bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒12‒05
27 papers selected by
Sean Rudd
Karolinska Institutet
  1. Activation of homologous recombination in G1 preserves centromeric integrity.
  2. Therapeutic vulnerability to PARP1,2 inhibition in RB1-mutant osteosarcoma.
  3. Oncometabolites as Regulators of DNA Damage Response and Repair.
  4. The FAcilitates Chromatin Transcription (FACT) complex: Its roles in DNA repair and implications for cancer therapy.
  5. The NUCKS1-SKP2-p21/p27 axis controls S phase entry.
  6. dCas9 binding inhibits the initiation of base excision repair in vitro.
  7. Radiotherapy-Poly(ADP-ribose) Polymerase Inhibitor Combinations: Progress to Date.
  8. The cytosolic iron-sulfur cluster assembly (CIA) pathway is required for replication stress tolerance of cancer cells to Chk1 and ATR inhibitors.
  9. MYCN recruits the nuclear exosome complex to RNA polymerase II to prevent transcription-replication conflicts.
  10. Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae.
  11. The chromatin remodeler RSF1 coordinates epigenetic marks for transcriptional repression and DSB repair.
  12. Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.
  13. Targeting the ATM Kinase to Enhance the Efficacy of Radiotherapy and Outcomes for Cancer Patients.
  14. BLM helicase inhibition synergizes with PARP inhibition to improve the radiosensitivity of olaparib resistant non-small cell lung cancer cells by inhibiting homologous recombination repair.
  15. MLK4 regulates DNA damage response and promotes triple-negative breast cancer chemoresistance.
  16. Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses.
  17. Transcriptome dataset of HEK293T cells depleted of one of the subunits of the DNA-PK complex: Ku70, Ku80 or DNA-PKcs.
  18. Blm helicase facilitates rapid replication of repetitive DNA sequences in early Drosophila development.
  19. Cryo-EM structures and biochemical insights into heterotrimeric PCNA regulation of DNA ligase.
  20. Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs.
  21. CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.
  22. Pan-cancer analysis of non-oncogene addiction to DNA repair.
  23. DNA repair inhibitors sensitize cells differently to high and low LET radiation.
  24. Deubiquitination of FBP1 by USP7 blocks FBP1-DNMT1 interaction and decreases the sensitivity of pancreatic cancer cells to PARP inhibitors.
  25. ASS1 and ASL suppress growth in clear cell renal cell carcinoma via altered nitrogen metabolism.
  26. Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure.
  27. Metabolite and thymocyte development defects in ADA-SCID mice receiving enzyme replacement therapy.
  1. Nature. 2021 Dec 01.
    Activation of homologous recombination in G1 preserves centromeric integrity.
    Duygu Yilmaz, Audrey Furst, Karen Meaburn, Aleksandra Lezaja, Yanlin Wen, Matthias Altmeyer, Bernardo Reina-San-Martin, Evi Soutoglou.
      Centromeric integrity is key for proper chromosome segregation during cell division1. Centromeres have unique chromatin features that are essential for centromere maintenance2. Although they are intrinsically fragile and represent hotspots for chromosomal rearrangements3, little is known about how centromere integrity in response to DNA damage is preserved. DNA repair by homologous recombination requires the presence of the sister chromatid and is suppressed in the G1 phase of the cell cycle4. Here we demonstrate that DNA breaks that occur at centromeres in G1 recruit the homologous recombination machinery, despite the absence of a sister chromatid. Mechanistically, we show that the centromere-specific histone H3 variant CENP-A and its chaperone HJURP, together with dimethylation of lysine 4 in histone 3 (H3K4me2), enable a succession of events leading to the licensing of homologous recombination in G1. H3K4me2 promotes DNA-end resection by allowing DNA damage-induced centromeric transcription and increased formation of DNA-RNA hybrids. CENP-A and HJURP interact with the deubiquitinase USP11, enabling formation of the RAD51-BRCA1-BRCA2 complex5 and rendering the centromeres accessible to RAD51 recruitment and homologous recombination in G1. Finally, we show that inhibition of homologous recombination in G1 leads to centromeric instability and chromosomal translocations. Our results support a model in which licensing of homologous recombination at centromeric breaks occurs throughout the cell cycle to prevent the activation of mutagenic DNA repair pathways and preserve centromeric integrity.
    DOI:  https://doi.org/10.1038/s41586-021-04200-z
  2. Nat Commun. 2021 Dec 03. 12(1): 7064
    Therapeutic vulnerability to PARP1,2 inhibition in RB1-mutant osteosarcoma.
    Georgia Zoumpoulidou, Carlos Alvarez-Mendoza, Caterina Mancusi, Ritika-Mahmuda Ahmed, Milly Denman, Christopher D Steele, Maxime Tarabichi, Errin Roy, Lauren R Davies, Jiten Manji, Camilla Cristalli, Katia Scotlandi, Nischalan Pillay, Sandra J Strauss, Sibylle Mittnacht.
      Loss-of-function mutations in the RB1 tumour suppressor are key drivers in cancer, including osteosarcoma. RB1 loss-of-function compromises genome-maintenance and hence could yield vulnerability to therapeutics targeting such processes. Here we demonstrate selective hypersensitivity to clinically-approved inhibitors of Poly-ADP-Polymerase1,2 inhibitors (PARPi) in RB1-defective cancer cells, including an extended panel of osteosarcoma-derived lines. PARPi treatment results in extensive cell death in RB1-defective backgrounds and prolongs survival of mice carrying human RB1-defective osteosarcoma grafts. PARPi sensitivity is not associated with canonical homologous recombination defect (HRd) signatures that predict PARPi sensitivity in cancers with BRCA1,2 loss, but is accompanied by rapid activation of DNA replication checkpoint signalling, and active DNA replication is a prerequisite for sensitivity. Importantly, sensitivity in backgrounds with natural or engineered RB1 loss surpasses that seen in BRCA-mutated backgrounds where PARPi have established clinical benefit. Our work provides evidence that PARPi sensitivity extends beyond cancers identifiable by HRd and advocates PARP1,2 inhibition as a personalised strategy for RB1-mutated osteosarcoma and other cancers.
    DOI:  https://doi.org/10.1038/s41467-021-27291-8
  3. Semin Radiat Oncol. 2022 Jan;pii: S1053-4296(21)00054-0. [Epub ahead of print]32(1): 82-94
    Oncometabolites as Regulators of DNA Damage Response and Repair.
    Susan E Gueble, Ranjit S Bindra.
      Dysregulation of DNA damage response and repair (DDR) contributes to oncogenesis, yet also generates the potential for targeted cancer therapies by exploiting synthetic lethal interactions. Oncometabolites, small intermediates of metabolism overproduced in certain cancers, have emerged as a new mechanism of DDR modulation through their effects on multiple DNA repair pathways. Increasing evidence suggests that oncometabolite-induced DDR defects may offer the opportunity for tumor-selective chemo- and radio-sensitization. Here we review the biology of oncometabolites and diverse mechanisms by which they impact DDR, with a focus on emerging therapeutic strategies and ongoing clinical trials targeting oncometabolite-induced DDR defects in cancer.
    DOI:  https://doi.org/10.1016/j.semradonc.2021.09.004
  4. DNA Repair (Amst). 2021 Nov 16. pii: S1568-7864(21)00202-0. [Epub ahead of print]109 103246
    The FAcilitates Chromatin Transcription (FACT) complex: Its roles in DNA repair and implications for cancer therapy.
    Kishor K Bhakat, Sutapa Ray.
      Genomic DNA in the nucleus is wrapped around nucleosomes, a repeating unit of chromatin. The nucleosome, consisting of octamer of core histones, is a barrier for several cellular processes that require access to the naked DNA. The FAcilitates Chromatin Transcription (FACT), a histone chaperone complex, is involved in nucleosome remodeling via eviction or assembly of histones during transcription, replication, and DNA repair. Increasing evidence suggests that FACT plays an important role in multiple DNA repair pathways including transcription-coupled nucleotide excision repair (TC-NER) of UV-induced damage, DNA single- and double-strand breaks (DSBs) repair, and base excision repair (BER) of oxidized or alkylated damaged bases. Further, studies have shown overexpression of FACT in multiple types of cancer and its association with drug resistance and patients' poor prognosis. In this review, we discuss how FACT is accumulated at the damage site and what functions it performs. We describe the known mechanisms by which FACT facilitates repair of different types of DNA damage. Further, we highlight the recent advances in a class of FACT inhibitors, called curaxins, which show promise as a new adjuvant therapy to sensitize multiple types of cancer to chemotherapy and radiation.
    Keywords:  APE1; Cancer Therapy; Curaxins/CBL0137; DNA Damage Repair; FACT; Histone Chaperone
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103246
  5. Nat Commun. 2021 Nov 29. 12(1): 6959
    The NUCKS1-SKP2-p21/p27 axis controls S phase entry.
    Samuel Hume, Claudia P Grou, Pauline Lascaux, Vincenzo D'Angiolella, Arnaud J Legrand, Kristijan Ramadan, Grigory L Dianov.
      Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homoeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCFSKP2 ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. The Cancer Genome Atlas (TCGA) data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation.
    DOI:  https://doi.org/10.1038/s41467-021-27124-8
  6. DNA Repair (Amst). 2021 Nov 20. pii: S1568-7864(21)00213-5. [Epub ahead of print]109 103257
    dCas9 binding inhibits the initiation of base excision repair in vitro.
    Jacob S Antony, Steven A Roberts, John J Wyrick, John M Hinz.
      Cas9 targets DNA during genome editing by forming an RNA:DNA heteroduplex (R-loop) between the Cas9-bound guide RNA and the targeted DNA strand. We have recently demonstrated that R-loop formation by catalytically inactive Cas9 (dCas9) is inherently mutagenic, in part, by promoting spontaneous cytosine deamination within the non-targeted single-stranded DNA of the dCas9-induced R-loop. However, the extent to which dCas9 binding and R-loop formation affect the subsequent repair of uracil lesions or other damaged DNA bases is unclear. Here, we show that DNA binding by dCas9 inhibits initiation of base excision repair (BER) for uracil lesions in vitro. Our data indicate that cleavage of uracil lesions by Uracil-DNA glycosylase (UDG) is generally inhibited at dCas9-bound DNA, in both the dCas9:sgRNA-bound target strand (TS) or the single-stranded non-target strand (NT). However, cleavage of a uracil lesion within the base editor window of the NT strand was less inhibited than at other locations, indicating that this site is more permissive to UDG activity. Furthermore, our data suggest that dCas9 binding to PAM sites can inhibit UDG activity. However, this non-specific inhibition can be relieved with the addition of an sgRNA lacking sequence complementarity to the DNA substrate. Moreover, we show that dCas9 binding also inhibits human single-strand selective monofunctional uracil-DNA glycosylase (SMUG1). Structural analysis of a Cas9-bound target site subsequently suggests a molecular mechanism for BER inhibition. Taken together, our results imply that dCas9 (or Cas9) binding may promote background mutagenesis by inhibiting the removal of DNA base lesions by BER.
    Keywords:  CRISPR; DNA glycosylase; DNA targeting; R-loop; Repair
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103257
  7. Semin Radiat Oncol. 2022 Jan;pii: S1053-4296(21)00055-2. [Epub ahead of print]32(1): 15-28
    Radiotherapy-Poly(ADP-ribose) Polymerase Inhibitor Combinations: Progress to Date.
    Sarah J Derby, Anthony J Chalmers, Ross D Carruthers.
      Radiation resistance remains a huge clinical problem for cancer patients and oncologists in the 21st century. In recent years, the mammalian DNA damage response (DDR) has been extensively characterized and shown to play a key role in determining cellular survival following ionizing radiation exposure. Genomic instability due to altered DDR is a hallmark of cancer, with many tumors exhibiting abnormal DNA repair or lack of redundancy in DDR. Targeting the abnormal DDR phenotype of tumor cells could lead to substantial gains in radiotherapy efficacy, improving local control and survival for patients with cancers that are refractory to current therapies. Poly(ADP-ribose) polymerase inhibitors (PARPi) are the most clinically advanced DDR inhibitors under investigation as radiosensitisers. Preclinical evidence suggests that PARPi may provide tumor specific radiosensitisation in certain contexts. In addition to inhibition of DNA single strand break repair, PARPi may offer other benefits in combination treatment including radiosensitisation of hypoxic cells and targeting of alternative repair pathways such as microhomology mediated end joining which are increasingly recognized to be upregulated in cancer. Several early phase clinical trials of PARPi with radiation have completed or are in progress. Early reports have highlighted tumor specific challenges, with tolerability dependent upon anatomical location and use of concomitant systemic therapies; these challenges were largely predicted by preclinical data. This review discusses the role of PARP in the cellular response to ionizing radiation, summarizes preclinical studies of PARPi in combination with radiotherapy and explores current early phase clinical trials that are evaluating these combinations.
    DOI:  https://doi.org/10.1016/j.semradonc.2021.09.005
  8. NPJ Breast Cancer. 2021 Dec 02. 7(1): 152
    The cytosolic iron-sulfur cluster assembly (CIA) pathway is required for replication stress tolerance of cancer cells to Chk1 and ATR inhibitors.
    Abena B Redwood, Xiaomei Zhang, Sahil B Seth, Zhongqi Ge, Wendy E Bindeman, Xinhui Zhou, Vidya C Sinha, Timothy P Heffernan, Helen Piwnica-Worms.
      The relationship between ATR/Chk1 activity and replication stress, coupled with the development of potent and tolerable inhibitors of this pathway, has led to the clinical exploration of ATR and Chk1 inhibitors (ATRi/Chk1i) as anticancer therapies for single-agent or combinatorial application. The clinical efficacy of these therapies relies on the ability to ascertain which patient populations are most likely to benefit, so there is intense interest in identifying predictive biomarkers of response. To comprehensively evaluate the components that modulate cancer cell sensitivity to replication stress induced by Chk1i, we performed a synthetic-lethal drop-out screen in a cell line derived from a patient with triple-negative breast cancer (TNBC), using a pooled barcoded shRNA library targeting ~350 genes involved in DNA replication, DNA damage repair, and cycle progression. In addition, we sought to compare the relative requirement of these genes when DNA fidelity is challenged by clinically relevant anticancer breast cancer drugs, including cisplatin and PARP1/2 inhibitors, that have different mechanisms of action. This global comparison is critical for understanding not only which agents should be used together for combinatorial therapies in breast cancer patients, but also the genetic context in which these therapies will be most effective, and when a single-agent therapy will be sufficient to provide maximum therapeutic benefit to the patient. We identified unique potentiators of response to ATRi/Chk1i and describe a new role for components of the cytosolic iron-sulfur assembly (CIA) pathway, MMS19 and CIA2B-FAM96B, in replication stress tolerance of TNBC.
    DOI:  https://doi.org/10.1038/s41523-021-00353-2
  9. Mol Cell. 2021 Nov 18. pii: S1097-2765(21)00952-7. [Epub ahead of print]
    MYCN recruits the nuclear exosome complex to RNA polymerase II to prevent transcription-replication conflicts.
    Dimitrios Papadopoulos, Daniel Solvie, Apoorva Baluapuri, Theresa Endres, Stefanie Anh Ha, Steffi Herold, Jacqueline Kalb, Celeste Giansanti, Christina Schülein-Völk, Carsten Patrick Ade, Cornelius Schneider, Abdallah Gaballa, Seychelle Vos, Utz Fischer, Matthias Dobbelstein, Elmar Wolf, Martin Eilers.
      The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.
    Keywords:  ATM; ATR; DCP1A; MYC; MYCN; Neuroblastoma; RNA Exosome; TFIIS; transcription-replication conflict.
    DOI:  https://doi.org/10.1016/j.molcel.2021.11.002
  10. G3 (Bethesda). 2021 Aug 07. pii: jkab205. [Epub ahead of print]11(8):
    Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae.
    Natasha C Koussa, Duncan J Smith.
      During lagging-strand synthesis, strand-displacement synthesis by DNA polymerase delta (Pol ∂), coupled to nucleolytic cleavage of DNA flap structures, produces a nick-translation reaction that replaces the DNA at the 5' end of the preceding Okazaki fragment. Previous work following depletion of DNA ligase I in Saccharomyces cerevisae suggests that DNA-bound proteins, principally nucleosomes and the transcription factors Abf1/Rap1/Reb1, pose a barrier to Pol ∂ synthesis and thereby limit the extent of nick translation in vivo. However, the extended ligase depletion required for these experiments could lead to ongoing, non-physiological nick translation. Here, we investigate nick translation by analyzing Okazaki fragments purified after transient nuclear depletion of DNA ligase I in synchronized or asynchronous Saccharomyces cerevisiae cultures. We observe that, even with a short ligase depletion, Okazaki fragment termini are enriched around nucleosomes and Abf1/Reb1/Rap1-binding sites. However, protracted ligase depletion leads to a global change in the location of these termini, moving them toward nucleosome dyads from a more upstream location and further enriching termini at Abf1/Reb1/Rap1-binding sites. In addition, we observe an under-representation of DNA derived from DNA polymerase alpha-the polymerase that initiates Okazaki fragment synthesis-around the sites of Okazaki termini obtained from very brief ligase depletion. Our data suggest that, while nucleosomes and transcription factors do limit strand-displacement synthesis by Pol ∂ in vivo, post-replicative nick translation can occur at unligated Okazaki fragment termini such that previous analyses represent an overestimate of the extent of nick translation occurring during normal lagging-strand synthesis.
    Keywords:  DNA ligase I; DNA polymerase delta; DNA replication; lagging-strand synthesis
    DOI:  https://doi.org/10.1093/g3journal/jkab205
  11. Nucleic Acids Res. 2021 Nov 25. pii: gkab1093. [Epub ahead of print]
    The chromatin remodeler RSF1 coordinates epigenetic marks for transcriptional repression and DSB repair.
    Sunwoo Min, Ho-Soo Lee, Jae-Hoon Ji, Yungyeong Heo, Yonghyeon Kim, Sunyoung Chae, Yong Won Choi, Ho-Chul Kang, Makoto Nakanishi, Hyeseong Cho.
      DNA lesions impact on local transcription and the damage-induced transcriptional repression facilitates efficient DNA repair. However, how chromatin dynamics cooperates with these two events remained largely unknown. We here show that histone H2A acetylation at K118 is enriched in transcriptionally active regions. Under DNA damage, the RSF1 chromatin remodeling factor recruits HDAC1 to DSB sites. The RSF1-HDAC1 complex induces the deacetylation of H2A(X)-K118 and its deacetylation is indispensable for the ubiquitination of histone H2A at K119. Accordingly, the acetylation mimetic H2A-K118Q suppressed the H2A-K119ub level, perturbing the transcriptional repression at DNA lesions. Intriguingly, deacetylation of H2AX at K118 also licenses the propagation of γH2AX and recruitment of MDC1. Consequently, the H2AX-K118Q limits DNA repair. Together, the RSF1-HDAC1 complex controls the traffic of the DNA damage response and transcription simultaneously in transcriptionally active chromatins. The interplay between chromatin remodelers and histone modifiers highlights the importance of chromatin versatility in the maintenance of genome integrity.
    DOI:  https://doi.org/10.1093/nar/gkab1093
  12. Nucleic Acids Res. 2021 Dec 01. pii: gkab1174. [Epub ahead of print]
    Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.
    Haixia Zhou, Manal S Zaher, Johannes C Walter, Alan Brown.
      When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.
    DOI:  https://doi.org/10.1093/nar/gkab1174
  13. Semin Radiat Oncol. 2022 Jan;pii: S1053-4296(21)00058-8. [Epub ahead of print]32(1): 3-14
    Targeting the ATM Kinase to Enhance the Efficacy of Radiotherapy and Outcomes for Cancer Patients.
    María E Guerra García, David G Kirsch, Zachary J Reitman.
      Targeting the DNA damage response represents a promising approach to improve the efficacy of radiation therapy. One appealing target for this approach is the serine/threonine kinase ataxia telangiectasia mutated (ATM), which is activated by DNA double strand breaks to orchestrate the cellular response to ionizing radiation. Small-molecule inhibitors targeting ATM have entered clinical trials testing their safety in combination with radiation therapy or in combination with other DNA damaging agents. Here, we review biochemical, genetic, and cellular functional studies of ATM, phenotypes associated with germline and somatic cancer mutations in ATM in humans, and experiments in genetically engineered mouse models that support a rationale for investigating ATM inhibitors as radiosensitizers for cancer therapy. These data identify important synthetic lethal relationships, which suggest that ATM inhibitors may be particularly effective in tumors with defects in other nodes of the DNA damage response. The potential for ATM inhibition to improve immunotherapy responses in preclinical models represents another emerging area of research. We summarize ongoing clinical trials of ATM inhibitors with radiotherapy. We also discuss critical ongoing areas of investigation that include discovery of biomarkers that predict for radiosensitization by ATM inhibitors and identification of effective combinations of ATM inhibitors, radiation therapy, other DNA damage response-directed therapies, and/or immunotherapies.
    DOI:  https://doi.org/10.1016/j.semradonc.2021.09.008
  14. Cancer Biol Med. 2021 Dec 01. pii: j.issn.2095-3941.2021.0178. [Epub ahead of print]
    BLM helicase inhibition synergizes with PARP inhibition to improve the radiosensitivity of olaparib resistant non-small cell lung cancer cells by inhibiting homologous recombination repair.
    Yangyang Kong, Chang Xu, Xiaohui Sun, Hao Sun, Xiaotong Zhao, Ningning He, Kaihua Ji, Qin Wang, Liqing Du, Jinhan Wang, Manman Zhang, Yang Liu, Yan Wang, Qiang Liu.
      OBJECTIVE: We aimed to investigate the radiosensitizing efficacy of the poly-ADP-ribose polymerase (PARP) inhibitor, olaparib, and the Bloom syndrome protein (BLM) helicase inhibitor, ML216, in non-small cell lung cancer (NSCLC) cells.METHODS: Radiosensitization of NSCLC cells was assessed by colony formation and tumor growth assays. Mechanistically, the effects of ML216, olaparib, and radiation on cell and tumor proliferation, DNA damage, cell cycle, apoptosis, homologous recombination (HR) repair, and non-homologous end joining (NHEJ) repair activity were determined.
    RESULTS: Both olaparib and ML216 enhanced the radiosensitivities of olaparib-sensitive H460 and H1299 cells, which was seen as decreased surviving fractions and Rad51 foci, increased total DNA damage, and γH2AX and 53BP1 foci (P < 0.05). The expressions of HR repair proteins were remarkably decreased in olaparib-treated H460 and H1299 cells after irradiation (P < 0.05), while olaparib combined with ML216 exerted a synergistic radiosensitization effect on olaparib-resistant A549 cells. In addition to increases of double strand break (DSB) damage and decreases of Rad51 foci, olaparib combined with ML216 also increased pDNA-PKcs (S2056) foci, abrogated G2 cell cycle arrest, and induced apoptosis in A549 lung cancer after irradiation in vitro and in vivo (P < 0.05). Moreover, Western blot showed that olaparib combined with ML216 and irradiation inhibited HR repair, promoted NHEJ repair, and inactivated cell cycle checkpoint signals both in vitro and in vivo (P < 0.05).
    CONCLUSIONS: Taken together, these results showed the efficacy of PARP and BLM helicase inhibitors for radiosensitizing NSCLC cells, and supported the model that BLM inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization, as well as providing the basis for the potential clinical development of this combination for tumors intrinsically resistant to PARP inhibitors and radiotherapy.
    Keywords:  BLM; NSCLC; PARP; homologous recombination repair; radiosensitization
    DOI:  https://doi.org/10.20892/j.issn.2095-3941.2021.0178
  15. Cell Death Dis. 2021 Nov 27. 12(12): 1111
    MLK4 regulates DNA damage response and promotes triple-negative breast cancer chemoresistance.
    Dawid Mehlich, Michał Łomiak, Aleksandra Sobiborowicz, Alicja Mazan, Dagmara Dymerska, Łukasz M Szewczyk, Anna Mehlich, Agnieszka Borowiec, Monika K Prełowska, Adam Gorczyński, Paweł Jabłoński, Ewa Iżycka-Świeszewska, Dominika Nowis, Anna A Marusiak.
      Chemoresistance constitutes a major challenge in the treatment of triple-negative breast cancer (TNBC). Mixed-Lineage Kinase 4 (MLK4) is frequently amplified or overexpressed in TNBC where it facilitates the aggressive growth and migratory potential of breast cancer cells. However, the functional role of MLK4 in resistance to chemotherapy has not been investigated so far. Here, we demonstrate that MLK4 promotes TNBC chemoresistance by regulating the pro-survival response to DNA-damaging therapies. We observed that MLK4 knock-down or inhibition sensitized TNBC cell lines to chemotherapeutic agents in vitro. Similarly, MLK4-deficient cells displayed enhanced sensitivity towards doxorubicin treatment in vivo. MLK4 silencing induced persistent DNA damage accumulation and apoptosis in TNBC cells upon treatment with chemotherapeutics. Using phosphoproteomic profiling and reporter assays, we demonstrated that loss of MLK4 reduced phosphorylation of key DNA damage response factors, including ATM and CHK2, and compromised DNA repair via non-homologous end-joining pathway. Moreover, our mRNA-seq analysis revealed that MLK4 is required for DNA damage-induced expression of several NF-кB-associated cytokines, which facilitate TNBC cells survival. Lastly, we found that high MLK4 expression is associated with worse overall survival of TNBC patients receiving anthracycline-based neoadjuvant chemotherapy. Collectively, these results identify a novel function of MLK4 in the regulation of DNA damage response signaling and indicate that inhibition of this kinase could be an effective strategy to overcome TNBC chemoresistance.
    DOI:  https://doi.org/10.1038/s41419-021-04405-0
  16. Angew Chem Int Ed Engl. 2021 Dec 01.
    Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses.
    Yong Woong Jun, Eddy Albarran, David L Wilson, Jun Ding, Eric T Kool.
      Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. Here we employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe ( UBER ) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.
    Keywords:  DNA damage and repair; dynamics; fluorescence probes; mitochondrial DNA
    DOI:  https://doi.org/10.1002/anie.202111829
  17. Data Brief. 2021 Dec;39 107596
    Transcriptome dataset of HEK293T cells depleted of one of the subunits of the DNA-PK complex: Ku70, Ku80 or DNA-PKcs.
    Andrey Anisenko, Olga Shadrina, Irina Garanina, Marina Gottikh.
      DNA-PK is a heterotrimeric complex that consists of Ku70 (XRCC6), Ku80 (XRCC5) and DNA-PKcs (PRKDC) subunits. The complex is a major player in the repair of DNA double strand break (DSB) via the non-homologous end joining (NHEJ) pathway. This process requires all DNA-PK subunits, since Ku70/Ku80 heterodimer firstly binds to DNA ends at DSB and then recruits DNA-PKcs. Recruitment of the DNA-PKcs subunit to DSB leads to phosphorylation events near DSB and recruitment of other NHEJ-related proteins that restore DNA integrity. However, today a lot of evidence demonstrates participation of the DNA-PK components in other cellular processes, e.g. telomere length maintenance, transcription, metabolism regulation, cytosolic DNA sensing, apoptosis, cellular movement and adhesion. It is important to note that not all the subunits of the DNA-PK complex are necessary for these processes, and the largest number of independent functions has been shown for the Ku70/Ku80 heterodimer and especially the Ku70 subunit. To better understand the role of each DNA-PK subunit in the cell life, we have analyzed transcriptome changes in HEK293T cells depleted of Ku70, Ku80 or DNA-PKcs using NGS-sequencing. Here, for the first time, we present the data obtained from the transcriptome analysis.
    Keywords:  DNA-PKcs; DNA-dependent protein kinase; Ku heterodimer; Ku70; Ku80; NHEJ-related proteins
    DOI:  https://doi.org/10.1016/j.dib.2021.107596
  18. Genetics. 2021 Nov 22. pii: iyab169. [Epub ahead of print]
    Blm helicase facilitates rapid replication of repetitive DNA sequences in early Drosophila development.
    Jolee M Ruchert, Morgan M Brady, Susan McMahan, Karly J Lacey, Leigh C Latta, Jeff Sekelsky, Eric P Stoffregen.
      The absence of functional BLM DNA helicase, a member of the RecQ family of helicases, is responsible for the rare human disorder Bloom Syndrome, which results in developmental abnormalities, DNA repair defects, genomic instability, and a predisposition to cancer. In Drosophila melanogaster, the orthologous Blm protein is essential during early development when the embryo is under the control of maternal gene products. We show that lack of functional maternal Blm during the syncytial cell cycles of Drosophila embryonic development results in severe nuclear defects and lethality. Amongst the small fraction of embryos from Blm mutant mothers that survive to adulthood, a prominent sex-bias favors the class that inherits less repetitive DNA content, which serves as an endogenous source of replication stress. This selection against repetitive DNA content reflects a role for Blm in facilitating replication through repetitive sequences during the rapid S-phases of syncytial cell cycles. During these syncytial cycles, Blm is not required for complex DNA double-strand break repair; however, the progeny sex-bias resulting from the absence of maternal Blm is exacerbated by repetitive DNA sequences and by the slowing of replication fork progression, suggesting that the essential role for Blm during this stage is to manage replication fork stress brought about by impediments to fork progression. Additionally, our data suggest that Blm is only required to manage this replication stress during embryonic development, and likely only during the early, rapid syncytial cell cycles, and not at later developmental stages. These results provide novel insights into Blm function throughout development.
    Keywords:   Drosophila melanogaster ; Blm DNA helicase; DNA replication; Y chromosome; repetitive DNA
    DOI:  https://doi.org/10.1093/genetics/iyab169
  19. Structure. 2021 Nov 22. pii: S0969-2126(21)00414-7. [Epub ahead of print]
    Cryo-EM structures and biochemical insights into heterotrimeric PCNA regulation of DNA ligase.
    Aleksandr Sverzhinsky, Alan E Tomkinson, John M Pascal.
      DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there are limited insights into the physical basis for this regulation. Here, we use single-particle cryoelectron microscopy (cryo-EM) to analyze an archaeal DNA ligase and heterotrimeric PCNA in complex with a single-strand DNA break. The cryo-EM structures highlight a continuous DNA-binding surface formed between DNA ligase and PCNA that supports the distorted conformation of the DNA break undergoing repair and contributes to PCNA stimulation of DNA ligation. DNA ligase is conformationally flexible within the complex, with its domains fully ordered only when encircling the repaired DNA to form a stacked ring structure with PCNA. The structures highlight DNA ligase structural transitions while docked on PCNA, changes in DNA conformation during ligation, and the potential for DNA ligase domains to regulate PCNA accessibility to other repair factors.
    Keywords:  DNA ligase; DNA ligation; PCNA; PIP motif; cryo-EM; protein-protein and protein-DNA interactions
    DOI:  https://doi.org/10.1016/j.str.2021.11.002
  20. Nat Commun. 2021 Dec 01. 12(1): 7013
    Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs.
    Nathalia Varejão, Jara Lascorz, Joan Codina-Fabra, Gemma Bellí, Helena Borràs-Gas, Jordi Torres-Rosell, David Reverter.
      Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response. Smc5/6 is a nuclear multi-subunit complex that participates in the recombinational DNA repair processes and is required in the maintenance of chromosome integrity. Nse2 is a subunit of the Smc5/6 complex that possesses SUMO E3 ligase activity by the presence of a SP-RING domain that activates the E2~SUMO thioester for discharge on the substrate. Here we present the crystal structure of the SUMO E3 ligase Nse2 in complex with an E2-SUMO thioester mimetic. In addition to the interface between the SP-RING domain and the E2, the complex reveals how two SIM (SUMO-Interacting Motif) -like motifs in Nse2 are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction. Both SIM interfaces are essential in the activity of Nse2 and are required to cope with DNA damage.
    DOI:  https://doi.org/10.1038/s41467-021-27301-9
  21. Cell Rep. 2021 Nov 30. pii: S2211-1247(21)01546-1. [Epub ahead of print]37(9): 110060
    CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.
    Jennifer E Klomp, Ye S Lee, Craig M Goodwin, Björn Papke, Jeff A Klomp, Andrew M Waters, Clint A Stalnecker, Jonathan M DeLiberty, Kristina Drizyte-Miller, Runying Yang, J Nathaniel Diehl, Hongwei H Yin, Mariaelena Pierobon, Elisa Baldelli, Meagan B Ryan, Siqi Li, Jackson Peterson, Amber R Smith, James T Neal, Aaron K McCormick, Calvin J Kuo, Christopher M Counter, Emanuel F Petricoin, Adrienne D Cox, Kirsten L Bryant, Channing J Der.
      We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.
    Keywords:  CHK1; DNA damage; ERK; KRAS; MYC; RIF1; pancreatic cancer
    DOI:  https://doi.org/10.1016/j.celrep.2021.110060
  22. Sci Rep. 2021 Dec 01. 11(1): 23264
    Pan-cancer analysis of non-oncogene addiction to DNA repair.
    Luis Bermúdez-Guzmán.
      Cancer cells usually depend on the aberrant function of one or few driver genes to initiate and promote their malignancy, an attribute known as oncogene addiction. However, cancer cells might become dependent on the normal cellular functions of certain genes that are not oncogenes but ensure cell survival (non-oncogene addiction). The downregulation or silencing of DNA repair genes and the consequent genetic and epigenetic instability is key to promote malignancy, but the activation of the DNA-damage response (DDR) has been shown to become a type of non-oncogene addiction that critically supports tumour survival. In the present study, a systematic evaluation of DNA repair addiction at the pan-cancer level was performed using data derived from The Cancer Dependency Map and The Cancer Genome Atlas (TCGA). From 241 DDR genes, 59 were identified as commonly essential in cancer cell lines. However, large differences were observed in terms of dependency scores in 423 cell lines and transcriptomic alterations across 18 cancer types. Among these 59 commonly essential genes, 14 genes were exclusively associated with better overall patient survival and 19 with worse overall survival. Notably, a specific molecular signature among the latter, characterized by DDR genes like UBE2T, RFC4, POLQ, BRIP1, and H2AFX showing the weakest dependency scores, but significant upregulation was strongly associated with worse survival. The present study supports the existence and importance of non-oncogenic addiction to DNA repair in cancer and may facilitate the identification of prognostic biomarkers and therapeutic opportunities.
    DOI:  https://doi.org/10.1038/s41598-021-02773-3
  23. Sci Rep. 2021 Dec 01. 11(1): 23257
    DNA repair inhibitors sensitize cells differently to high and low LET radiation.
    Kristina Bannik, Balázs Madas, Sabrina Jarke, Andreas Sutter, Gerhard Siemeister, Christoph Schatz, Dominik Mumberg, Sabine Zitzmann-Kolbe.
      The aim of this study was to investigate effects of high LET α-radiation in combination with inhibitors of DDR (DNA-PK and ATM) and to compare the effect with the radiosensitizing effect of low LET X-ray radiation. The various cell lines were irradiated with α-radiation and with X-ray. Clonogenic survival, the formation of micronuclei and cell cycle distribution were studied after combining of radiation with DDR inhibitors. The inhibitors sensitized different cancer cell lines to radiation. DNA-PKi affected survival rates in combination with α-radiation in selected cell lines. The sensitization enhancement ratios were in the range of 1.6-1.85 in cancer cells. ATMi sensitized H460 cells and significantly increased the micronucleus frequency for both radiation qualities. ATMi in combination with α-radiation reduced survival of HEK293. A significantly elicited cell cycle arrest in G2/M phase after co-treatment of ATMi with α-radiation and X-ray. The most prominent treatment effect was observed in the HEK293 by combining α-radiation and inhibitions. ATMi preferentially sensitized cancer cells and normal HEK293 cells to α-radiation. DNA-PKi and ATMi can sensitize cancer cells to X-ray, but the effectiveness was dependent on cancer cells itself. α-radiation reduced proliferation in primary fibroblast without G2/M arrest.
    DOI:  https://doi.org/10.1038/s41598-021-02719-9
  24. Mol Oncol. 2021 Dec 01.
    Deubiquitination of FBP1 by USP7 blocks FBP1-DNMT1 interaction and decreases the sensitivity of pancreatic cancer cells to PARP inhibitors.
    Xiang Cheng, Bin Zhang, Feng Guo, Heshui Wu, Xin Jin.
      Poly [ADP-ribose] polymerase (PARP) inhibitors can block DNA single-strand damage repair and subsequently increase double-stranded breaks (DSBs) by reducing the activity of the PARP1 protease and preventing the PARP1 protein from dissociating from chromatin. Tumors with the BRCA mutation are particularly sensitive to PARP inhibitors. So far, PARP inhibitors (olaparib) have been used to treat pancreatic cancer patients with BRCA mutation. However, these patients are prone to PARP-inhibitor resistance. Our previous studies suggest that fructose-1,6-bisphosphatase 1 (FBP1) is responsible for the sensitivity to various anticancer agents, such as gemcitabine or mitogen-activated protein kinase kinase (MEK) inhibitors. In this study, we demonstrate that FBP1 regulates the sensitivity to PARP inhibitors in pancreatic cancer. Then, we showed that nuclear FBP1 is responsible for this process by interacting with DNA (cytosine-5)-methyltransferase 1 (DNMT1) and trapping PARP1 in chromatin. Moreover, we revealed that ubiquitin carboxyl-terminal hydrolase 7 (USP7) binds to and induces the deubiquitination of FBP1, which prevented FBP1 from translocating to the nucleus. Finally, we demonstrated that USP7 inhibitors enhanced the antitumor effect of PARP inhibitors in an FBP1-dependent manner. Collectively, our results identify a novel USP7-FBP1-DNMT1 signaling axis in pancreatic cancer, which might indicate that USP7 inhibitors and PARP inhibitors might have more powerful antitumor effects than PARP inhibitors alone in pancreatic cancer patients.
    Keywords:  DNMT1; FBP1; PARP inhibitors; USP7; pancreatic cancer
    DOI:  https://doi.org/10.1002/1878-0261.13149
  25. Cancer Metab. 2021 Dec 03. 9(1): 40
    ASS1 and ASL suppress growth in clear cell renal cell carcinoma via altered nitrogen metabolism.
    Sanika Khare, Laura C Kim, Graham Lobel, Paschalis-Thomas Doulias, Harry Ischiropoulos, Itzhak Nissim, Brian Keith, M Celeste Simon.
      BACKGROUND: Kidney cancer is a common adult malignancy in the USA. Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, is characterized by widespread metabolic changes. Urea metabolism is one such altered pathway in ccRCC. The aim of this study was to elucidate the contributions of urea cycle enzymes, argininosuccinate synthase 1 (ASS1), and argininosuccinate lyase (ASL) towards ccRCC progression.METHODS: We employed a combination of computational, genetic, and metabolomic tools along with in vivo animal models to establish a tumor-suppressive role for ASS1 and ASL in ccRCC.
    RESULTS: We show that the mRNA and protein expression of urea cycle enzymes ASS1 and ASL are reduced in ccRCC tumors when compared to the normal kidney. Furthermore, the loss of ASL in HK-2 cells (immortalized renal epithelial cells) promotes growth in 2D and 3D growth assays, while combined re-expression of ASS1 and ASL in ccRCC cell lines suppresses growth in 2D, 3D, and in vivo xenograft models. We establish that this suppression is dependent on their enzymatic activity. Finally, we demonstrate that conservation of cellular aspartate, regulation of nitric oxide synthesis, and pyrimidine production play pivotal roles in ASS1+ASL-mediated growth suppression in ccRCC.
    CONCLUSIONS: ccRCC tumors downregulate the components of the urea cycle including the enzymes argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL). These cytosolic enzymes lie at a critical metabolic hub in the cell and are involved in aspartate catabolism and arginine and nitric oxide biosynthesis. Loss of ASS1 and ASL helps cells redirect aspartate towards pyrimidine synthesis and support enhanced proliferation. Additionally, reduced levels of ASS1 and ASL might help regulate nitric oxide (NO) generation and mitigate its cytotoxic effects. Overall, our work adds to the understanding of urea cycle enzymes in a context-independent of ureagenesis, their role in ccRCC progression, and uncovers novel potential metabolic vulnerabilities in ccRCC.
    Keywords:  Argininosuccinate lyase; Argininosuccinate synthase 1; Aspartate; DNA synthesis; Nitric oxide metabolism; Urea cycle; ccRCC
    DOI:  https://doi.org/10.1186/s40170-021-00271-8
  26. PLoS Biol. 2021 Dec 03. 19(12): e3001468
    Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure.
    Gabriel Piedrafita, Sreejith J Varma, Cecilia Castro, Christoph Messner, Lukasz Szyrwiel, Julian L Griffin, Markus Ralser.
      The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken-egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a "metabolism first" theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network.
    DOI:  https://doi.org/10.1371/journal.pbio.3001468
  27. Sci Rep. 2021 Dec 01. 11(1): 23221
    Metabolite and thymocyte development defects in ADA-SCID mice receiving enzyme replacement therapy.
    Federico A Moretti, Giuliana Giardino, Teresa C H Attenborough, Athina Soragia Gkazi, Ben K Margetts, Giancarlo la Marca, Lynette Fairbanks, Tessa Crompton, H Bobby Gaspar.
      Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme intrinsic to the purine salvage pathway, leads to severe combined immunodeficiency (SCID) both in humans and mice. Lack of ADA results in the intracellular accumulation of toxic metabolites which have effects on T cell development and function. While untreated ADA-SCID is a fatal disorder, there are different therapeutic options available to restore ADA activity and reconstitute a functioning immune system, including enzyme replacement therapy (ERT). Administration of ERT in the form of pegylated bovine ADA (PEG-ADA) has proved a life-saving though non-curative treatment for ADA-SCID patients. However, in many patients treated with PEG-ADA, there is suboptimal immune recovery with low T and B cell numbers. Here, we show reduced thymus cellularity in ADA-SCID mice despite weekly PEG-ADA treatment. This was associated with lack of effective adenosine (Ado) detoxification in the thymus. We also show that thymocyte development in ADA-deficient thymi is arrested at the DN3-to-DN4 stage transition with thymocytes undergoing dATP-induced apoptosis rather than defective TCRβ rearrangement or β-selection. Our studies demonstrate at a detailed level that exogenous once-a-week enzyme replacement does not fully correct intra-thymic metabolic or immunological abnormalities associated with ADA deficiency.
    DOI:  https://doi.org/10.1038/s41598-021-02572-w
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