (PARP) inhibitors in patients with homologous recombination-deficient tumours provides one of the best examples of a targeted therapy that has been successfully translated into the clinic. The success of this approach has so far led to the approval of four different PARP inhibitors for the treatment of several types of cancers and a total of seven different compounds are currently under clinical investigation for various indications. Clinical trials have demonstrated promising response rates among patients receiving PARP inhibitors, although the majority will inevitably develop resistance. Preclinical and clinical data have revealed multiple mechanisms of resistance and current efforts are focused on developing strategies to address this challenge. In this Review, we summarize the diverse processes underlying resistance to PARP inhibitors and discuss the potential strategies that might overcome these mechanisms such as combinations with chemotherapies, targeting the acquired vulnerabilities associated with resistance to PARP inhibitors or suppressing genomic instability.

DOI: https://doi.org/10.1038/s41571-021-00532-x
Cancers (Basel). 2021 Jul 11. pii: 3470. [Epub ahead of print]13(14):

The BET Inhibitor JQ1 Augments the Antitumor Efficacy of Gemcitabine in Preclinical Models of Pancreatic Cancer.

Aubrey L Miller, Patrick L Garcia, Samuel C Fehling, Tracy L Gamblin, Rebecca B Vance, Leona N Council, Dongquan Chen, Eddy S Yang, Robert C A M van Waardenburg, Karina J Yoon.

  Gemcitabine is used to treat pancreatic cancer (PC), but is not curative. We sought to determine whether gemcitabine + a BET bromodomain inhibitor was superior to gemcitabine, and identify proteins that may contribute to the efficacy of this combination. This study was based on observations that cell cycle dysregulation and DNA damage augment the efficacy of gemcitabine. BET inhibitors arrest cells in G1 and allow increases in DNA damage, likely due to inhibition of expression of DNA repair proteins Ku80 and RAD51. BET inhibitors (JQ1 or I-BET762) + gemcitabine were synergistic in vitro, in Panc1, MiaPaCa2 and Su86 PC cell lines. JQ1 + gemcitabine was more effective in vivo than either drug alone in patient-derived xenograft models (P < 0.01). Increases in the apoptosis marker cleaved caspase 3 and DNA damage marker γH2AX paralleled antitumor efficacy. Notably, RNA-seq data showed that JQ1 + gemcitabine selectively inhibited HMGCS2 and APOC1 ~6-fold, compared to controls. These proteins contribute to cholesterol biosynthesis and lipid metabolism, and their overexpression supports tumor cell proliferation. IPA data indicated that JQ1 + gemcitabine selectively inhibited the LXR/RXR activation pathway, suggesting the hypothesis that this inhibition may contribute to the observed in vivo efficacy of JQ1 + gemcitabine.

Keywords:  APOC1; BET bromodomain inhibitor; HMGCS2; JQ1; LXR/RXR; RNA-seq; combination therapy; gemcitabine; pancreatic ductal adenocarcinoma; patient-derived xenograft

DOI:  https://doi.org/10.3390/cancers13143470
Eur J Drug Metab Pharmacokinet. 2021 Jul 18.

Differential Inhibition of Equilibrative Nucleoside Transporter 1 (ENT1) Activity by Tyrosine Kinase Inhibitors.

Elodie Jouan, Amélie Moreau, Arnaud Bruyere, Karima Alim, Claire Denizot, Yannick Parmentier, Olivier Fardel.

BACKGROUND AND OBJECTIVES: Equilibrative nucleoside transporter (ENT) 1 is a widely-expressed drug transporter, handling nucleoside analogues as well as endogenous nucleosides. ENT1 has been postulated to be inhibited by some marketed tyrosine kinase inhibitors (TKIs). To obtain insights into this point, the interactions of 24 TKIs with ENT1 activity have been analyzed.
METHODS: Inhibition of ENT1 activity was investigated in vitro through quantifying the decrease of [3H]-uridine uptake caused by TKIs in HAP1 ENT2-knockout cells, exhibiting selective ENT1 expression. TKI effects towards ENT1-mediated transport were additionally characterized in terms of their in vivo relevance and of their relationship to TKI molecular descriptors. Putative transport of the TKI lorlatinib by ENT1/ENT2 was analyzed by LC-MS/MS.
RESULTS: Of 24 TKIs, 12 of them, each used at 10 µM, were found to behave as moderate or strong inhibitors of ENT1, i.e., they decreased ENT1 activity by at least 35%. This inhibition was concentration-dependent for at least the strongest ones (IC50 less than 10 µM) and was correlated with some molecular descriptors, especially with atom-type E-state indices. Lorlatinib was notably a potent in vitro inhibitor of ENT1/ENT2 (IC50 values around 1.0-2.5 µM) and was predicted to inhibit these nucleoside transporters at relevant clinical concentrations, without, however, being a substrate for them.
CONCLUSION: Our data unambiguously add ENT1 to the list of drug transporters inhibited by TKIs, especially by lorlatinib. This point likely merits attention in terms of possible drug-drug interactions, notably for nucleoside analogues, whose ENT1-mediated uptake into their target cells may be hampered by co-administrated TKIs such as lorlatinib.

DOI: https://doi.org/10.1007/s13318-021-00703-2
Elife. 2021 Jul 19. pii: e57436. [Epub ahead of print]10

KSHV-encoded vCyclin can modulate HIF1α levels to promote DNA replication in hypoxia.

Rajnish Kumar Singh, Yonggang Pei, Dipayan Bose, Zachary L Lamplugh, Kunfeng Sun, Yan Yuan, Paul Lieberman, Jianxin You, Erle S Robertson.

  The cellular adaptive response to hypoxia, mediated by high HIF1α levels includes metabolic reprogramming, restricted DNA replication and cell division. In contrast to healthy cells, the genome of cancer cells, and Kaposi's sarcoma associated herpesvirus (KSHV) infected cells maintains replication in hypoxia. We show that KSHV infection, despite promoting expression of HIF1α in normoxia, can also restrict transcriptional activity, and promoted its degradation in hypoxia. KSHV-encoded vCyclin, expressed in hypoxia, mediated HIF1a cytosolic translocation, and its degradation through a non-canonical lysosomal pathway. Attenuation of HIF1α levels by vCyclin allowed cells to bypass the block to DNA replication and cell proliferation in hypoxia. These results demonstrated that KSHV utilizes a unique strategy to balance HIF1α levels to overcome replication arrest and induction of the oncogenic phenotype, which are dependent on the levels of oxygen in the microenvironment.

Keywords:  infectious disease; microbiology; viruses

DOI:  https://doi.org/10.7554/eLife.57436
Cancers (Basel). 2021 Jul 02. pii: 3333. [Epub ahead of print]13(13):

Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma.

Kaoru Onidani, Nami Miura, Yuki Sugiura, Yuichi Abe, Yukio Watabe, Takanori Kakuya, Taisuke Mori, Seiichi Yoshimoto, Jun Adachi, Takao Kiyoi, Yasuaki Kabe, Makoto Suematsu, Takeshi Tomonaga, Takahiko Shibahara, Kazufumi Honda.

  The epidermal growth factor receptor is the only available tyrosine kinase molecular target for treating oral cancer. To improve the prognosis of tongue squamous cell carcinoma (TSCC) patients, a novel molecular target for tyrosine kinases is thus needed. We examined the expression of interleukin-2-inducible T-cell kinase (ITK) using immunohistochemistry, and the biological function of ITK was investigated using biochemical, phosphoproteomic, and metabolomic analyses. We found that ITK is overexpressed in TSCC patients with poor outcomes. The proliferation of oral cancer cell lines expressing ITK via transfection exhibited significant increases in three-dimensional culture assays and murine inoculation models with athymic male nude mice as compared with mock control cells. Suppressing the kinase activity using chemical inhibitors significantly reduced the increase in cell growth induced by ITK expression. Phosphoproteomic analyses revealed that ITK expression triggered phosphorylation of a novel tyrosine residue in trifunctional purine biosynthetic protein adenosine-3, an enzyme in the purine biosynthesis pathway. A significant increase in de novo biosynthesis of purines was observed in cells expressing ITK, which was abolished by the ITK inhibitor. ITK thus represents a potentially useful target for treating TSCC through modulation of purine biosynthesis.

Keywords:  IL2-inducible T-cell kinase (ITK); tongue squamous cell carcinoma; trifunctional purine biosynthetic protein adenosine-3 (GART)

DOI:  https://doi.org/10.3390/cancers13133333
Cancers (Basel). 2021 Jul 01. pii: 3317. [Epub ahead of print]13(13):

A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.

Eric Moeglin, Dominique Desplancq, Audrey Stoessel, Christian Massute, Jeremy Ranniger, Alastair G McEwen, Gabrielle Zeder-Lutz, Mustapha Oulad-Abdelghani, Manuela Chiper, Pierre Lafaye, Barbara Di Ventura, Pascal Didier, Arnaud Poterszman, Etienne Weiss.

  Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

Keywords:  H2AX; cancer cells; genotoxicity assay in live cells; imaging; nanobody; one-step detection; phosphorylation; replication stress

DOI:  https://doi.org/10.3390/cancers13133317
Proc Natl Acad Sci U S A. 2021 Jul 27. pii: e2026621118. [Epub ahead of print]118(30):

Structural basis for ligand binding modes of CTP synthase.

Xian Zhou, Chen-Jun Guo, Chia-Chun Chang, Jiale Zhong, Huan-Huan Hu, Guang-Ming Lu, Ji-Long Liu.

  Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo-electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

Keywords:  CTP synthase; allosteric regulation; cryo–electron microscopy; cytoophidium

DOI:  https://doi.org/10.1073/pnas.2026621118
Bioengineered. 2021 Dec;12(1): 3924-3933

Knockdown of DNA polymerase ζ relieved the chemoresistance of glioma via inhibiting the PI3K/AKT signaling pathway.

Junbao Yang, Weilong Ding, Xiangyu Wang, Yongsheng Xiang.

  Previous reports suggest that DNA polymerase ζ is highly expressed in glioma tissues. The present study aimed to investigate the roles of the REV7 subunit of DNA polymerase ζ in glioma cell chemoresistance and its underlying mechanisms. The bioinformatics method was used to compare the expression of REV7 in glioma and normal tissues. The expression of REV7 in glioma tumor samples and the adjacent tissue was examined by reverse transcription polymerase chain reaction. Moreover, an in vitro analysis using glioma cells was used to test the effects of REV7 siRNA on the proliferation and apoptosis of glioma cell line U251 cells, and the effect of REV7 siRNA on the sensitivity of the U251 cells to cisplatin was also explored. The expression of REV7 in glioma tumors was significantly increased. Moreover, the knockdown of REV7 in glioma cells decreased the proliferation and increased the apoptosis of U251 cells; moreover, REV7 siRNA also increased the sensitivity of U251 cells to cisplatin. Finally, REV7 may regulate the proliferation, apoptosis, and chemosensitivity of U251 cells by affecting phosphoinositide 3-kinase signaling. Our data suggest that REV7 is involved in the chemosensitivity of glioma cells and provides a theoretical basis for targeting DNA polymerase ζ to improve the sensitivity of glioma cells to chemotherapy.

Keywords:  DNA polymerase ζ; REV7; chemoresistance; glioma

DOI:  https://doi.org/10.1080/21655979.2021.1944027
Curr Opin Genet Dev. 2021 Jul 17. pii: S0959-437X(21)00084-8. [Epub ahead of print]71 34-38

The WRN helicase: resolving a new target in microsatellite unstable cancers.

Niek van Wietmarschen, William J Nathan, André Nussenzweig.

One of the goals of precision medicine is to uncover selective vulnerabilities in various cancers. A notable success has been the development of PARP inhibitors for the treatment of breast and ovarian cancers with mutations in BRCA genes. Only two years ago, it was discovered that cancers with microsatellite instability (MSI) were selectively dependent on the RecQ DNA helicase WRN. Subsequently, the molecular mechanism underlying WRN dependency in MSI cancers was uncovered. Here, we review how these developments have led to a promising new drug target in MSI cancers.

DOI: https://doi.org/10.1016/j.gde.2021.06.014
Transl Lung Cancer Res. 2021 Jun;10(6): 2523-2538

One carbon metabolism in human lung cancer.

Sha Yao, Luogen Peng, Omar Elakad, Stefan Küffer, Marc Hinterthaner, Bernhard C Danner, Alexander von Hammerstein-Equord, Philipp Ströbel, Hanibal Bohnenberger.

   Background: Lung cancer remains the major cause of cancer related death worldwide. The discovery of targeted therapies against activating mutations in genes like EGFR considerably improved the prognosis for a subgroup of patients but still leaves a large part without a targeted therapy. One carbon metabolism (1CM) has been investigated in several cancer entities and its increased activity has been linked to higher tumor aggressiveness and reduced prognosis. In spite of 1CM enzymes role and correlation to cancer cells progression, comprehensive analysis for the diagnostic and functional role of the complete 1CM enzymes in lung cancer has not been conducted so far.
Methods: We investigated the prognostic and functional relevance of five major 1CM factors (MTHFD2, PGDH3, SHMT2, MTHFD1 and TYMS) in the three major subclasses of lung cancer [pulmonary adenocarcinoma (AC), squamous cell lung cancer (SQCLC) and small cell lung cancer (SCLC)]. We analyzed 1CM enzymes expression and clinicopathological correlation in patient derived tissue samples of 103 AC, 183 SQCLC and 37 SCLC patients by immunohistochemistry. Furthermore, the effect of 1CM enzymes expression on lung cancer cell proliferation and the response to chemotherapy was investigated in 15 representative AC, SQCLC and SCLC cell lines.
Results: Expression of MTHFD2 and PGDH3 was significantly correlated to a worse overall survival only in AC patients. Cell proliferation assays resolved that all 1CM enzymes have a significant impact on cell growth in AC cell lines and are partially involved in cell proliferation in SQCLC and SCLC cell lines. In addition, expression of MTHFD2 correlated significantly with an increased pemetrexed chemoresistance.
Conclusions: Expression of MTHFD2 significantly reduces the prognosis of AC patients. Furthermore, MTHFD2 expression is crucial for survival of AC cell lines and its expression correlates with resistance against Pemetrexed. As MTHFD2 is almost not expressed in healthy adult tissue, we therefore suggest that the inhibition of MTHFD2 might be a potential therapeutic strategy to surround pemetrexed resistance in AC.

Keywords:  MTHFD2; One carbon metabolism (1CM); chemoresistance; lung cancer; pemetrexed

DOI:  https://doi.org/10.21037/tlcr-20-1039
Sci Rep. 2021 Jul 19. 11(1): 14736

A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition.

Manuel Kaulich, Verena M Link, John D Lapek, Yeon J Lee, Christopher K Glass, David J Gonzalez, Steven F Dowdy.

During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.

DOI: https://doi.org/10.1038/s41598-021-94200-w
Cell Rep. 2021 Jul 20. pii: S2211-1247(21)00825-1. [Epub ahead of print]36(3): 109412

Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.

Marine Bruand, David Barras, Marco Mina, Eleonora Ghisoni, Matteo Morotti, Evripidis Lanitis, Noémie Fahr, Mathieu Desbuisson, Alizée Grimm, Hualing Zhang, Chloe Chong, Julien Dagher, Sora Chee, Theodora Tsianou, Julien Dorier, Brian J Stevenson, Christian Iseli, Catherine Ronet, Sara Bobisse, Raphael Genolet, Josephine Walton, Michal Bassani-Sternberg, Lana E Kandalaft, Bing Ren, Iain McNeish, Elizabeth Swisher, Alexandre Harari, Mauro Delorenzi, Giovanni Ciriello, Melita Irving, Sylvie Rusakiewicz, Periklis G Foukas, Fabio Martinon, Denarda Dangaj Laniti, George Coukos.

  In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1mut) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53-/-Brca1-/- but not Brca1+/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers.

Keywords:  BRCA1; CTLA-4; DNA sensing; ICB; PARPi; PD-L1; STING; T cells; VEGF-A; angiogenesis; dual immune checkpoint blockade; ovarian cancer; type I IFN

DOI:  https://doi.org/10.1016/j.celrep.2021.109412
Science. 2021 Jul 22. pii: eabc6506. [Epub ahead of print]

A DNA-repair pathway can affect transcriptional noise to promote cell fate transitions.

Ravi V Desai, Xinyue Chen, Benjamin Martin, Sonali Chaturvedi, Dong Woo Hwang, Weihan Li, Chen Yu, Sheng Ding, Matt Thomson, Robert H Singer, Robert A Coleman, Maike M K Hansen, Leor S Weinberger.

Stochastic fluctuations in gene expression ('noise') are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here that DNA base-excision repair amplifies transcriptional noise to facilitate cellular reprogramming. Specifically, the DNA-repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean-expression levels. This amplified expression noise originates from shorter duration, higher intensity, transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. This mechanism, which we term Discordant Transcription through Repair (DiThR; pronounced /'dither'/), potentiates cellular reprogramming and differentiation. Our study reveals a potential functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease.

DOI: https://doi.org/10.1126/science.abc6506
Cell Death Discov. 2021 Jul 20. 7(1): 184

Comparable radiation sensitivity in p53 wild-type and p53 deficient tumor cells associated with different cell death modalities.

Ping Li, Xiongxiong Liu, Ting Zhao, Feifei Li, Qiqi Wang, Pengcheng Zhang, Ryoichi Hirayama, Weiqiang Chen, Xiaodong Jin, Xiaogang Zheng, Zhen Wang, Qiang Li.

Studies of radiation interaction with tumor cells often take apoptosis as the desired results. However, mitotic catastrophe and senescence are also promoted by clinically relevant doses of radiation. Furthermore, p53 is a well-known transcription factor that is closely associated with radiosensitivity and radiation-induced cell death. Therefore, we aimed to investigate the involvement of radiosensitivity, cell death modalities and p53 status in response to carbon-ion radiation (CIR) here. Isogenic human colorectal cancer cell lines HCT116 (p53+/+ and p53-/-) were irradiated with high-LET carbon ions. Cell survival was determined by the standard colony-forming assay. 53BP1 foci were visualized to identify the repair kinetics of DNA double-strand breaks (DSBs). Cellular senescence was measured by SA-β-Gal and Ki67 staining. Mitotic catastrophe was determined with DAPI staining. Comparable radiosensitivities of p53+/+ and p53-/- HCT116 colorectal cells induced by CIR were demonstrated, as well as persistent 53BP1 foci indicated DNA repair deficiency in both cell lines. Different degree of premature senescence in isogenic HCT116 colorectal cancer cells suggested that CIR-induced premature senescence was more dependent on p21 but not p53. Sustained upregulation of p21 played multifunctional roles in senescence enhancement and apoptosis inhibition in p53+/+ cells. p21 inhibition further increased radiosensitivity of p53+/+ cells. Complex cell death modalities rather than single cell death were induced in both p53+/+ and p53-/- cells after 5 Gy CIR. Mitotic catastrophe was predominant in p53-/- cells due to inefficient activation of Chk1 and Chk2 phosphorylation in combination with p53 null. Senescence was the major cell death mechanism in p53+/+ cells via p21-dependent pathway. Taken together, p21-mediated premature senescence might be used by tumor cells to escape from CIR-induced cytotoxicity, at least for a time.

DOI: https://doi.org/10.1038/s41420-021-00570-5