bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2022–07–24
28 papers selected by
Sean Rudd, Karolinska Institutet
  1. The mechanism of replication stalling and recovery within repetitive DNA.
  2. Extrinsic proofreading.
  3. BLM Sumoylation Is Required for Replication Stability and Normal Fork Velocity During DNA Replication.
  4. PHIP variants associated with Chung-Jansen syndrome disrupt replication fork stability and genome integrity.
  5. Extended DNA binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks.
  6. Replication stress impairs chromosome segregation and preimplantation development in human embryos.
  7. The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis.
  8. Chromatin Ubiquitination Guides DNA Double Strand Break Signaling and Repair.
  9. Targeting DNA-Protein Crosslinks via Post-Translational Modifications.
  10. Targeting Mre11 overcomes platinum resistance and induces synthetic lethality in XRCC1 deficient epithelial ovarian cancers.
  11. TDP1-independent pathways in the process and repair of TOP1-induced DNA damage.
  12. And-1 coordinates with the FANCM complex to regulate Fanconi Anemia signaling and cisplatin resistance.
  13. The functional analysis of the ubiquitin ligase Brl2 in the repair of DNA double-strand breaks.
  14. Accumulation of oncometabolite D-2-Hydroxyglutarate by SLC25A1 inhibition: A metabolic strategy for induction of HR-ness and radiosensitivity.
  15. Polymerases and DNA Repair in Neurons: Implications in Neuronal Survival and Neurodegenerative Diseases.
  16. DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.
  17. LncRNA CTBP1-DT-encoded microprotein DDUP sustains DNA damage response signalling to trigger dual DNA repair mechanisms.
  18. Implications of N7-hydrogen and C8-keto on the base pairing, mutagenic potential and repair of 8-oxo-2'-deoxy-adenosine: Investigation by nucleotide analogues.
  19. Acetylation of p62 regulates base excision repair through interaction with APE1.
  20. Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice.
  21. Selenocystine induces oxidative-mediated DNA damage via impairing homologous recombination repair of DNA double-strand breaks in human hepatoma cells.
  22. RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.
  23. Efficient replication of human nuclear DNA.
  24. G6PD-mediated increase in de novo NADP+ biosynthesis promotes antioxidant defense and tumor metastasis.
  25. Venetoclax in combination with nucleoside analogs in acute myelogenous leukemia.
  26. Ribonucleotide reductase: Implications of thiol S-nitrosylation and tyrosine nitration for different subunits.
  27. Leukemia-initiating HSCs in chronic lymphocytic leukemia reveal clonal leukemogenesis and differential drug sensitivity.
  28. Decitabine mildly attenuates MLL-rearranged acute lymphoblastic leukemia in vivo, and represents a poor chemo-sensitizer.
  1. Nat Commun. 2022 Jul 19. 13(1): 3953
    The mechanism of replication stalling and recovery within repetitive DNA.
    Corella S Casas-Delucchi, Manuel Daza-Martin, Sophie L Williams, Gideon Coster.
      Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstitute replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We find that structure-prone repeats are sufficient to impair replication. Whilst template unwinding is unaffected, leading strand synthesis is inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats is rescued by replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats requires an extrinsic accessory helicase. DNA-induced fork stalling is mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress may also be applied to DNA-induced replication stalling.
    DOI:  https://doi.org/10.1038/s41467-022-31657-x
  2. DNA Repair (Amst). 2022 Jul 04. pii: S1568-7864(22)00102-1. [Epub ahead of print]117 103369
    Extrinsic proofreading.
    Zhi-Xiong Zhou, Thomas A Kunkel.
      The high fidelity of replication of the nuclear DNA genome in eukaryotes involves three processes. Correct rather than incorrect dNTPs are almost always incorporated by the three major replicases, DNA polymerases α, δ and ε. When an incorrect base is occasionally inserted, the latter Pols δ and ε also have a 3 ´ to 5 ´ exonuclease activity that can remove the mismatch to allow correct DNA synthesis to proceed. Lastly, rare mismatches that escape proofreading activity and are present in newly replicated DNA can be removed by DNA mismatch repair. In this review, we consider evidence supporting the hypothesis that the second mechanism, proofreading, can operate in two different ways. Primer terminal mismatches made by either Pol δ or Pol ε can be 'intrinsically' proofread. This mechanism occurs by direct transfer of a misinserted base made at the polymerase active site to the exonuclease active site that is located a short distance away. Intrinsic proofreading allows mismatch excision without intervening enzyme dissociation. Alternatively, considerable evidence suggests that mismatches made by any of the three replicases can also be proofread by 'extrinsic' proofreading by Pol δ. Extrinsic proofreading occurs when a mismatch made by any of the three replicases is initially abandoned, thereby allowing the exonuclease active site of Pol δ to bind directly to and remove the mismatch before replication continues. Here we review the evidence that extrinsic proofreading significantly enhances the fidelity of nuclear DNA replication, and we then briefly consider the implications of this process for evolution and disease.
    Keywords:  Base substitutions; DNA polymerase; Genome stability; Mismatches; Proofreading; Replication fidelity
    DOI:  https://doi.org/10.1016/j.dnarep.2022.103369
  3. Front Mol Biosci. 2022 ;9 875102
    BLM Sumoylation Is Required for Replication Stability and Normal Fork Velocity During DNA Replication.
    Christelle de Renty, Kelvin W Pond, Mary K Yagle, Nathan A Ellis.
      BLM is sumoylated in response to replication stress. We have studied the role of BLM sumoylation in physiologically normal and replication-stressed conditions by expressing in BLM-deficient cells a BLM with SUMO acceptor-site mutations, which we refer to as SUMO-mutant BLM cells. SUMO-mutant BLM cells exhibited multiple defects in both stressed and unstressed DNA replication conditions, including, in hydroxyurea-treated cells, reduced fork restart and increased fork collapse and, in untreated cells, slower fork velocity and increased fork instability as assayed by track-length asymmetry. We further showed by fluorescence recovery after photobleaching that SUMO-mutant BLM protein was less dynamic than normal BLM and comprised a higher immobile fraction at collapsed replication forks. BLM sumoylation has previously been linked to the recruitment of RAD51 to stressed forks in hydroxyurea-treated cells. An important unresolved question is whether the failure to efficiently recruit RAD51 is the explanation for replication stress in untreated SUMO-mutant BLM cells.
    Keywords:  BLM; Bloom syndrome; DNA replication; replication fork stalling; sumoylation
    DOI:  https://doi.org/10.3389/fmolb.2022.875102
  4. Cold Spring Harb Mol Case Stud. 2022 Jul 21. pii: mcs.a006212. [Epub ahead of print]
    PHIP variants associated with Chung-Jansen syndrome disrupt replication fork stability and genome integrity.
    Neysha Tirado-Class, Caitlin Hathaway, Wendy K Chung, Huzefa Dungrawala.
      Chung-Jansen syndrome (CJS) is a rare, autosomal dominant disorder characterized by developmental delay, intellectual disability/cognitive impairment, behavioral challenges, obesity, and dysmorphic features. CJS is associated with heterozygous variants in PHIP (Pleckstrin-Homology Interacting Protein), a gene that encodes one of several substrate receptors for Cullin4-RING (CRL4) E3 ubiquitin ligase complex. Full length PHIP, also called DCAF14, was recently identified to function as a replication stress response protein. Herein, we report the identification of two PHIP missense variants identified by exome sequencing in unrelated individuals with CJS. The variants p.D488V and p.E963G occur in different functional elements of DCAF14- WD40 repeat domain and pleckstrin homology-binding region (PBR), respectively. Using DNA fiber assays, we reveal that cells expressing either variant exhibit defective replication fork progression in conditions of replication stress. Furthermore, unlike wild type DCAF14, both variants fail to accomplish DNA replication after exposure to genotoxic stress indicating a critical role of DCAF14 in protecting stalled replication forks. Thus, we have identified replication defects associated with CJS variants and predict replication-associated genome instability with CJS syndrome.
    Keywords:  2-3 toe syndactyly; Abdominal obesity; Generalized neonatal hypotonia; Intellectual disability, moderate; Mild global developmental delay
    DOI:  https://doi.org/10.1101/mcs.a006212
  5. J Biol Chem. 2022 Jul 15. pii: S0021-9258(22)00710-4. [Epub ahead of print] 102268
    Extended DNA binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks.
    Alexandra S Weinheimer, YiTing Paung, Julie Rageul, Arafat Khan, Natalie Lo, Brian Ho, Michael Tong, Sébastien Alphonse, Markus A Seeliger, Hyungjin Kim.
      Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator SDE2 is known to bind to TIMELESS (TIM), a protein of the fork protection complex (FPC), and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA binding activity of SDE2 is not well defined. Here, we structurally and functionally characterize a new conserved DNA binding motif related to the SAP (SAF-A/B, Acinus, PIAS) domain in human SDE2 and establish its preference for single-stranded DNA (ssDNA). Our nuclear magnetic resonance solution structure of the SDE2SAP domain reveals a helix-extended loop-helix core with the helices aligned parallel to each other, consistent with known canonical SAP folds. Notably, we shown the DNA interaction of this SAP domain extends beyond the core SAP domain and is augmented by two lysine residues in the C-terminal tail, which is uniquely positioned adjacent to the SAP motif and conserved in the pre-mRNA splicing factor SF3A3. Furthermore, we found mutation in the SAP domain and extended C-terminus not only disrupts ssDNA binding but also impairs TIM localization at replication forks, thus inhibiting efficient fork progression. Taken together, our results establish SDE2SAP as an essential element for SDE2 to exert its role in preserving replication fork integrity via FPC regulation and highlights the structural diversity of the DNA-protein interactions achieved by a specialized DNA binding motif.
    Keywords:  DNA binding protein; DNA damage; DNA replication; Genomic instability; SDE2; TIMELESS; nuclear magnetic resonance (NMR)
    DOI:  https://doi.org/10.1016/j.jbc.2022.102268
  6. Cell. 2022 Jul 14. pii: S0092-8674(22)00780-2. [Epub ahead of print]
    Replication stress impairs chromosome segregation and preimplantation development in human embryos.
    Katherine L Palmerola, Selma Amrane, Alejandro De Los Angeles, Shuangyi Xu, Ning Wang, Joao de Pinho, Michael V Zuccaro, Angelo Taglialatela, Dashiell J Massey, Jenna Turocy, Alex Robles, Anisa Subbiah, Bob Prosser, Rogerio Lobo, Alberto Ciccia, Amnon Koren, Timour Baslan, Dieter Egli.
      Human cleavage-stage embryos frequently acquire chromosomal aneuploidies during mitosis due to unknown mechanisms. Here, we show that S phase at the 1-cell stage shows replication fork stalling, low fork speed, and DNA synthesis extending into G2 phase. DNA damage foci consistent with collapsed replication forks, DSBs, and incomplete replication form in G2 in an ATR- and MRE11-dependent manner, followed by spontaneous chromosome breakage and segmental aneuploidies. Entry into mitosis with incomplete replication results in chromosome breakage, whole and segmental chromosome errors, micronucleation, chromosome fragmentation, and poor embryo quality. Sites of spontaneous chromosome breakage are concordant with sites of DNA synthesis in G2 phase, locating to gene-poor regions with long neural genes, which are transcriptionally silent at this stage of development. Thus, DNA replication stress in mammalian preimplantation embryos predisposes gene-poor regions to fragility, and in particular in the human embryo, to the formation of aneuploidies, impairing developmental potential.
    Keywords:  chromosomal mosaicism; chromosome fragility; developmental arrest; double-strand break; gene-poor regions; incomplete replication; micronuclei; preimplantation development; replication stress; stalled replication forks
    DOI:  https://doi.org/10.1016/j.cell.2022.06.028
  7. Nat Commun. 2022 Jul 16. 13(1): 4143
    The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis.
    Mara De Marco Zompit, Mònica Torres Esteban, Clémence Mooser, Salomé Adam, Silvia Emma Rossi, Alain Jeanrenaud, Pia-Amata Leimbacher, Daniel Fink, Ann-Marie K Shorrocks, Andrew N Blackford, Daniel Durocher, Manuel Stucki.
      The accurate repair of DNA double-strand breaks (DSBs), highly toxic DNA lesions, is crucial for genome integrity and is tightly regulated during the cell cycle. In mitosis, cells inactivate DSB repair in favor of a tethering mechanism that stabilizes broken chromosomes until they are repaired in the subsequent cell cycle phases. How this is achieved mechanistically is not yet understood, but the adaptor protein TOPBP1 is critically implicated in this process. Here, we identify CIP2A as a TOPBP1-interacting protein that regulates TOPBP1 localization specifically in mitosis. Cells lacking CIP2A display increased radio-sensitivity, micronuclei formation and chromosomal instability. CIP2A is actively exported from the cell nucleus in interphase but, upon nuclear envelope breakdown at the onset of mitosis, gains access to chromatin where it forms a complex with MDC1 and TOPBP1 to promote TOPBP1 recruitment to sites of mitotic DSBs. Collectively, our data uncover CIP2A-TOPBP1 as a mitosis-specific genome maintenance complex.
    DOI:  https://doi.org/10.1038/s41467-022-31865-5
  8. Front Cell Dev Biol. 2022 ;10 928113
    Chromatin Ubiquitination Guides DNA Double Strand Break Signaling and Repair.
    Ksenia G Kolobynina, Alexander Rapp, M Cristina Cardoso.
      Chromatin is the context for all DNA-based molecular processes taking place in the cell nucleus. The initial chromatin structure at the site of the DNA damage determines both, lesion generation and subsequent activation of the DNA damage response (DDR) pathway. In turn, proceeding DDR changes the chromatin at the damaged site and across large fractions of the genome. Ubiquitination, besides phosphorylation and methylation, was characterized as an important chromatin post-translational modification (PTM) occurring at the DNA damage site and persisting during the duration of the DDR. Ubiquitination appears to function as a highly versatile "signal-response" network involving several types of players performing various functions. Here we discuss how ubiquitin modifiers fine-tune the DNA damage recognition and response and how the interaction with other chromatin modifications ensures cell survival.
    Keywords:  DNA damage response; DNA repair; chromatin; double-strand breaks; ubiquitination
    DOI:  https://doi.org/10.3389/fcell.2022.928113
  9. Front Mol Biosci. 2022 ;9 944775
    Targeting DNA-Protein Crosslinks via Post-Translational Modifications.
    Xueyuan Leng, Julien P Duxin.
      Covalent binding of proteins to DNA forms DNA-protein crosslinks (DPCs), which represent cytotoxic DNA lesions that interfere with essential processes such as DNA replication and transcription. Cells possess different enzymatic activities to counteract DPCs. These include enzymes that degrade the adducted proteins, resolve the crosslinks, or incise the DNA to remove the crosslinked proteins. An important question is how DPCs are sensed and targeted for removal via the most suited pathway. Recent advances have shown the inherent role of DNA replication in triggering DPC removal by proteolysis. However, DPCs are also efficiently sensed and removed in the absence of DNA replication. In either scenario, post-translational modifications (PTMs) on DPCs play essential and versatile roles in orchestrating the repair routes. In this review, we summarize the current knowledge of the mechanisms that trigger DPC removal via PTMs, focusing on ubiquitylation, small ubiquitin-related modifier (SUMO) conjugation (SUMOylation), and poly (ADP-ribosyl)ation (PARylation). We also briefly discuss the current knowledge gaps and emerging hypotheses in the field.
    Keywords:  DNA repair; DNA replication; DNA-protein crosslink (DPC); poly(ADP-ribosyl)ation; post-translational modifications (PTMs); small ubiquitin-related modifier (SUMO); ubiquitylation
    DOI:  https://doi.org/10.3389/fmolb.2022.944775
  10. NPJ Precis Oncol. 2022 Jul 19. 6(1): 51
    Targeting Mre11 overcomes platinum resistance and induces synthetic lethality in XRCC1 deficient epithelial ovarian cancers.
    Adel Alblihy, Reem Ali, Mashael Algethami, Ahmed Shoqafi, Michael S Toss, Juliette Brownlie, Natalie J Tatum, Ian Hickson, Paloma Ordonez Moran, Anna Grabowska, Jennie N Jeyapalan, Nigel P Mongan, Emad A Rakha, Srinivasan Madhusudan.
      Platinum resistance is a clinical challenge in ovarian cancer. Platinating agents induce DNA damage which activate Mre11 nuclease directed DNA damage signalling and response (DDR). Upregulation of DDR may promote chemotherapy resistance. Here we have comprehensively evaluated Mre11 in epithelial ovarian cancers. In clinical cohort that received platinum- based chemotherapy (n = 331), Mre11 protein overexpression was associated with aggressive phenotype and poor progression free survival (PFS) (p = 0.002). In the ovarian cancer genome atlas (TCGA) cohort (n = 498), Mre11 gene amplification was observed in a subset of serous tumours (5%) which correlated highly with Mre11 mRNA levels (p < 0.0001). Altered Mre11 levels was linked with genome wide alterations that can influence platinum sensitivity. At the transcriptomic level (n = 1259), Mre11 overexpression was associated with poor PFS (p = 0.003). ROC analysis showed an area under the curve (AUC) of 0.642 for response to platinum-based chemotherapy. Pre-clinically, Mre11 depletion by gene knock down or blockade by small molecule inhibitor (Mirin) reversed platinum resistance in ovarian cancer cells and in 3D spheroid models. Importantly, Mre11 inhibition was synthetically lethal in platinum sensitive XRCC1 deficient ovarian cancer cells and 3D-spheroids. Selective cytotoxicity was associated with DNA double strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. We conclude that pharmaceutical development of Mre11 inhibitors is a viable clinical strategy for platinum sensitization and synthetic lethality in ovarian cancer.
    DOI:  https://doi.org/10.1038/s41698-022-00298-0
  11. Nat Commun. 2022 Jul 22. 13(1): 4240
    TDP1-independent pathways in the process and repair of TOP1-induced DNA damage.
    Huimin Zhang, Yun Xiong, Dan Su, Chao Wang, Mrinal Srivastava, Mengfan Tang, Xu Feng, Min Huang, Zhen Chen, Junjie Chen.
      Anticancer drugs, such as camptothecin (CPT), trap topoisomerase I (TOP1) on DNA and form TOP1 cleavage complexes (TOP1cc). Alternative repair pathways have been suggested in the repair of TOP1cc. However, how these pathways work with TDP1, a key repair enzyme that specifically hydrolyze the covalent bond between TOP1 catalytic tyrosine and the 3'-end of DNA and contribute to the repair of TOP1cc is poorly understood. Here, using unbiased whole-genome CRISPR screens and generation of co-deficient cells with TDP1 and other genes, we demonstrate that MUS81 is an important factor that mediates the generation of excess double-strand breaks (DSBs) in TDP1 KO cells. APEX1/2 are synthetic lethal with TDP1. However, deficiency of APEX1/2 does not reduce DSB formation in TDP1 KO cells. Together, our data suggest that TOP1cc can be either resolved directly by TDP1 or be converted into DSBs and repaired further by the Homologous Recombination (HR) pathway.
    DOI:  https://doi.org/10.1038/s41467-022-31801-7
  12. Cancer Res. 2022 Jul 22. pii: CAN-22-0769. [Epub ahead of print]
    And-1 coordinates with the FANCM complex to regulate Fanconi Anemia signaling and cisplatin resistance.
    Yi Zhang, Jing Li, Yuan Zhou, Zhuqing Li, Changmin Peng, Huadong Pei, Wenge Zhu.
      The Fanconi anemia (FA) pathway is essential for repairing DNA inter-strand crosslinks (ICL). ICLs induce stalled DNA replication forks and trigger activation of the FA pathway by promoting recruitment of the FANCM/FAAP24/MHF complex to ICL sites. Given that stalled replication forks are proximal to ICL sites, fork-associated proteins may coordinate with FA factors to rapidly sense ICLs for activation of FA signaling. Here we report that And-1, a replisome protein, is critical for activation of the FA pathway by sensing ICL-stalled forks and recruiting the FANCM/FAAP24 complex to ICLs. In response to ICLs, And-1 rapidly accumulated at ICL-stalled forks in a manner dependent on ATR-induced phosphorylation at T826. And-1 phosphorylation triggered an intramolecular change that promoted the interaction of And-1 with FANCM/FAAP24, resulting in recruitment of the FANCM/FAAP24 complex to ICLs. Furthermore, p-T826 And-1 was elevated in cisplatin-resistant ovarian cancer cells, and activated And-1 contributed to cisplatin resistance. Collectively, these studies elucidate a mechanism by which And-1 regulates FA signaling and identify And-1 as a potential target for developing therapeutic approaches to treat platinum-resistant ovarian cancer.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-0769
  13. Yi Chuan. 2022 Jul 20. 44(7): 609-617
    The functional analysis of the ubiquitin ligase Brl2 in the repair of DNA double-strand breaks.
    Xiao-Qin Liu, Fei-Ran Chang, Si-Jie Liu, Fei Wu, Dao-Chun Kong.
      Mono-ubiquitination of histone H2B plays a critical role in the regulation of gene transcription, DNA replication, and DNA damage repair. In Schizosaccharomyces pombe, Brl2 is an E3 ubiquitin ligase and required for the ubiquitination of H2B at lysine residue 119. Currently, there are few studies related to the function of Brl2 in DNA damage repair. Using camptothecin (CPT) to induce DNA double-strand breaks (DSBs) in S. pombe, we investigated the effect of Brl2 on DSB repair, and found that brl2-null mutants showed greater sensitivity to CPT when compared with wild-type (WT) cells, as well as having a drastically reduced spontaneous recombinant frequency. The fluorescent analysis demonstrated that Brl2 was co-localized with the recombination factor Rad52 at DSBs. Moreover, Brl2 promoted the recruitment of Rad52 to DSBs. Under CPT-induced DSBs, Brl2 was phosphorylated. These findings indicate that Brl2 plays a critical role in DNA homologous recombination and its mediated repair of DSBs.
    Keywords:  Brl2; CPT; DSB repair; S. pombe; histone H2B
    DOI:  https://doi.org/10.16288/j.yczz.22-031
  14. Cell Death Dis. 2022 Jul 22. 13(7): 641
    Accumulation of oncometabolite D-2-Hydroxyglutarate by SLC25A1 inhibition: A metabolic strategy for induction of HR-ness and radiosensitivity.
    Kexu Xiang, Christian Kalthoff, Corinna Münch, Verena Jendrossek, Johann Matschke.
      Oncogenic mutations in metabolic genes and associated oncometabolite accumulation support cancer progression but can also restrict cellular functions needed to cope with DNA damage. For example, gain-of-function mutations in isocitrate dehydrogenase (IDH) and the resulting accumulation of the oncometabolite D-2-hydroxyglutarate (D-2-HG) enhanced the sensitivity of cancer cells to inhibition of poly(ADP-ribose)-polymerase (PARP)1 and radiotherapy (RT). In our hand, inhibition of the mitochondrial citrate transport protein (SLC25A1) enhanced radiosensitivity of cancer cells and this was associated with increased levels of D-2-HG and a delayed repair of radiation-induced DNA damage. Here we aimed to explore the suggested contribution of D-2-HG-accumulation to disturbance of DNA repair, presumably homologous recombination (HR) repair, and enhanced radiosensitivity of cancer cells with impaired SLC25A1 function. Genetic and pharmacologic inhibition of SLC25A1 (SLC25A1i) increased D-2-HG-levels and sensitized lung cancer and glioblastoma cells to the cytotoxic action of ionizing radiation (IR). SLC25A1i-mediated radiosensitization was abrogated in MEFs with a HR-defect. D-2-HG-accumulation was associated with increased DNA damage and delayed resolution of IR-induced γH2AX and Rad51 foci. Combining SLC25A1i with PARP- or the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs)-inhibitors further potentiated IR-induced DNA damage, delayed DNA repair kinetics resulting in radiosensitization of cancer cells. Importantly, proof of concept experiments revealed that combining SLC25A1i with IR without and with PARPi also reduced tumor growth in the chorioallantoic membrane (CAM) model in vivo. Thereby SLC25A1i offers an innovative strategy for metabolic induction of context-dependent lethality approaches in combination with RT and clinically relevant inhibitors of complementary DNA repair pathways.
    DOI:  https://doi.org/10.1038/s41419-022-05098-9
  15. Front Cell Neurosci. 2022 ;16 852002
    Polymerases and DNA Repair in Neurons: Implications in Neuronal Survival and Neurodegenerative Diseases.
    Xiaoling Li, Guanghui Cao, Xiaokang Liu, Tie-Shan Tang, Caixia Guo, Hongmei Liu.
      Most of the neurodegenerative diseases and aging are associated with reactive oxygen species (ROS) or other intracellular damaging agents that challenge the genome integrity of the neurons. As most of the mature neurons stay in G0/G1 phase, replication-uncoupled DNA repair pathways including BER, NER, SSBR, and NHEJ, are pivotal, efficient, and economic mechanisms to maintain genomic stability without reactivating cell cycle. In these progresses, polymerases are prominent, not only because they are responsible for both sensing and repairing damages, but also for their more diversified roles depending on the cell cycle phase and damage types. In this review, we summarized recent knowledge on the structural and biochemical properties of distinct polymerases, including DNA and RNA polymerases, which are known to be expressed and active in nervous system; the biological relevance of these polymerases and their interactors with neuronal degeneration would be most graphically illustrated by the neurological abnormalities observed in patients with hereditary diseases associated with defects in DNA repair; furthermore, the vicious cycle of the trinucleotide repeat (TNR) and impaired DNA repair pathway is also discussed. Unraveling the mechanisms and contextual basis of the role of the polymerases in DNA damage response and repair will promote our understanding about how long-lived postmitotic cells cope with DNA lesions, and why disrupted DNA repair contributes to disease origin, despite the diversity of mutations in genes. This knowledge may lead to new insight into the development of targeted intervention for neurodegenerative diseases.
    Keywords:  DNA polymerase; DNA repair pathway; RNA polymerase; neurodegenerative diseases; postmitotic cells
    DOI:  https://doi.org/10.3389/fncel.2022.852002
  16. Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00891-9. [Epub ahead of print]40(3): 111089
    DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.
    Wen-Ling Lin, Jung-Kuei Chen, Xuemei Wen, Wei He, Geovanny A Zarceno, Yutian Chen, Shi Chen, Tanya T Paull, Hung-Wen Liu.
      R loops occur frequently in genomes and contribute to fundamental biological processes at multiple levels. Consequently, understanding the molecular and cellular biology of R loops has become an emerging area of research. Here, it is shown that poly(ADP-ribose) polymerase-1 (PARP-1) can mediate the association of DDX18, a putative RNA helicase, with R loops thereby modulating R-loop homeostasis in endogenous R-loop-prone and DNA lesion regions. DDX18 depletion results in aberrant endogenous R-loop accumulation, which leads to DNA-replication defects. In addition, DDX18 depletion renders cells more sensitive to DNA-damaging agents and reduces RPA32 and RAD51 foci formation in response to irradiation. Notably, DDX18 depletion leads to γH2AX accumulation and genome instability, and RNase H1 overexpression rescues all the DNA-repair defects caused by DDX18 depletion. Taken together, these studies uncover a function of DDX18 in R-loop-mediated events and suggest a role for PARP-1 in mediating the binding of specific DDX-family proteins with R loops in cells.
    Keywords:  CP: Molecular biology; DDX18; DNA damage; PARP-1; R loops; genome integrity
    DOI:  https://doi.org/10.1016/j.celrep.2022.111089
  17. Nucleic Acids Res. 2022 Jul 18. pii: gkac611. [Epub ahead of print]
    LncRNA CTBP1-DT-encoded microprotein DDUP sustains DNA damage response signalling to trigger dual DNA repair mechanisms.
    Ruyuan Yu, Yameng Hu, Shuxia Zhang, Xincheng Li, Miaoling Tang, Meisongzhu Yang, Xingui Wu, Ziwen Li, Xinyi Liao, Yingru Xu, Man Li, Suwen Chen, Wanying Qian, Li-Yun Gong, Libing Song, Jun Li.
      Sustaining DNA damage response (DDR) signalling via retention of DDR factors at damaged sites is important for transmitting damage-sensing and repair signals. Herein, we found that DNA damage provoked the association of ribosomes with IRES region in lncRNA CTBP1-DT, which overcame the negative effect of upstream open reading frames (uORFs), and elicited the novel microprotein DNA damage-upregulated protein (DDUP) translation via a cap-independent translation mechanism. Activated ATR kinase-mediated phosphorylation of DDUP induced a drastic 'dense-to-loose' conformational change, which sustained the RAD18/RAD51C and RAD18/PCNA complex at damaged sites and initiated RAD51C-mediated homologous recombination and PCNA-mediated post-replication repair mechanisms. Importantly, treatment with ATR inhibitor abolished the effect of DDUP on chromatin retention of RAD51C and PCNA, thereby leading to hypersensitivity of cancer cells to DNA-damaging chemotherapeutics. Taken together, our results uncover a plausible mechanism underlying the DDR sustaining and might represent an attractive therapeutic strategy in improvement of DNA damage-based anticancer therapies.
    DOI:  https://doi.org/10.1093/nar/gkac611
  18. Bioorg Chem. 2022 Jul 16. pii: S0045-2068(22)00435-7. [Epub ahead of print]127 106029
    Implications of N7-hydrogen and C8-keto on the base pairing, mutagenic potential and repair of 8-oxo-2'-deoxy-adenosine: Investigation by nucleotide analogues.
    Junjie Wu, Mengmeng Zhang, Lulu Song, Yahong Tan, Yosuke Taniguchi, Christopher John Hipolito, Youming Zhang, Yizhen Yin.
      Oxidative lesions, such as 8-oxo-dG and 8-oxo-dA, are continuously generated from exposure to reactive oxygen species. While 8-oxo-dG has been extensively studied, 8-oxo-dA has not received as much attention until recently. Herein, we report the synthesis of duplex DNAs incorporating dA, 8-oxo-dA, 7-deaza-dA, 8-Br-dA, and 8-Br-7-deaza-dA, which have different substitutions at 7- and 8-position, for the investigation into the implications of N7-hydrogen and C8-keto on the base pairing preference, mutagenic potential and repair of 8-oxo-dA. Base pairing study suggested that the polar N7-hydrogen and C8-keto of 8-oxo-dA, rather than the syn-preference, might be essential for 8-oxo-dA to form a stable base pair with dG. Insertion and extension studies using KF-exo- and human DNA polymerase β indicated that the efficient dGTP insertion opposite 8-oxo-dA and extension past 8-oxo-dA:dG are contingent upon not only the stable base pair with dG, but also the flexibility of the active site in polymerase. The N7-hydrogen in 8-oxo-dA or C7-hydrogen in 7-deaza-dA and 8-Br-7-deaza-dA was suggested to be important for the recognition by hOGG1, although the excision efficiencies of 7-deaza-dA and 8-Br-7-deaza-dA were much lower than 8-oxo-dA. This study provides an insight into the structure-function relationship of 8-oxo-dA by nucleotide analogues.
    Keywords:  8-oxo-dA; Base pairing; Mutagenic potential; Nucleotide analogue; Repair
    DOI:  https://doi.org/10.1016/j.bioorg.2022.106029
  19. Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00922-6. [Epub ahead of print]40(3): 111116
    Acetylation of p62 regulates base excision repair through interaction with APE1.
    Meiting Li, Jiannan Xiong, Liqian Yang, Jie Huang, Yu Zhang, Minghui Liu, Lina Wang, Jianguo Ji, Ying Zhao, Wei-Guo Zhu, Jianyuan Luo, Haiying Wang.
      p62, a well-known adaptor of autophagy, plays multiple functions in response to various stresses. Here, we report a function for p62 in base excision repair that is distinct from its known functions. Loss of p62 impairs base excision repair capacity and increases the sensitivity of cancer cells to alkylating and oxidizing agents. In response to alkylative and oxidative damage, p62 is accumulated in the nucleus,acetylated by hMOF,and deacetylated by SIRT7, and acetylated p62 is recruited to chromatin. The chromatin-enriched p62 directly interacts with APE1, a key enzyme of the BER pathway, and promotes its endonuclease activity, which facilitates BER and cell survival. Collectively, our findings demonstrate that p62 is a regulator of BER and provide further rationale for targeting p62 as a cancer therapeutic strategy.
    Keywords:  APE1; CP: Molecular biology; SIRT7; acetylation; base excision repair; hMOF; p62
    DOI:  https://doi.org/10.1016/j.celrep.2022.111116
  20. Nucleic Acids Res. 2022 Jul 18. pii: gkac616. [Epub ahead of print]
    Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice.
    Shanzhi Wang, Kyeryoung Lee, Stephen Gray, Yongwei Zhang, Catherine Tang, Rikke B Morrish, Elena Tosti, Johanna van Oers, Mohammad Ruhul Amin, Paula E Cohen, Thomas MacCarthy, Sergio Roa, Matthew D Scharff, Winfried Edelmann, Richard Chahwan.
      DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.
    DOI:  https://doi.org/10.1093/nar/gkac616
  21. Chem Biol Interact. 2022 Jul 18. pii: S0009-2797(22)00251-4. [Epub ahead of print] 110046
    Selenocystine induces oxidative-mediated DNA damage via impairing homologous recombination repair of DNA double-strand breaks in human hepatoma cells.
    Eva Ari Wahyuni, Chin-Yuan Yii, Hsiao-Lan Liang, Yueh-Hsia Luo, Sheng-Hua Yang, Pei-Yi Wu, Wei-Lun Hsu, Chung-Yi Nien, Ssu-Ching Chen.
      Selenocystine (SeC) has been identified as a novel compound with broad-spectrum anticancer activity. However, the effects of SeC on modifying DNA repair mechanism were less addressed. In this study, we demonstrated that SeC selectively induced cytotoxicity and genotoxicity against HepG2 hepatoma cell line. Comet assay revealed SeC-induced DNA damage in HepG2 cells, particularly in the form of DNA double strand breaks (DSBs), corroborated by the increase expression of the DSB marker, gamma-H2AX. We further demonstrated that SeC suppressed DNA homologous recombination repair, exacerbating DNA damage accumulation. Such effects on DNA damage and cell viability inhibition were alleviated by antioxidants, glutathione and Trolox, suggesting the involvement of reactive oxygen species (ROS). High levels of intracellular and mitochondrial ROS were detected in SeC-treated HepG2. In addition, SeC impaired the expression of antioxidant enzymes (superoxidase mutases and catalase), prompting the imbalance between antioxidant protection and excessive ROS formation and eliciting DSBs and cellular death. Decreased procaspase-3, 7, and 9 and Bcl-2 proteins and an increased Bax/Bcl-2 ratio, were observed after SeC treatment, but could be reversed by Torlox, confirming the action of SeC on ROS-induced apoptosis. In vivo, the xenograft tumor model of HepG2 cells validated the inhibition of SeC on tumor growth, and the induction of DSBs and apoptosis. In summary, SeC has the capability to induce ROS-dependent DNA damage and impeded DBS repair in HepG2 cells. Thus, SeC holds great promise as a therapeutic or adjuvant agent targeting DNA repair for cancer treatment.
    Keywords:  Apoptosis; DDR; Homologous recombination repair; ROS; Selenocysteine
    DOI:  https://doi.org/10.1016/j.cbi.2022.110046
  22. Sci Adv. 2022 Jul 15. 8(28): eabn1382
    RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.
    Carolina Nunes, Lisa Depestel, Liselot Mus, Kaylee M Keller, Louis Delhaye, Amber Louwagie, Muhammad Rishfi, Alex Whale, Neesha Kara, Simon R Andrews, Filemon Dela Cruz, Daoqi You, Armaan Siddiquee, Camila Takeno Cologna, Sam De Craemer, Emmy Dolman, Christoph Bartenhagen, Fanny De Vloed, Ellen Sanders, Aline Eggermont, Sarah-Lee Bekaert, Wouter Van Loocke, Jan Willem Bek, Givani Dewyn, Siebe Loontiens, Gert Van Isterdael, Bieke Decaesteker, Laurentijn Tilleman, Filip Van Nieuwerburgh, Vanessa Vermeirssen, Christophe Van Neste, Bart Ghesquiere, Steven Goossens, Sven Eyckerman, Katleen De Preter, Matthias Fischer, Jon Houseley, Jan Molenaar, Bram De Wilde, Stephen S Roberts, Kaat Durinck, Frank Speleman.
      High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.
    DOI:  https://doi.org/10.1126/sciadv.abn1382
  23. Cell Res. 2022 Jul 20.
    Efficient replication of human nuclear DNA.
    Thomas A Kunkel.
      
    DOI:  https://doi.org/10.1038/s41422-022-00690-2
  24. Sci Adv. 2022 Jul 22. 8(29): eabo0404
    G6PD-mediated increase in de novo NADP+ biosynthesis promotes antioxidant defense and tumor metastasis.
    Yang Zhang, Yi Xu, Wenyun Lu, Jinyang Li, Sixiang Yu, Eric J Brown, Ben Z Stanger, Joshua D Rabinowitz, Xiaolu Yang.
      Metastasizing cancer cells are able to withstand high levels of oxidative stress through mechanisms that are poorly understood. Here, we show that under various oxidative stress conditions, pancreatic cancer cells markedly expand NADPH and NADP+ pools. This expansion is due to up-regulation of glucose-6-phosphate dehydrogenase (G6PD), which stimulates the cytoplasmic nicotinamide adenine dinucleotide kinase (NADK1) to produce NADP+ while converting NADP+ to NADPH. G6PD is activated by the transcription factor TAp73, which is, in turn, regulated by two pathways. Nuclear factor-erythroid 2 p45-related factor-2 suppresses expression of the ubiquitin ligase PIRH2, stabilizing the TAp73 protein. Checkpoint kinases 1/2 and E2F1 induce expression of the TAp73 gene. Levels of G6PD and its upstream activators are elevated in metastatic pancreatic cancer. Knocking down G6PD impedes pancreatic cancer metastasis, whereas forced G6PD expression promotes it. These findings reveal an intracellular network that maintains redox homeostasis through G6PD-mediated increase in de novo NADP+ biosynthesis, which may be co-opted by tumor cells to enable metastasis.
    DOI:  https://doi.org/10.1126/sciadv.abo0404
  25. Curr Opin Oncol. 2022 Jul 16.
    Venetoclax in combination with nucleoside analogs in acute myelogenous leukemia.
    Brian J Ball, Paul B Koller, Vinod Pullarkat.
       PURPOSE OF REVIEW: Venetoclax in combination with nucleoside analogs such as hypomethylating agents (HMA) and low-dose cytarabine (LDAC) has led to unprecedented response and survival outcomes in patients with acute myeloid leukemia (AML). This has spurred the development of regimens combining venetoclax with other nucleoside analogs with distinct mechanisms of action. Here, we review older and newer nucleoside analogs, the rationale for their combination with venetoclax, and clinical evidence for the combination when available.
    RECENT FINDINGS: Venetoclax with HMA prolonged survival in a phase 3 study. Additionally, biologic correlates of response and resistance to venetoclax with HMA have been identified. The addition of venetoclax to standard intensive regimens containing higher doses of cytarabine and purine nucleoside analogs are safe and induce very high rates of remission and measurable residual disease negativity (MRD) negativity in newly diagnosed and relapsed/refractory AML. Investigational nucleoside analogs aim to improve upon the safety, bioavailability, or efficacy of approved venetoclax combinations and are currently being evaluated in clinical studies.
    SUMMARY: The development of venetoclax with HMA has transformed care for elderly adults with AML and opened the door for novel combinations of venetoclax with other nucleoside analogs. Further clinical studies are needed to see if these novel combinations further improve outcomes in AML particularly for patients with high-risk disease.
    DOI:  https://doi.org/10.1097/CCO.0000000000000868
  26. Nitric Oxide. 2022 Jul 15. pii: S1089-8603(22)00072-6. [Epub ahead of print]127 26-43
    Ribonucleotide reductase: Implications of thiol S-nitrosylation and tyrosine nitration for different subunits.
    Surupa Chakraborty, Prerona Mukherjee, Rajib Sengupta.
      Ribonucleotide reductase (RNR) is a multi-subunit enzyme responsible for catalyzing the rate-limiting step in the production of deoxyribonucleotides essential for DNA synthesis and repair. The active RNR complex is composed of multimeric R1 and R2 subunits. The RNR catalysis involves the formation of tyrosyl radicals in R2 subunits and thiyl radicals in R1 subunits. Despite the quaternary structure and cofactor diversity, all the three classes of RNR have a conserved cysteine residue at the active site which is converted into a thiyl radical that initiates the substrate turnover, suggesting that the catalytic mechanism is somewhat similar for all three classes of the RNR enzyme. Increased RNR activity has been associated with malignant transformation, cancer cell growth, and tumorigenesis. Efforts concerning the understanding of RNR inhibition in designing potent RNR inhibitors/drugs as well as developing novel approaches for antibacterial, antiviral treatments, and cancer therapeutics with improved radiosensitization have been made in clinical research. This review highlights the precise and potent roles of NO in RNR inhibition by targeting both the subunits. Under nitrosative stress, the thiols of the R1 subunits have been found to be modified by S-nitrosylation and the tyrosyl radicals of the R2 subunits have been modified by nitration. In view of the recent advances and progresses in the field of nitrosative modifications and its fundamental role in signaling with implications in health and diseases, the present article focuses on the regulations of RNR activity by S-nitrosylation of thiols (R1 subunits) and nitration of tyrosyl residues (R2 subunits) which will further help in designing new drugs and therapies.
    DOI:  https://doi.org/10.1016/j.niox.2022.07.002
  27. Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00921-4. [Epub ahead of print]40(3): 111115
    Leukemia-initiating HSCs in chronic lymphocytic leukemia reveal clonal leukemogenesis and differential drug sensitivity.
    Chi-Ling Chiang, Eileen Y Hu, Lingqian Chang, Jadwiga Labanowska, Kevan Zapolnik, Xiaokui Mo, Junfeng Shi, Tzyy-Jye Doong, Arletta Lozanski, Pearlly S Yan, Ralf Bundschuh, Logan A Walker, Daniel Gallego-Perez, Wu Lu, Meixiao Long, Sanggu Kim, Nyla A Heerema, Gerard Lozanski, Jennifer A Woyach, John C Byrd, Ly James Lee, Natarajan Muthusamy.
      The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.
    Keywords:  CP: Cancer; GATA2; IKZF1; LC-FACSeq; chronic lymphocytic leukemia; fludarabine; lenalidomide; leukemia initiating cells; single-cell analysis; stem cells
    DOI:  https://doi.org/10.1016/j.celrep.2022.111115
  28. EJHaem. 2020 Nov;1(2): 527-536
    Decitabine mildly attenuates MLL-rearranged acute lymphoblastic leukemia in vivo, and represents a poor chemo-sensitizer.
    Pauline Schneider, Patricia Garrido Castro, Sandra M Pinhanços, Mark Kerstjens, Eddy H van Roon, Anke H W Essing, M Emmy M Dolman, Jan J Molenaar, Rob Pieters, Ronald W Stam.
      MLL-rearranged acute lymphoblastic leukemia (ALL) represents a highly aggressive ALL subtype, characterized by aberrant DNA methylation patterns. DNA methyltransferase inhibitors, such as decitabine have previously been demonstrated to be effective in eradicating MLL-rearranged ALL cells in vitro. Here, we assessed the in vivo anti-leukemic potential of low-dose DNA methyltransferase inhibitor decitabine using a xenograft mouse model of human MLL-rearranged ALL. Furthermore, we explored whether prolonged exposure to low-dose decitabine could chemo-sensitize MLL-rearranged ALL cells toward conventional chemotherapy as well as other known epigenetic-based and anti-neoplastic compounds. Our data reveal that decitabine prolonged survival in xenograft mice of MLL-rearranged ALL by 8.5 days (P = .0181), but eventually was insufficient to prevent leukemia out-growth, based on the examination of the MLLAF4 cell line SEM. Furthermore, we observe that prolonged pretreatment of low-dose decitabine mildly sensitized toward the conventional drugs prednisolone, vincristine, daunorubicin, asparaginase, and cytarabine in a panel of MLL-rearranged cell lines. Additionally, we assessed synergistic effects of decitabine with other epigenetic-based or anticancer drugs using high-throughput drug library screens. Validation of the top hits, including histone deacetylase inhibitor panobinostat, BCL2 inhibitor Venetoclax, MEK inhibitor pimasertib, and receptor tyrosine kinase foretinib, revealed additive and moderate synergistic effects for the combination of each drug together with decitabine in a cell line-dependent manner.
    Keywords:  DNA demethylating agent; KMT2A; MLL; acute lymphoblastic leukemia; chemo‐sensitizer; decitabine; xenograft mouse models
    DOI:  https://doi.org/10.1002/jha2.81
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