bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒05‒30
twenty-six papers selected by
Sean Rudd
Karolinska Institutet
  1. Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors.
  2. p97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation.
  3. Novel and highly potent ATR inhibitor M4344 kills cancer cells with replication stress and enhances the chemotherapeutic activity of widely used DNA damaging agents.
  4. NEIL3 prevents senescence in hepatocellular carcinoma by repairing oxidative lesions at telomeres during mitosis.
  5. Replication Fork Reversal and Protection.
  6. Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro.
  7. PARP1-mediated PARylation of TonEBP prevents R-loop-associated DNA damage.
  8. The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing.
  9. LRR1-mediated replisome disassembly promotes DNA replication by recycling replisome components.
  10. Coping with RNA damage with a focus on APE1, a BER enzyme at the crossroad between DNA damage repair and RNA processing/decay.
  11. DDRugging glioblastoma: understanding and targeting the DNA damage response to improve future therapies.
  12. Aurora kinase A, a synthetic lethal target for precision cancer medicine.
  13. Targeting one-carbon metabolism requires mTOR inhibition: a new therapeutic approach in osteosarcoma.
  14. The H3K4 methyltransferase SETD1A is required for proliferation of non-small cell lung cancer cells by promoting S-phase progression.
  15. Cornelia de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect.
  16. Studying PAR-Dependent Chromatin Remodeling to Tackle PARPi Resistance.
  17. Phosphorylation of Ago2 is required for its role in DNA double-strand break repair.
  18. Cytidine Deaminase APOBEC3A Regulates PD-L1 Expression in Cancer Cells in a JNK/c-JUN-dependent Manner.
  19. Radiotherapy is associated with a deletion signature that contributes to poor outcomes in patients with cancer.
  20. R-loop-Mediated ssDNA Breaks Accumulate Following Short-Term Exposure to the HDAC Inhibitor Romidepsin.
  21. Structural basis of antifolate recognition and transport by PCFT.
  22. BKM120 sensitizes glioblastoma to the PARP inhibitor rucaparib by suppressing homologous recombination repair.
  23. IMPDH2 and HPRT expression and a prognostic significance in preoperative and postoperative patients with osteosarcoma.
  24. Hexose-6-phosphate dehydrogenase blockade reverses prostate cancer drug resistance in xenograft models by glucocorticoid inactivation.
  25. Phase 1 study of the ATR inhibitor berzosertib in combination with cisplatin in patients with advanced solid tumours.
  26. Chromosomal copy number heterogeneity predicts survival rates across cancers.
  1. NAR Cancer. 2021 Jun;3(2): zcab018
    Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors.
    Amrita Sule, Jinny Van Doorn, Ranjini K Sundaram, Sachita Ganesa, Juan C Vasquez, Ranjit S Bindra.
      Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as 'BRCAness', which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.
    DOI:  https://doi.org/10.1093/narcan/zcab018
  2. Cell Rep. 2021 May 25. pii: S2211-1247(21)00495-2. [Epub ahead of print]35(8): 109153
    p97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation.
    Susan Kilgas, Abhay Narayan Singh, Salome Paillas, Chee-Kin Then, Ignacio Torrecilla, Judith Nicholson, Lisa Browning, Iolanda Vendrell, Rebecca Konietzny, Benedikt M Kessler, Anne E Kiltie, Kristijan Ramadan.
      The ATPase p97 is a central component of the ubiquitin-proteasome degradation system. p97 uses its ATPase activity and co-factors to extract ubiquitinated substrates from different cellular locations, including DNA lesions, thereby regulating DNA repair pathway choice. Here, we find that p97 physically and functionally interacts with the MRE11-RAD50-NBS1 (MRN) complex on chromatin and that inactivation of p97 blocks the disassembly of the MRN complex from the sites of DNA damage upon ionizing radiation (IR). The inhibition of p97 function results in excessive 5'-DNA end resection mediated by MRE11 that leads to defective DNA repair and radiosensitivity. In addition, p97 inhibition by the specific small-molecule inhibitor CB-5083 increases tumor cell killing following IR both in vitro and in vivo. Mechanistically, this is mediated via increased MRE11 nuclease accumulation. This suggests that p97 inhibitors might be exploited to improve outcomes for radiotherapy patients.
    Keywords:  CB-5083; DNA damage; DNA double-strand break repair; IR; MRE11; bladder cancer; homologous recombination; ionizing radiation; p97; single-strand annealing
    DOI:  https://doi.org/10.1016/j.celrep.2021.109153
  3. Mol Cancer Ther. 2021 May 27. pii: molcanther.1026.2020. [Epub ahead of print]
    Novel and highly potent ATR inhibitor M4344 kills cancer cells with replication stress and enhances the chemotherapeutic activity of widely used DNA damaging agents.
    Ukhyun Jo, Ilya S Senatorov, Astrid Zimmermann, Liton Kumar Saha, Yasuhisa Murai, Se Hyun Kim, Vinodh N Rajapakse, Fathi Elloumi, Nobuyuki Takahashi, Christopher W Schultz, Anish Thomas, Frank T Zenke, Yves Pommier.
      PURPOSE: Although several ATR inhibitors are in development, there are unresolved questions regarding their differential potency, molecular signatures of cancer patients for predicting activity and most effective therapeutic combinations. Here, we elucidate how to improve ATR-based chemotherapy with the newly developed ATR inhibitor, M4344 using in vitro and in vivo models.EXPERIMENTAL DESIGN: The potency of M4344 was compared with the clinically developed ATR inhibitors BAY1895344, berzosertib, and ceralasertib. The anticancer activity of M4344 was investigated as monotherapy and combination with clinical DNA damaging agents in multiple cancer cell lines, patient-derived tumor organoids and mouse xenograft models. We also elucidated the anticancer mechanisms and potential biomarkers for M4344.
    RESULTS: M4344 is highly potent among the clinically developed ATR inhibitors. Replication stress (RepStress) and neuroendocrine (NE) gene expression signatures are significantly associated with a response to M4344 treatment. M4344 kills cancer cells by inducing cellular catastrophe and DNA damage. M4344 is highly synergistic with a broad range of DNA-targeting anticancer agents. It significantly synergizes with topotecan and irinotecan in patient-derived tumor organoids and xenograft models.
    CONCLUSIONS: M4344 is a promising and highly potent ATR inhibitor. It enhances the activity of clinical DNA damaging agents commonly used in cancer treatment including topoisomerase inhibitors, gemcitabine, cisplatin and talazoparib. RepStress and NE gene expression signatures can be exploited as predictive markers for M4344.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-20-1026
  4. Cancer Res. 2021 May 27. pii: canres.1028.2020. [Epub ahead of print]
    NEIL3 prevents senescence in hepatocellular carcinoma by repairing oxidative lesions at telomeres during mitosis.
    Zhenjun Zhao, Helge Gad, Carlos Benitez-Buelga, Kumar Sanjiv, Hua Xiangwei, He Kang, Mingxuan Feng, Zhicong Zhao, Ulrika Warpman Berglund, Qiang Xia, Thomas Helleday.
      Hepatocellular carcinoma (HCC) patients suffer from few treatment options and poor survival rates. Here we report that endonuclease VIII-like protein 3 (NEIL3) is overexpressed in HCC and correlates with poor survival. All six HCC cell lines investigated were dependent on NEIL3 catalytic activity for survival and prevention of senescence, while NEIL3 was dispensable for non-transformed cells. NEIL3-depleted HCC cell lines accumulated oxidative DNA lesions specifically at telomeres, resulting in telomere dysfunctional foci and 53BP1 foci formation. Following oxidative DNA damage during mitosis, NEIL3 relocated to telomeres and recruited apurinic endonuclease 1 (APE1), indicating activation of base excision repair. META-FISH revealed that NEIL3, but not NEIL1 or NEIL2, is required to initiate APE1 and Polβ-dependent base excision repair at oxidized telomeres. Repeated exposure of NEIL3-depleted cells to oxidizing damage induced chromatin bridges and damaged telomeres. These results demonstrate a novel function for NEIL3 in repair of oxidative DNA damage at telomeres in mitosis, which is important to prevent senescence of HCC cells. Furthermore, these data suggest that NEIL3 could be a target for therapeutic intervention for HCC.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-1028
  5. Front Cell Dev Biol. 2021 ;9 670392
    Replication Fork Reversal and Protection.
    Shan Qiu, Guixing Jiang, Liping Cao, Jun Huang.
      During genome replication, replication forks often encounter obstacles that impede their progression. Arrested forks are unstable structures that can give rise to collapse and rearrange if they are not properly processed and restarted. Replication fork reversal is a critical protective mechanism in higher eukaryotic cells in response to replication stress, in which forks reverse their direction to form a Holliday junction-like structure. The reversed replication forks are protected from nuclease degradation by DNA damage repair proteins, such as BRCA1, BRCA2, and RAD51. Some of these molecules work cooperatively, while others have unique functions. Once the stress is resolved, the replication forks can restart with the help of enzymes, including human RECQ1 helicase, but restart will not be considered here. Here, we review research on the key factors and mechanisms required for the remodeling and protection of stalled replication forks in mammalian cells.
    Keywords:  DNA translocase; genome instability; replication fork reversal; replication fork stalling; replication stress
    DOI:  https://doi.org/10.3389/fcell.2021.670392
  6. Nucleic Acids Res. 2021 May 28. pii: gkab416. [Epub ahead of print]
    Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro.
    Max E Douglas, John F X Diffley.
      Telomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process.
    DOI:  https://doi.org/10.1093/nar/gkab416
  7. DNA Repair (Amst). 2021 May 11. pii: S1568-7864(21)00088-4. [Epub ahead of print]104 103132
    PARP1-mediated PARylation of TonEBP prevents R-loop-associated DNA damage.
    Byeong Jin Ye, Hyun Je Kang, Whaseon Lee-Kwon, Hyug Moo Kwon, Soo Youn Choi.
      Lack of coordination between the DNA replication and transcription machineries can increase the frequency of transcription-replication conflicts, leading ultimately to DNA damage and genomic instability. A major source of these conflicts is the formation of R-loops, which consist of a transcriptionally generated RNA-DNA hybrid and the displaced single-stranded DNA. R-loops play important physiological roles and have been implicated in human diseases. Although these structures have been extensively studied, many aspects of R-loop biology and R-loop-mediated genome instability remain unclear. We found that in cancer cells, tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) interacted with PARP1 and localized to R-loops in response to DNA-damaging agent camptothecin (CPT), which is associated with R-loop formation. PARP1-mediated PARylation was required for recruitment of TonEBP to the sites of R-loop-associated DNA damage. Loss of TonEBP increased levels of R-loop accumulation and DNA damage, and promoted cell death in response to CPT. These findings suggest that TonEBP mediates resistance to CPT-induced cell death by preventing R-loop accumulation in cancer cells.
    Keywords:  Camptothecin; Cancer cell; DNA damage response; DNA repair; Genome instability; NFAT5; PARylation
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103132
  8. Nat Commun. 2021 May 26. 12(1): 3153
    The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing.
    Evi Goulielmaki, Maria Tsekrekou, Nikos Batsiotos, Mariana Ascensão-Ferreira, Eleftheria Ledaki, Kalliopi Stratigi, Georgia Chatzinikolaou, Pantelis Topalis, Theodore Kosteas, Janine Altmüller, Jeroen A Demmers, Nuno L Barbosa-Morais, George A Garinis.
      RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.
    DOI:  https://doi.org/10.1038/s41467-021-23505-1
  9. J Cell Biol. 2021 Aug 02. pii: e202009147. [Epub ahead of print]220(8):
    LRR1-mediated replisome disassembly promotes DNA replication by recycling replisome components.
    Yilin Fan, Marielle S Köberlin, Nalin Ratnayeke, Chad Liu, Madhura Deshpande, Jeannine Gerhardt, Tobias Meyer.
      After two converging DNA replication forks meet, active replisomes are disassembled and unloaded from chromatin. A key process in replisome disassembly is the unloading of CMG helicases (CDC45-MCM-GINS), which is initiated in Caenorhabditis elegans and Xenopus laevis by the E3 ubiquitin ligase CRL2LRR1. Here, we show that human cells lacking LRR1 fail to unload CMG helicases and accumulate increasing amounts of chromatin-bound replisome components as cells progress through S phase. Markedly, we demonstrate that the failure to disassemble replisomes reduces the rate of DNA replication increasingly throughout S phase by sequestering rate-limiting replisome components on chromatin and blocking their recycling. Continued binding of CMG helicases to chromatin during G2 phase blocks mitosis by activating an ATR-mediated G2/M checkpoint. Finally, we provide evidence that LRR1 is an essential gene for human cell division, suggesting that CRL2LRR1 enzyme activity is required for the proliferation of cancer cells and is thus a potential target for cancer therapy.
    DOI:  https://doi.org/10.1083/jcb.202009147
  10. DNA Repair (Amst). 2021 May 13. pii: S1568-7864(21)00089-6. [Epub ahead of print]104 103133
    Coping with RNA damage with a focus on APE1, a BER enzyme at the crossroad between DNA damage repair and RNA processing/decay.
    Matilde Clarissa Malfatti, Giulia Antoniali, Marta Codrich, Gianluca Tell.
      Interest in RNA damage as a novel threat associated with several human pathologies is rapidly increasing. Knowledge on damaged RNA recognition, repair, processing and decay is still scanty. Interestingly, in the last few years, more and more evidence put a bridge between DNA damage repair enzymes and the RNA world. The Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) was firstly identified as a crucial enzyme of the base excision repair (BER) pathway preserving genome stability toward non-distorting DNA lesion-induced damages. Later, an unsuspected role of APE1 in controlling gene expression was discovered and its pivotal involvement in several human pathologies, ranging from tumor progression to neurodegenerative diseases, has emerged. Recent novel findings indicate a role of APE1 in RNA metabolism, particularly in processing activities of damaged (abasic and oxidized) RNA and in the regulation of oncogenic microRNAs (miRNAs). Even though the role of miRNAs in human pathologies is well-known, the mechanisms underlying their quality control are still totally unexplored. A detailed knowledge of damaged RNA decay processes in human cells is crucial in order to understand the molecular processes involved in multiple pathologies. This cutting-edge perspective article will highlight these emerging aspects of damaged RNA processing and decay, focusing the attention on the involvement of APE1 in RNA world.
    Keywords:  8-oxo-7,8-dihydroguanosine; APE1; DNA repair; LLPS; Neurodegeneration; miRNA
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103133
  11. Mol Oncol. 2021 May 26.
    DDRugging glioblastoma: understanding and targeting the DNA damage response to improve future therapies.
    Ola Rominiyi, Spencer J Collis.
      Glioblastoma is the most frequently diagnosed type of primary brain tumour in adults. These aggressive tumours are characterised by inherent treatment resistance and disease progression, contributing to ~190,000 brain-tumour related deaths globally each year. Current therapeutic interventions consist of surgical resection followed by radiotherapy and temozolomide chemotherapy, but average survival is typically around 1 year, with less than 10% of patients surviving more than 5 years. Recently, a fourth treatment modality of intermediate-frequency low-intensity electric fields [called tumour-treating fields ( TTFields)] was clinically approved for glioblastoma in some countries after it was found to increase median overall survival rates by ~5 months in a phase III randomised clinical trial. However, beyond these treatments, attempts to establish more effective therapies have yielded little improvement in survival for patients over the last 50 years. This is in contrast to many other types of cancer and highlights glioblastoma as a recognised tumour of unmet clinical need. Previous work has revealed that glioblastomas contain stem-cell-like subpopulations that exhibit heightened expression of DNA damage response (DDR) factors, contributing to therapy resistance and disease relapse. Given that radiotherapy, chemotherapy and TTFields-based therapies all impact DDR mechanisms, this Review will focus on our current knowledge of the role of the DDR in glioblastoma biology and treatment. We also discuss the potential of effective multi-modal targeting of the DDR combined with standard-of-care therapies, as well as emerging therapeutic targets, in providing much-needed improvements in survival rates for patients.
    Keywords:  Chemotherapy; DNA damage response (DDR); Glioblastoma; Radiotherapy; Synthetic lethality; Tumour treating fields (TTFields)
    DOI:  https://doi.org/10.1002/1878-0261.13020
  12. Exp Mol Med. 2021 May 28.
    Aurora kinase A, a synthetic lethal target for precision cancer medicine.
    Pui Kei Mou, Eun Ju Yang, Changxiang Shi, Guowen Ren, Shishi Tao, Joong Sup Shim.
      Recent advances in high-throughput sequencing technologies and data science have facilitated the development of precision medicine to treat cancer patients. Synthetic lethality is one of the core methodologies employed in precision cancer medicine. Synthetic lethality describes the phenomenon of the interplay between two genes in which deficiency of a single gene does not abolish cell viability but combined deficiency of two genes leads to cell death. In cancer treatment, synthetic lethality is leveraged to exploit the dependency of cancer cells on a pathway that is essential for cell survival when a tumor suppressor is mutated. This approach enables pharmacological targeting of mutant tumor suppressors that are theoretically undruggable. Successful clinical introduction of BRCA-PARP synthetic lethality in cancer treatment led to additional discoveries of novel synthetic lethal partners of other tumor suppressors, including p53, PTEN, and RB1, using high-throughput screening. Recent work has highlighted aurora kinase A (AURKA) as a synthetic lethal partner of multiple tumor suppressors. AURKA is a serine/threonine kinase involved in a number of central biological processes, such as the G2/M transition, mitotic spindle assembly, and DNA replication. This review introduces synthetic lethal interactions between AURKA and its tumor suppressor partners and discusses the potential of AURKA inhibitors in precision cancer medicine.
    DOI:  https://doi.org/10.1038/s12276-021-00635-6
  13. Mol Cell Oncol. 2021 Mar 25. 8(3): 1902250
    Targeting one-carbon metabolism requires mTOR inhibition: a new therapeutic approach in osteosarcoma.
    Richa Rathore, Brian Van Tine.
      The rate-limiting enzyme of serine biosynthesis, 3-phosphoglycerate dehydrogenase (PHGDH), contributes to rapid growth and proliferation when it is overexpressed in cancer. We recently described the metabolic adaptations that occur upon PHGDH inhibition in osteosarcoma. PHGDH inhibition causes metabolite accumulation that activates the mechanistic target of rapamycin (mTOR) signaling, sensitizing osteosarcoma to non-rapalog mTOR inhibition.
    Keywords:  PHGDH; mTORC1; methotrexate; osteosarcoma; perhexiline; serine
    DOI:  https://doi.org/10.1080/23723556.2021.1902250
  14. Biochem Biophys Res Commun. 2021 May 20. pii: S0006-291X(21)00797-X. [Epub ahead of print]561 120-127
    The H3K4 methyltransferase SETD1A is required for proliferation of non-small cell lung cancer cells by promoting S-phase progression.
    Joo-Young Kang, Jin Woo Park, Yusang Hwang, Ja Young Hahm, Junyoung Park, Kwon-Sik Park, Sang-Beom Seo.
      Epigenetic dysregulation has been strongly implicated in carcinogenesis and is one of the mechanisms that contribute to the development of lung cancer. Using genome-wide CRISPR/Cas9 library screening, we showed SET domain-containing protein 1A (SETD1A) is an essential epigenetic modifier of the proliferation of NSCLC H1299 cells. Depletion of SETD1A strikingly inhibited the proliferation of NSCLC cells. IHC staining and bioinformatics showed that SETD1A is upregulated in lung cancer. Kaplan-Meier survival analysis indicated that high expression of SETD1A is associated with poor prognosis of patients with NSCLC. We revealed that loss of SETD1A inhibits DNA replication and induces replication stress accompanied by impaired fork progression. In addition, transcription of CDC7 and TOP1, which are involved in replication origin activation and fork progression, respectively, was significantly reduced by knockdown of SETD1A. Taken together, these findings demonstrated SETD1A is a critical epigenetic modifier of NSCLC cell proliferation by promoting the transcription of a subset of DNA replication-associated genes.
    Keywords:  CRISPR screen; H3K4me3; Non-small cell lung cancer; Replication; SETD1A
    DOI:  https://doi.org/10.1016/j.bbrc.2021.05.026
  15. Nat Commun. 2021 May 25. 12(1): 3127
    Cornelia de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect.
    Gabrielle Olley, Madapura M Pradeepa, Graeme R Grimes, Sandra Piquet, Sophie E Polo, David R FitzPatrick, Wendy A Bickmore, Charlene Boumendil.
      Cornelia de Lange syndrome is a multisystem developmental disorder typically caused by mutations in the gene encoding the cohesin loader NIPBL. The associated phenotype is generally assumed to be the consequence of aberrant transcriptional regulation. Recently, we identified a missense mutation in BRD4 associated with a Cornelia de Lange-like syndrome that reduces BRD4 binding to acetylated histones. Here we show that, although this mutation reduces BRD4-occupancy at enhancers it does not affect transcription of the pluripotency network in mouse embryonic stem cells. Rather, it delays the cell cycle, increases DNA damage signalling, and perturbs regulation of DNA repair in mutant cells. This uncovers a role for BRD4 in DNA repair pathway choice. Furthermore, we find evidence of a similar increase in DNA damage signalling in cells derived from NIPBL-deficient individuals, suggesting that defective DNA damage signalling and repair is also a feature of typical Cornelia de Lange syndrome.
    DOI:  https://doi.org/10.1038/s41467-021-23500-6
  16. Trends Mol Med. 2021 May 21. pii: S1471-4914(21)00119-2. [Epub ahead of print]
    Studying PAR-Dependent Chromatin Remodeling to Tackle PARPi Resistance.
    Christina Andronikou, Sven Rottenberg.
      Histone eviction and chromatin relaxation are important processes for efficient DNA repair. Poly(ADP) ribose (PAR) polymerase 1 (PARP1) is a key mediator of this process, and disruption of PARP1 activity has a direct impact on chromatin structure. PARP inhibitors (PARPis) have been established as a treatment for BRCA1- or BRCA2-deficient tumors. Unfortunately, PARPi resistance occurs in many patients and the underlying mechanisms are not fully understood. In particular, it remains unclear how chromatin remodelers and histone chaperones compensate for the loss of the PARylation signal. In this Opinion article, we summarize currently known mechanisms of PARPi resistance. We discuss how the study of PARP1-mediated chromatin remodeling may help in further understanding PARPi resistance and finding new therapeutic approaches to overcome it.
    Keywords:  DNA damage response; PARP inhibition; chromatin remodeling; drug resistance; poly(ADP) ribosylation polymerase
    DOI:  https://doi.org/10.1016/j.molmed.2021.04.010
  17. J Genet Genomics. 2021 Apr 24. pii: S1673-8527(21)00091-6. [Epub ahead of print]
    Phosphorylation of Ago2 is required for its role in DNA double-strand break repair.
    Xiaolu Hu, Yan Li, Tianfang Zhang, Lin Li, She Chen, Xiaohong Wu, Haijun Li, Binjie Qi, Zuobing Chen.
      Repair of DNA double-strand break (DSB) is critical for the maintenance of genome integrity. A class of DSB-induced small RNAs (diRNAs) has been shown to play an important role in DSB repair. In humans, diRNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination (HR). Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions. However, the role of Ago2 phosphorylation in DNA damage repair is unexplored. Here, we show that S672, S828, T830, and S831 of human Ago2 are phosphorylated in response to ionizing radiation (IR). S672A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency. We further show that defective association of Ago2 S672A variant with DSB sites, instead of defects in diRNA and Rad51 binding, may account for decreased Rad51 foci formation and HR efficiency. Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.
    Keywords:  Ago2; DNA repair; Phosphorylation; Small RNA; diRNA
    DOI:  https://doi.org/10.1016/j.jgg.2021.03.011
  18. Mol Cancer Res. 2021 May 27. pii: molcanres.MCR-21-0219-A.2021. [Epub ahead of print]
    Cytidine Deaminase APOBEC3A Regulates PD-L1 Expression in Cancer Cells in a JNK/c-JUN-dependent Manner.
    Kailiang Zhao, Qiang Zhang, Sheryl Flanagan, Xueting Lang, Long Jiang, Leslie A Parsels, Joshua D Parsels, Weiping Zou, Theodore S Lawrence, Rémi Buisson, Michael D Green, Meredith A Morgan.
      Programmed death-ligand 1 (PD-L1) promotes tumor immune evasion by engaging the PD-1 receptor and inhibiting T-cell activity. While the regulation of PD-L1 expression is not fully understood, its expression is associated with tumor mutational burden and response to immune checkpoint therapy. Here, we report that Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) is an important regulator of PD-L1 expression. Using an APOBEC3A inducible expression system as well as siRNA against endogenous APOBECA, we found that APOBEC3A regulates PD-L1 mRNA and protein levels as well as PD-L1 cell surface expression in cancer. Mechanistically, APOBEC3A-induced PD-L1 expression was dependent on APOBEC3A catalytic activity as catalytically dead APOBEC3A mutants (E72A) failed to induce PD-L1 expression. Furthermore, APOBEC3A-induced PD-L1 expression was dependent on replication-associated DNA damage and JNK/c-JUN signaling but not interferon signaling. In addition, we confirmed the relevance of these finding in patient tumors as APOBEC3A expression and mutational signature correlated with PD-L1 expression in multiple patient cancer types. These data provide a novel link between APOBECA, its DNA mutagenic activity and PD-L1-mediated anti-tumoral immunity. This work nominates APOBEC3A as a mechanism of immune evasion and a potential biomarker for the therapeutic efficacy of immune checkpoint blockade. Implications: APOBEC3A catalytic activity induces replication-associated DNA damage to promote PD-L1 expression implying that APOBEC3A-driven mutagenesis represents both a mechanism of tumor immune evasion and a therapeutically targetable vulnerability in cancer cells.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-0219
  19. Nat Genet. 2021 May 27.
    Radiotherapy is associated with a deletion signature that contributes to poor outcomes in patients with cancer.
    Emre Kocakavuk, Kevin J Anderson, Frederick S Varn, Kevin C Johnson, Samirkumar B Amin, Erik P Sulman, Martijn P Lolkema, Floris P Barthel, Roel G W Verhaak.
      Ionizing radiation causes DNA damage and is a mainstay for cancer treatment, but understanding of its genomic impact is limited. We analyzed mutational spectra following radiotherapy in 190 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium and 3,693 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified radiotherapy-associated significant increases in the burden of small deletions (5-15 bp) and large deletions (20+ bp to chromosome-arm length). Small deletions were characterized by a larger span size, lacking breakpoint microhomology and were genomically more dispersed when compared to pre-existing deletions and deletions in non-irradiated tumors. Mutational signature analysis implicated classical non-homologous end-joining-mediated DNA damage repair and APOBEC mutagenesis following radiotherapy. A high radiation-associated deletion burden was associated with worse clinical outcomes, suggesting that effective repair of radiation-induced DNA damage is detrimental to patient survival. These results may be leveraged to predict sensitivity to radiation therapy in recurrent cancer.
    DOI:  https://doi.org/10.1038/s41588-021-00874-3
  20. Mol Cancer Res. 2021 May 28. pii: molcanres.0833.2020. [Epub ahead of print]
    R-loop-Mediated ssDNA Breaks Accumulate Following Short-Term Exposure to the HDAC Inhibitor Romidepsin.
    Maryam Safari, Thomas Litman, Robert Robey, Andres Aguilera, Arup R Chakraborty, William Reinhold, Agnes Basseville, Lubov Petrukhin, Luigi Scotto, Owen A O'Connor, Yves Pommier, Antonio T Fojo, Susan E Bates.
      Histone deacetylase inhibitors (HDACis) induce hyperacetylation of histones by blocking HDAC catalytic sites. Despite regulatory approvals in hematological malignancies, limited solid tumor clinical activity has constrained their potential, arguing for better understanding of mechanisms of action. Multiple activities of HDAC inhibitors have been demonstrated, dependent on cell context, beyond the canonical induction of gene expression. Here, using a clinically relevant exposure duration, we established DNA damage as the dominant signature using the NCI-60 cell line database and then focused on the mechanism by which hyperacetylation induces DNA damage. We identified accumulation of DNA-RNA hybrids (R-loops) following romidepsin-induced histone hyperacetylation, with single-stranded DNA breaks detected by single cell electrophoresis. Our data suggest that transcription-coupled base excision repair (BER) is involved in resolving ssDNA breaks that, when overwhelmed, evolve to lethal dsDNA breaks. We show that inhibition of BER proteins such as PARP will increase dsDNA breaks in this context. These studies establish accumulation of R-loops as a consequence of romidepsin-mediated histone hyperacetylation. We believe that the insights provided will inform design of more effective combination therapy with HDACis for treatment of solid tumors. Implications: Key HDAC inhibitor mechanisms of action remain unknown; we identify accumulation of DNA-RNA hybrids (R-loops) due to chromatin hyperacetylation that provokes single-stranded DNA damage as a first step toward cell death.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0833
  21. Nature. 2021 May 26.
    Structural basis of antifolate recognition and transport by PCFT.
    Joanne L Parker, Justin C Deme, Gabriel Kuteyi, Zhiyi Wu, Jiandong Huo, I David Goldman, Raymond J Owens, Philip C Biggin, Susan M Lea, Simon Newstead.
      Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.
    DOI:  https://doi.org/10.1038/s41586-021-03579-z
  22. Cell Death Dis. 2021 May 26. 12(6): 546
    BKM120 sensitizes glioblastoma to the PARP inhibitor rucaparib by suppressing homologous recombination repair.
    Shaolu Zhang, Xin Peng, Xiaofei Li, Hongyan Liu, Baoquan Zhao, Moshe Elkabets, Yao Liu, Wei Wang, Ran Wang, Yuxu Zhong, Dexin Kong.
      PARP inhibitors have been approved for the therapy of cancers with homologous recombination (HR) deficiency based on the concept of "synthetic lethality". However, glioblastoma (GBM) patients have gained little benefit from PARP inhibitors due to a lack of BRCA mutations. Herein, we demonstrated that concurrent treatment with the PARP inhibitor rucaparib and the PI3K inhibitor BKM120 showed synergetic anticancer effects on GBM U251 and U87MG cells. Mechanistically, BKM120 decreased expression of HR molecules, including RAD51 and BRCA1/2, and reduced HR repair efficiency in GBM cells, therefore increasing levels of apoptosis induced by rucaparib. Furthermore, we discovered that the two compounds complemented each other in DNA damage response and drug accumulation. Notably, in the zebrafish U87MG-RFP orthotopic xenograft model, nude mouse U87MG subcutaneous xenograft model and U87MG-Luc orthotopic xenograft model, combination showed obviously increased antitumor efficacy compared to each monotherapy. Immunohistochemical analysis of tumor tissues indicated that the combination obviously reduced expression of HR repair molecules and increased the DNA damage biomarker γ-H2AX, consistent with the in vitro results. Collectively, our findings provide new insight into combined blockade of PI3K and PARP, which might represent a promising therapeutic approach for GBM.
    DOI:  https://doi.org/10.1038/s41419-021-03805-6
  23. Sci Rep. 2021 May 25. 11(1): 10887
    IMPDH2 and HPRT expression and a prognostic significance in preoperative and postoperative patients with osteosarcoma.
    Parunya Chaiyawat, Areerak Phanphaisarn, Nutnicha Sirikaew, Jeerawan Klangjorhor, Viraporn Thepbundit, Pimpisa Teeyakasem, Phichayut Phinyo, Dumnoensun Pruksakorn, Jongkolnee Settakorn.
      Osteosarcoma is one of the most aggressive bone tumors in children and adolescents. Development of effective therapeutic options is still lacking due to the complexity of the genomic background. In previous work, we applied a proteomics-guided drug repurposing to explore potential treatments for osteosarcoma. Our follow-up study revealed an FDA-approved immunosuppressant drug, mycophenolate mofetil (MMF) targeting inosine-5'-phosphate dehydrogenase (IMPDH) enzymes, has an anti-tumor effect that appeared promising for further investigation and clinical trials. Profiling of IMPDH2 and hypoxanthine-guanine phosphoribosyltransferase (HPRT), key purine-metabolizing enzymes, could deepen understanding of the importance of purine metabolism in osteosarcoma and provide evidence for expanded use of MMF in the clinic. In the present study, we investigated levels of IMPDH2, and HPRT in biopsy of 127 cases and post-chemotherapy tissues in 20 cases of high-grade osteosarcoma patients using immunohistochemical (IHC) analysis. Cox regression analyses were performed to determine prognostic significance of all enzymes. The results indicated that low levels of HPRT were significantly associated with a high Enneking stage (P = 0.023) and metastatic status (P = 0.024). Univariate and multivariate analyses revealed that patients with low HPRT expression have shorter overall survival times [HR 1.70 (1.01-2.84), P = 0.044]. Furthermore, high IMPDH2/HPRT ratios were similarly associated with shorter overall survival times [HR 1.67 (1.02-2.72), P = 0.039]. Levels of the enzymes were also examined in post-chemotherapy tissues. The results showed that high IMPDH2 expression was associated with shorter metastasis-free survival [HR 7.42 (1.22-45.06), P = 0.030]. These results suggest a prognostic value of expression patterns of purine-metabolizing enzymes for the pre- and post-chemotherapy period of osteosarcoma treatment.
    DOI:  https://doi.org/10.1038/s41598-021-90456-4
  24. Sci Transl Med. 2021 May 26. pii: eabe8226. [Epub ahead of print]13(595):
    Hexose-6-phosphate dehydrogenase blockade reverses prostate cancer drug resistance in xenograft models by glucocorticoid inactivation.
    Jianneng Li, Michael Berk, Mohammad Alyamani, Navin Sabharwal, Christopher Goins, Joseph Alvarado, Mehdi Baratchian, Ziqi Zhu, Shaun Stauffer, Eric A Klein, Nima Sharifi.
      Prostate cancer resistance to next-generation hormonal treatment with enzalutamide is a major problem and eventuates into disease lethality. Biologically active glucocorticoids that stimulate glucocorticoid receptor (GR) have an 11β-OH moiety, and resistant tumors exhibit loss of 11β-HSD2, the oxidative (11β-OH → 11-keto) enzyme that normally inactivates glucocorticoids, allowing elevated tumor glucocorticoids to drive resistance by stimulating GR. Here, we show that up-regulation of hexose-6-phosphate dehydrogenase (H6PD) protein occurs in prostate cancer tissues of men treated with enzalutamide, human-derived cell lines, and patient-derived prostate tissues treated ex vivo with enzalutamide. Genetically silencing H6PD blocks NADPH generation, which inhibits the usual reductive directionality of 11β-HSD1, to effectively replace 11β-HSD2 function in human-derived cell line models, suppress the concentration of biologically active glucocorticoids in prostate cancer, and reverse enzalutamide resistance in mouse xenograft models. Similarly, pharmacologic blockade of H6PD with rucaparib normalizes tumor glucocorticoid metabolism in human cell lines and reinstates responsiveness to enzalutamide in mouse xenograft models. Our data show that blockade of H6PD, which is essential for glucocorticoid synthesis in humans, normalizes glucocorticoid metabolism and reverses enzalutamide resistance in mouse xenograft models. We credential H6PD as a pharmacologic vulnerability for treatment of next-generation androgen receptor antagonist-resistant prostate cancer by depleting tumor glucocorticoids.
    DOI:  https://doi.org/10.1126/scitranslmed.abe8226
  25. Br J Cancer. 2021 May 26.
    Phase 1 study of the ATR inhibitor berzosertib in combination with cisplatin in patients with advanced solid tumours.
    Geoffrey I Shapiro, Robert Wesolowski, Craig Devoe, Simon Lord, John Pollard, Bart S Hendriks, Martin Falk, Ivan Diaz-Padilla, Ruth Plummer, Timothy A Yap.
      BACKGROUND: Berzosertib (formerly M6620, VX-970) is a highly potent and selective, first-in-class ataxia telangiectasia-mutated and Rad3-related protein kinase (ATR) inhibitor. We assessed the safety, tolerability, pharmacokinetics, and preliminary efficacy of berzosertib plus cisplatin.METHODS: Adult patients with advanced solid tumours refractory or resistant to standard of care therapies received ascending doses of cisplatin (day 1) and berzosertib (days 2 and 9) every 3 weeks (Q3W).
    RESULTS: Thirty-one patients received berzosertib (90-210 mg/m2) and cisplatin (40-75 mg/m2) across seven dose levels. The most common grade ≥3 treatment-emergent adverse events were neutropenia (20.0%) and anaemia (16.7%). There were two dose-limiting toxicities: a grade 3 hypersensitivity reaction and a grade 3 increase in alanine aminotransferase. Berzosertib 140 mg/m2 (days 2 and 9) and cisplatin 75 mg/m2 (day 1) Q3W was determined as the recommended Phase 2 dose. Cisplatin had no apparent effect on berzosertib pharmacokinetics. Of the 31 patients, four achieved a partial response (two confirmed and two unconfirmed) despite having previously experienced disease progression following platinum-based chemotherapy.
    CONCLUSIONS: Berzosertib plus cisplatin is well tolerated and shows preliminary clinical activity in patients with advanced solid tumours, warranting further evaluation in a Phase 2 setting.
    CLINICAL TRIALS IDENTIFIER: NCT02157792.
    DOI:  https://doi.org/10.1038/s41416-021-01406-w
  26. Nat Commun. 2021 May 27. 12(1): 3188
    Chromosomal copy number heterogeneity predicts survival rates across cancers.
    Erik van Dijk, Tom van den Bosch, Kristiaan J Lenos, Khalid El Makrini, Lisanne E Nijman, Hendrik F B van Essen, Nico Lansu, Michiel Boekhout, Joris H Hageman, Rebecca C Fitzgerald, Cornelis J A Punt, Jurriaan B Tuynman, Hugo J G Snippert, Geert J P L Kops, Jan Paul Medema, Bauke Ylstra, Louis Vermeulen, Daniël M Miedema.
      Survival rates of cancer patients vary widely within and between malignancies. While genetic aberrations are at the root of all cancers, individual genomic features cannot explain these distinct disease outcomes. In contrast, intra-tumour heterogeneity (ITH) has the potential to elucidate pan-cancer survival rates and the biology that drives cancer prognosis. Unfortunately, a comprehensive and effective framework to measure ITH across cancers is missing. Here, we introduce a scalable measure of chromosomal copy number heterogeneity (CNH) that predicts patient survival across cancers. We show that the level of ITH can be derived from a single-sample copy number profile. Using gene-expression data and live cell imaging we demonstrate that ongoing chromosomal instability underlies the observed heterogeneity. Analysing 11,534 primary cancer samples from 37 different malignancies, we find that copy number heterogeneity can be accurately deduced and predicts cancer survival across tissues of origin and stages of disease. Our results provide a unifying molecular explanation for the different survival rates observed between cancer types.
    DOI:  https://doi.org/10.1038/s41467-021-23384-6
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