bims-numges 2022-04-17 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–04–17
28 papers selected by
Sean Rudd, Karolinska Institutet

Filling in the gaps in PARP inhibitor-induced synthetic lethality.
Flap endonuclease 1 and DNA-PKcs synergistically participate in stabilizing replication fork to encounter replication stress in glioma cells.
Novel insights into the roles of Cdc7 in response to replication stress.
HDAC Inhibitor Sodium Butyrate Attenuates the DNA Repair in Transformed but Not in Normal Fibroblasts.
Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region.
The PARP1 Inhibitor Niraparib Represses DNA Damage Repair and Synergizes with Temozolomide for Antimyeloma Effects.
Cyclin-Dependent Kinase Synthetic Lethality Partners in DNA Damage Response.
RAD18 confers radioresistance of esophagus squamous cell carcinoma through regulating p-DNA-PKcs.
A new class of selective ATM inhibitors as combination partners of DNA double-strand break inducing cancer therapies.
Role and Regulation of Pif1 Family Helicases at the Replication Fork.
Microarray screening reveals two non-conventional SUMO-binding modules linked to DNA repair by non-homologous end-joining.
Targeting the DNA Damage Response to Increase Anthracycline-Based Chemotherapy Cytotoxicity in T-Cell Lymphoma.
Multi-omics data integration analysis identifies the spliceosome as a key regulator of DNA double-strand break repair.
IKZF1 selectively enhances homologous recombination repair by interacting with CtIP and USP7 in multiple myeloma.
Turning end-joining upside down in mitosis.
BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents.
A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis.
Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication.
LSD1 is required for euchromatic origin firing and replication timing.
Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids.
High replication stress and limited Rad51-mediated DNA repair capacity, but not oxidative stress, underlie oligodendrocyte precursor cell radiosensitivity.
Cinobufagin restrains the growth and triggers DNA damage of human hepatocellular carcinoma cells via proteasome-dependent degradation of thymidylate synthase.
The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase.
TLR8 is activated by 5'-methylthioinosine, a Plasmodium falciparum-derived intermediate of the purine salvage pathway.
SHMT2 promotes cell viability and inhibits ROS-dependent, mitochondrial-mediated apoptosis via the intrinsic signaling pathway in bladder cancer cells.
Multiple-turnover single nucleotide primer extension reactions to detect 8-oxo-2'-deoxyguanosine in DNA.
Inosine monophosphate dehydrogenase type1 sustains tumor growth in hepatocellular carcinoma.
Deep White Matter Cysts in a Patient with Aicardi-Goutières Syndrome and SAMHD1 Variants.

Mol Cell Oncol. 2021 ;8(6): 2010512

Filling in the gaps in PARP inhibitor-induced synthetic lethality.

Mariana Paes Dias, Jos Jonkers.

  Tumors with loss of breast cancer type 1 susceptibility protein (BRCA1) are homologous recombination (HR) deficient and hypersensitive to poly(ADP-ribose) polymerase inhibitors (PARPi). However, these tumors may restore HR and acquire PARPi resistance via loss of end-protection of DNA double-strand breaks. We found that loss of nuclear DNA ligase III resensitizes HR-restored BRCA1-deficient cells to PARPi by exposing post-replicative single-stranded DNA (ssDNA) gaps. Our work, and that of others, identifies ssDNA gaps as a key determinant of PARPi response.

Keywords:  BRCA1/2; PARP inhibitors; drug resistance; ssDNA gaps

DOI:  https://doi.org/10.1080/23723556.2021.2010512
J Exp Clin Cancer Res. 2022 Apr 12. 41(1): 140

Flap endonuclease 1 and DNA-PKcs synergistically participate in stabilizing replication fork to encounter replication stress in glioma cells.

Jing Zhang, Mu Chen, Ying Pang, Meng Cheng, Bingsong Huang, Siyi Xu, Min Liu, Hao Lian, Chunlong Zhong.

   BACKGROUND: Selectively utilizing alternative mechanisms to repair damaged DNA in essential factors deficient cancer facilitates tumor genetic evolution and contributes to treatment resistance. Synthetic lethality strategies provide a novel scenario to anticancer therapy with DNA repair protein mutation, such as glioma with DNA-PKcs-deficiency, a core factor crucial for non-homologous end joining (NHEJ) mediated DNA damage repair. Nevertheless, the clinical significance and molecular mechanisms of synthetic lethality function by interfering tumor DNA replication remain largely unexplored.
METHODS: Cancer clinic treatment resistance-related replication core factors were identified through bioinformatics analysis and RNA-sequencing and verified in clinical specimens by immunoblotting and in situ Proximity Ligation Analysis (PLA). Then, in vitro and in vivo experiments, including visible single molecular tracking system were performed to determine functional roles, the molecular mechanisms and clinical significance of synthetic lethality on glioma tumors.
RESULTS: Hyperactive DNA replication and regulator Flap endonuclease 1 (FEN1) provides high efficiency DNA double strand breaks (DSB) repair abilities preventing replication forks collapse during DNA replication which facilitate adaptation to selective pressures. DNA-PKcs deficient glioma cells are highly dependent on FEN1/BRCA1/RAD51 to survival and counteract replication stress. FEN1 protects perturbed forks from erroneous over-resection by MRE11 through regulating of BRCA1-RAD51 and WRN helicase, uncovering an essential genetic interaction between FEN1 and DNA-PKcs in mitigating replication-stress induced tumor genomic instability. Therapeutically, genetic depletion or molecular inhibition of FEN1 and DNA-PKcs perturb glioma progression.
CONCLUSIONS: Our findings highlight an unanticipated synthetic interaction between FEN1/BRCA1/RAD51 and DNA-PKcs when dysfunction leads to incompatible with cell survival under conditions of interrupted replication progression by disrupting addictive alternative tumor evolution and demonstrate the applicability of combined FEN1 and DNA-PKcs targeting in the treatment of glioma.

Keywords:  DNA damage; DNA replication; Genome instability; Glioma; Synthetic lethality; Tumor genetic evolution

DOI:  https://doi.org/10.1186/s13046-022-02334-0
FEBS J. 2022 Apr 09.

Novel insights into the roles of Cdc7 in response to replication stress.

Cristina González-Garrido, Félix Prado.

  Cdc7 and its regulator Dbf4 (Dbf4-dependent kinase; DDK) form an essential complex due to its function in replication initiation, which is carried out by phosphorylating different residues at the helicase MCM during the G1/S transition. In response to replication stress, late origins are inhibited to prevent cell cycle progression until the problems are resolved. In yeast, this inhibition is partially achieved by attenuating DDK activity. In addition, evidence from yeast to human shows that Cdc7 is required for a successful DNA damage response by coordinating multiple processes dealing with replication stress (replication checkpoint, DNA damage tolerance and break-induced replication) through mechanisms that go beyond its role in origin activation. These studies reveal the importance of getting a better understanding of the spatiotemporal regulation of DDK. Here, we will discuss how DDK operates in these processes and its putative role in controlling the activity of replication and repair factors at specific nuclease-resistant nucleoprotein scaffolds.

Keywords:  Cdc7; DDK; Dbf4; checkpoint; homologous recombination; replication; translesion synthesis

DOI:  https://doi.org/10.1111/febs.16456
Int J Mol Sci. 2022 Mar 23. pii: 3517. [Epub ahead of print]23(7):

HDAC Inhibitor Sodium Butyrate Attenuates the DNA Repair in Transformed but Not in Normal Fibroblasts.

Olga O Gnedina, Alisa V Morshneva, Elena V Skvortsova, Maria V Igotti.

  Many cancer therapy strategies cause DNA damage leading to the death of tumor cells. The DNA damage response (DDR) modulators are considered as promising candidates for use in combination therapy to enhance the efficacy of DNA-damage-mediated cancer treatment. The inhibitors of histone deacetylases (HDACis) exhibit selective antiproliferative effects against transformed and tumor cells and could enhance tumor cell sensitivity to genotoxic agents, which is partly attributed to their ability to interfere with DDR. Using the comet assay and host-cell reactivation of transcription, as well as γH2AX staining, we have shown that sodium butyrate inhibited DNA double-strand break (DSB) repair of both endo- and exogenous DNA in transformed but not in normal cells. According to our data, the dysregulation of the key repair proteins, especially the phosphorylated Mre11 pool decrease, is the cause of DNA repair impairment in transformed cells. The inability of HDACis to obstruct DSB repair in normal cells shown in this work demonstrates the advantages of HDACis in combination therapy with genotoxic agents to selectively enhance their cytotoxic activity in cancer cells.

Keywords:  DNA damage response; DSB repair; MRN complex; cancer; histone deacetylase inhibitor (HDACi)

DOI:  https://doi.org/10.3390/ijms23073517
Elife. 2022 Apr 13. pii: e75047. [Epub ahead of print]11

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region.

Sandhya Balasubramanian, Matteo Andreani, Júlia Goncalves Andrade, Tannishtha Saha, Devakumar Sundaravinayagam, Javier Garzón, Wenzhu Zhang, Oliver Popp, Shin-Ichiro Hiraga, Ali Rahjouei, Daniel B Rosen, Philipp Mertins, Brian T Chait, Anne D Donaldson, Michela Di Virgilio.

  RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

Keywords:  DNA replication fork protection; DSB resection inhibition; RIF1; SQ motifs; cancer biology; genetics; genomics; intrinsically disordered region; mouse

DOI:  https://doi.org/10.7554/eLife.75047
J Oncol. 2022 ;2022 2800488

The PARP1 Inhibitor Niraparib Represses DNA Damage Repair and Synergizes with Temozolomide for Antimyeloma Effects.

Hong-Yuan Shen, Hai-Long Tang, Yan-Hua Zheng, Juan Feng, Bao-Xia Dong, Xie-Qun Chen.

Purpose: Poly(ADP-ribose) polymerase 1 (PARP1) is necessary for single-strand break (SSB) repair by sensing DNA breaks and facilitating DNA repair through poly ADP-ribosylation of several DNA-binding and repair proteins. Inhibition of PARP1 results in collapsed DNA replication fork and double-strand breaks (DSBs). Accumulation of DSBs goes beyond the capacity of DNA repair response, ultimately resulting in cell death. This work is aimed at assessing the synergistic effects of the DNA-damaging agent temozolomide (TMZ) and the PARP inhibitor niraparib (Nira) in human multiple myeloma (MM) cells.
Materials and Methods: MM RPMI8226 and NCI-H929 cells were administered TMZ and/or Nira for 48 hours. CCK-8 was utilized for cell viability assessment. Cell proliferation and apoptosis were detected flow-cytometrically. Immunofluorescence was performed for detecting γH2A.X expression. Soft-agar colony formation assay was applied to evaluate the antiproliferative effect. The amounts of related proteins were obtained by immunoblot. The combination index was calculated with the CompuSyn software. A human plasmacytoma xenograft model was established to assess the anti-MM effects in vivo. The anti-MM activities of TMZ and/or Nira were evaluated by H&E staining, IHC, and the TUNEL assay.
Results: The results demonstrated that cotreatment with TMZ and Nira promoted DNA damage, cell cycle arrest, and apoptotic death in cultured cells but also reduced MM xenograft growth in nude mice, yielding highly synergistic effects. Immunoblot revealed that TMZ and Nira cotreatment markedly increased the expression of p-ATM, p-CHK2, RAD51, and γH2A.X, indicating the suppression of DNA damage response (DDR) and elevated DSB accumulation.
Conclusion: Inhibition of PARP1 sensitizes genotoxic agents and represents an important therapeutic approach for MM. These findings provide preliminary evidence for combining PARP1 inhibitors with TMZ for MM treatment.

DOI: https://doi.org/10.1155/2022/2800488
Int J Mol Sci. 2022 Mar 24. pii: 3555. [Epub ahead of print]23(7):

Cyclin-Dependent Kinase Synthetic Lethality Partners in DNA Damage Response.

Mateusz Kciuk, Adrianna Gielecińska, Somdutt Mujwar, Mariusz Mojzych, Renata Kontek.

  Cyclin-dependent kinases (CDKs) are pivotal mediators and effectors of the DNA damage response (DDR) that regulate both the pathway components and proteins involved in repair processes. Synthetic lethality (SL) describes a situation in which two genes are linked in such a way that the lack of functioning of just one maintains cell viability, while depletion of both triggers cell death. Synthetic lethal interactions involving CDKs are now emerging, and this can be used to selectively target tumor cells with DNA repair defects. In this review, SL interactions of CDKs with protooncogene products MYC, poly (ADP-ribose) polymerase (PARP-1), and cellular tumor antigen p53 (TP53) are discussed. The individual roles of each of the SL partners in DDR are described.

Keywords:  DNA damage response (DDR); MYC oncogene; cyclin-dependent kinase (CDK); inhibitor; poly (ADP-ribose) polymerase 1 (PARP-1); synthetic lethality

DOI:  https://doi.org/10.3390/ijms23073555
Cancer Med. 2022 Apr 14.

RAD18 confers radioresistance of esophagus squamous cell carcinoma through regulating p-DNA-PKcs.

Xiaoqing Li, Shitao Zou, Liangsu Zhou, Aidi Gao, Jing Xu, Chao He, Jundong Zhou, Shuhua Wu, Yihong Chen.

   BACKGROUND: Radiotherapy has recently become more common for the treatment of esophageal squamous cell carcinoma (ESCC). Radioresistance, on the other hand, continues to be a major issue because it interferes with the effectiveness of ESCC radiation. It has been demonstrated that RAD18, an E3 ubiquitin-protein ligase that regulates translesion DNA synthesis (TLS), is implicated in the regulation of genomic integrity and DNA damage response.
METHODS: In the present study, immunohistochemical staining and western blotting were utilized to determine RAD18 expression in ESCC tissues and cells. ESCC cell proliferation was determined using a colony formation assay. Immunofluorescence staining, comet assay, and homologous recombination (HR)/non-homologous end-joining (NHEJ) assays were conducted to examine the effect of RAD18 on the DNA damage response in ESCC cells.
RESULTS: We found that high RAD18 expression was positively associated with a poorer prognosis in patients with ESCC who received radiotherapy. Downregulation of RAD18 expression significantly increased the sensitivity of ESCC cells to irradiation. Moreover, RAD18 knockdown prolonged the repair kinetics of γH2AX foci and resulted in longer comet tails. Furthermore, loss of RAD18 expression markedly decreased non-homologous end-joining (NHEJ) activity, but it did not affect homologous recombination (HR)-mediated double-strand break repair in ESCC cells. RAD18 upregulated p-DNA-dependent protein kinase complex (p-DNA-PKc) expression in vivo and in vitro.
CONCLUSIONS: These data indicated that RAD18 may regulate radioresistance by facilitating NHEJ via phosphorylation of DNA-PKcs in ESCC cells, providing a novel radiotherapy target for ESCC.

Keywords:  NHEJ; RAD18; esophageal squamous cell carcinoma; p-DNA-PKcs; radioresistance

DOI:  https://doi.org/10.1002/cam4.4754
Mol Cancer Ther. 2022 Apr 11. pii: molcanther.0934.2021. [Epub ahead of print]

A new class of selective ATM inhibitors as combination partners of DNA double-strand break inducing cancer therapies.

Astrid Zimmermann, Frank T Zenke, Li-Ya Chiu, Heike Dahmen, Ulrich Pehl, Thomas Fuchss, Thomas Grombacher, Beatrix Blume, Lyubomir T Vassilev, Andree Blaukat.

Radiation and chemical DNA damaging agents are among the most widely used classes of cancer therapeutics today. Double strand breaks (DSBs) induced by many of these treatments are lethal to cancer cells if left unrepaired. Ataxia telangiectasia mutated (ATM) kinase plays a key role in the DNA damage response by driving DSB repair and cell cycle checkpoints to protect cancer cells. Inhibitors of ATM catalytic activity have been shown to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiation therapy and other DSB-inducing modalities. Here, we describe the pharmacological activities of two highly potent and selective ATM inhibitors from a new chemical class, M3541 and M4076. In biochemical assays, they inhibited ATM kinase activity with a sub-nanomolar potency and showed remarkable selectivity against other protein kinases. In cancer cells, the ATM inhibitors suppressed DSB repair, clonogenic cancer cell growth and potentiated antitumor activity of ionizing radiation in cancer cell lines. Oral administration of M3541 and M4076 to nude mice bearing human tumor xenografts with a clinically relevant radiation regimen strongly enhanced the antitumor activity, leading to complete tumor regressions. The efficacy correlated with the inhibition of ATM activity and modulation of its downstream targets in the xenograft tissues. In vitro and in vivo experiments demonstrated strong combination potential with PARP and topoisomerase I inhibitors. M4076 is currently under clinical investigation.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0934
Int J Mol Sci. 2022 Mar 29. pii: 3736. [Epub ahead of print]23(7):

Role and Regulation of Pif1 Family Helicases at the Replication Fork.

Emory G Malone, Matthew D Thompson, Alicia K Byrd.

  Pif1 helicases are a multifunctional family of DNA helicases that are important for many aspects of genomic stability in the nucleus and mitochondria. Pif1 helicases are conserved from bacteria to humans. Pif1 helicases play multiple roles at the replication fork, including promoting replication through many barriers such as G-quadruplex DNA, the rDNA replication fork barrier, tRNA genes, and R-loops. Pif1 helicases also regulate telomerase and promote replication termination, Okazaki fragment maturation, and break-induced replication. This review highlights many of the roles and regulations of Pif1 at the replication fork that promote cellular health and viability.

Keywords:  DNA helicase; G-quadruplexes; Pif1 helicase; replication fork; replication fork barriers; telomerase

DOI:  https://doi.org/10.3390/ijms23073736
Nucleic Acids Res. 2022 Apr 14. pii: gkac237. [Epub ahead of print]

Microarray screening reveals two non-conventional SUMO-binding modules linked to DNA repair by non-homologous end-joining.

Maria Jose Cabello-Lobato, Matthew Jenner, Metztli Cisneros-Aguirre, Kira Brüninghoff, Zac Sandy, Isabelle C da Costa, Thomas A Jowitt, Christian M Loch, Stephen P Jackson, Qian Wu, Henning D Mootz, Jeremy M Stark, Matthew J Cliff, Christine K Schmidt.

SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, the interaction of SUMO2 and XRCC4 is incompatible with XRCC4 binding to three other proteins important for NHEJ-mediated DSB repair. These findings are consistent with SUMO2 forming a redundant NHEJ layer with the potential to regulate different NHEJ complexes at distinct levels including, but not limited to, XRCC4 interactions with XLF, LIG4 and IFFO1. Regulation of NHEJ is not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. In addition to providing molecular insights into NHEJ, this work uncovers a conserved SUMO-binding module and provides a rich resource on direct SUMO binders exploitable towards uncovering SUMOylation pathways in a wide array of cellular processes.

DOI: https://doi.org/10.1093/nar/gkac237
Int J Mol Sci. 2022 Mar 30. pii: 3834. [Epub ahead of print]23(7):

Targeting the DNA Damage Response to Increase Anthracycline-Based Chemotherapy Cytotoxicity in T-Cell Lymphoma.

Martina Magni, Chiara Paolizzi, Chiara Monfrini, Cristina Vella, Paolo Corradini, Cristiana Carniti.

  Mature T-cell lymphomas (MTCLs) represent a heterogeneous group of aggressive non-Hodgkin lymphomas comprising different entities. Anthracycline-based regimens are considered the standard of care in the front-line treatment. However, responses to these approaches have been neither adequate nor durable, and new treatment strategies are urgently needed to improve survival. Genomic instability is a common feature of cancer cells and can be caused by aberrations in the DNA damage response (DDR) and DNA repair mechanisms. Consistently, molecules involved in DDR are being targeted to successfully sensitize cancer cells to chemotherapy. Recent studies showed that some hematological malignancies display constitutive DNA damage and intrinsic DDR activation, but these features have not been investigated yet in MTCLs. In this study, we employed a panel of malignant T cell lines, and we report for the first time the characterization of intrinsic DNA damage and basal DDR activation in preclinical models in T-cell lymphoma. Moreover, we report the efficacy of targeting the apical kinase ATM using the inhibitor AZD0156, in combination with standard chemotherapy to promote apoptotic cell death. These findings suggest that DDR is an attractive pathway to be pharmacologically targeted when developing novel therapies and improving MTCL patients' outcomes.

Keywords:  ATM inhibition; AZD0156; DNA damage response; T-cell lymphoma; anthracycline-based chemotherapy; drug combination

DOI:  https://doi.org/10.3390/ijms23073834
NAR Cancer. 2022 Jun;4(2): zcac013

Multi-omics data integration analysis identifies the spliceosome as a key regulator of DNA double-strand break repair.

Dana Sherill-Rofe, Oded Raban, Steven Findlay, Dolev Rahat, Irene Unterman, Arash Samiei, Amber Yasmeen, Zafir Kaiser, Hellen Kuasne, Morag Park, William D Foulkes, Idit Bloch, Aviad Zick, Walter H Gotlieb, Yuval Tabach, Alexandre Orthwein.

DNA repair by homologous recombination (HR) is critical for the maintenance of genome stability. Germline and somatic mutations in HR genes have been associated with an increased risk of developing breast (BC) and ovarian cancers (OvC). However, the extent of factors and pathways that are functionally linked to HR with clinical relevance for BC and OvC remains unclear. To gain a broader understanding of this pathway, we used multi-omics datasets coupled with machine learning to identify genes that are associated with HR and to predict their sub-function. Specifically, we integrated our phylogenetic-based co-evolution approach (CladePP) with 23 distinct genetic and proteomic screens that monitored, directly or indirectly, DNA repair by HR. This omics data integration analysis yielded a new database (HRbase) that contains a list of 464 predictions, including 76 gold standard HR genes. Interestingly, the spliceosome machinery emerged as one major pathway with significant cross-platform interactions with the HR pathway. We functionally validated 6 spliceosome factors, including the RNA helicase SNRNP200 and its co-factor SNW1. Importantly, their RNA expression correlated with BC/OvC patient outcome. Altogether, we identified novel clinically relevant DNA repair factors and delineated their specific sub-function by machine learning. Our results, supported by evolutionary and multi-omics analyses, suggest that the spliceosome machinery plays an important role during the repair of DNA double-strand breaks (DSBs).

DOI: https://doi.org/10.1093/narcan/zcac013
Int J Biol Sci. 2022 ;18(6): 2515-2526

IKZF1 selectively enhances homologous recombination repair by interacting with CtIP and USP7 in multiple myeloma.

Meng Liu, Ying Zhang, Yunzhao Wu, Jin Jin, Yang Cao, Zhixiao Fang, Lou Geng, Li Yang, Miao Yu, Zhilei Bu, Yanjie Ji, Huizhuang Shan, Zhihui Zou, Ligen Liu, Yingying Wang, Youping Zhang, Yin Tong, Hanzhang Xu, Hu Lei, Wei Liu, Fenghou Gao, Yingli Wu.

  Rationale: In multiple myeloma (MM), the activities of non-homologous end joining (NHEJ) and homologous recombination repair (HR) are increased compared with healthy controls. Whether and how IKZF1 as an enhancer of MM participates in the DNA repair pathway of tumor cells remains elusive. Methods: We used an endonuclease AsiSI-based system and quantitative chromatin immunoprecipitation assay (qChIP) analysis to test whether IKZF1 is involved in DNA repair. Immunopurification and mass spectrometric (MS) analysis were performed in MM1.S cells to elucidate the molecular mechanism that IKZF1 promotes DNA damage repair. The combination effect of lenalidomide or USP7 inhibitor with PARP inhibitor on cell proliferation was evaluated using MM cells in vitro and in vivo. Results: We demonstrate that IKZF1 specifically promotes homologous recombination DNA damage repair in MM cells, which is regulated by its interaction with CtIP and USP7. In this process, USP7 could regulate the stability of IKZF1 through its deubiquitinating activity. The N-terminal zinc finger domains of IKZF1 and the ubiquitin-like domain of USP7 are necessary for their interaction. Furthermore, targeted inhibition IKZF1 or USP7 could sensitize MM cells to PARP inhibitor treatment in vitro and in vivo. Conclusions: Our findings identify USP7 as a deubiquitinating enzyme for IKZF1 and uncover a new function of IKZF1 in DNA damage repair. In translational perspective, the combination inhibition of IKZF1 or USP7 with PARP inhibitor deserves further evaluation in clinical trials for the treatment of MM.

Keywords:  DNA repair; HR; IKZF1; USP7; multiple myeloma

DOI:  https://doi.org/10.7150/ijbs.70960
Mol Cell Oncol. 2021 ;8(6): 2007029

Turning end-joining upside down in mitosis.

Marta Llorens-Agost, Michael Ensminger, Hang Phuong Le, Wolf-Dietrich Heyer, Markus Löbrich.

  How cells deal with DNA breaks during mitosis is not well understood. While canonical non-homologous end-joining predominates in interphase, it is inhibited in mitosis to avoid telomere fusions. DNA polymerase θ mediated end-joining appears to be repressed in interphase, but promotes break repair in mitosis. The nature and induction time of breaks might determine their fate during mitosis.

Keywords:  DNA repair; TMEJ; c-NHEJ; mitosis; tethering

DOI:  https://doi.org/10.1080/23723556.2021.2007029
Int J Mol Sci. 2022 Apr 06. pii: 4060. [Epub ahead of print]23(7):

BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents.

Zida Zhu, Taisuke Kitano, Masami Morimatsu, Arisa Tanaka, Ryo Morioka, Xianghui Lin, Koichi Orino, Yasunaga Yoshikawa.

  Breast cancer type 2 susceptibility (BRCA2) protein is crucial for initiating DNA damage repair after chemotherapy with DNA interstrand crosslinking agents or X-ray irradiation, which induces DNA double-strand breaks. BRCA2 contains a C-terminal RAD51-binding domain (CTRBD) that interacts with RAD51 oligomer-containing nucleofilaments. In this study, we investigated CTRBD expression in cells exposed to X-ray irradiation and mitomycin C treatment. Surprisingly, BRCA2 CTRBD expression in HeLa cells increased their resistance to X-ray irradiation and mitomycin C. Under endogenous BRCA2 depletion using shRNA, the sensitivities of the BRCA2-depleted cells with and without the CTRBD did not significantly differ. Thus, the resistance to X-ray irradiation conferred by an exogenous CTRBD required endogenous BRCA2 expression. BRCA2 CTRBD-expressing cells demonstrated effective RAD51 foci formation and increased homologous recombination efficiency, but not nonhomologous end-joining efficiency. To the best of our knowledge, our study is the first to report the ability of the BRCA2 functional domain to confer resistance to X-ray irradiation and mitomycin C treatment by increased homologous recombination efficiency. Thus, this peptide may be useful for protecting cells against X-ray irradiation or chemotherapeutic agents.

Keywords:  BRCA2; C-terminal RAD51-binding domain; DNA damage repair; X-ray irradiation; homologous recombination repair; mitomycin C

DOI:  https://doi.org/10.3390/ijms23074060
Int J Cancer. 2022 Apr 12.

A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis.

Umar Khalid, Milena Simovic, Linda A Hammann, Murat Iskar, John Kl Wong, Rithu Kumar, Manfred Jugold, Martin Sill, Michiel Bolkestein, Thorsten Kolb, Michaela Hergt, Frauke Devens, Jonas Ecker, Marcel Kool, Till Milde, Frank Westermann, Axel Benner, Joe Lewis, Sascha Dietrich, Stefan M Pfister, Peter Lichter, Marc Zapatka, Aurélie Ernst.

  Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses, and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

Keywords:  Chromothripsis; HDAC inhibitor; PARP inhibitor; Synergy; Synthetic lethality

DOI:  https://doi.org/10.1002/ijc.34027
BMC Biol. 2022 Apr 13. 20(1): 87

Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication.

Steff Horemans, Matthaios Pitoulias, Alexandria Holland, Emilie Pateau, Christophe Lechaplais, Dariy Ekaterina, Alain Perret, Panos Soultanas, Laurent Janniere.

   BACKGROUND: In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system Bacillus subtilis.
RESULTS: On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions.
CONCLUSIONS: We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis.

Keywords:  Allosteric regulation; Cell cycle; Central carbon metabolism; Glycolytic enzymes; PEPut domain; Protein phosphorylation; Replication enzymes; Replication timing; Signaling

DOI:  https://doi.org/10.1186/s12915-022-01278-3
Signal Transduct Target Ther. 2022 Apr 13. 7(1): 102

LSD1 is required for euchromatic origin firing and replication timing.

Yue Wang, Yunchao Huang, Edith Cheng, Xinhua Liu, Yu Zhang, Jianguo Yang, Jordan T F Young, Grant W Brown, Xiaohan Yang, Yongfeng Shang.

The chromatin-based rule governing the selection and activation of replication origins remains to be elucidated. It is believed that DNA replication initiates from open chromatin domains; thus, replication origins reside in open and active chromatin. However, we report here that lysine-specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation favoring chromatin condensation, interacts with the DNA replication machinery in human cells. We find that LSD1 level peaks in early S phase, when it is required for DNA replication by facilitating origin firing in euchromatic regions. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for the pre-replicative complex (pre-RC) binding. Remarkably, LSD1 deficiency leads to a genome-wide switch of replication from early to late. We show that LSD1-engaged DNA replication is mechanistically linked to the loading of TopBP1-Interacting Checkpoint and Replication Regulator (TICRR) onto the pre-RC and subsequent recruitment of CDC45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing, highlighting the importance of epigenetic regulation in the activation of replication origins. As selective inhibitors of LSD1 are being exploited as potential cancer therapeutics, our study supports the importance of leveraging an appropriate level of LSD1 to curb the side effects of anti-LSD1 therapy.

DOI: https://doi.org/10.1038/s41392-022-00927-x
Sci Rep. 2022 Apr 13. 12(1): 6169

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids.

Sribalasubashini Muralimanoharan, Ross Shamby, Nicholas Stansbury, Robert Schenken, Barbara de la Pena Avalos, Samin Javanmardi, Eloise Dray, Patrick Sung, Thomas G Boyer.

Uterine fibroid (UF) driver mutations in Mediator complex subunit 12 (MED12) trigger genomic instability and tumor development through unknown mechanisms. Herein, we show that MED12 mutations trigger aberrant R-loop-induced replication stress, suggesting a possible route to genomic instability and a novel therapeutic vulnerability in this dominant UF subclass. Immunohistochemical analyses of patient-matched tissue samples revealed that MED12 mutation-positive UFs, compared to MED12 mutation-negative UFs and myometrium, exhibited significantly higher levels of R-loops and activated markers of Ataxia Telangiectasia and Rad3-related (ATR) kinase-dependent replication stress signaling in situ. Single molecule DNA fiber analysis revealed that primary cells from MED12 mutation-positive UFs, compared to those from patient-matched MED12 mutation-negative UFs and myometrium, exhibited defects in replication fork dynamics, including reduced fork speeds, increased and decreased numbers of stalled and restarted forks, respectively, and increased asymmetrical bidirectional forks. Notably, these phenotypes were recapitulated and functionally linked in cultured uterine smooth muscle cells following chemical inhibition of Mediator-associated CDK8/19 kinase activity that is known to be disrupted by UF driver mutations in MED12. Thus, Mediator kinase inhibition triggered enhanced R-loop formation and replication stress leading to an S-phase cell cycle delay, phenotypes that were rescued by overexpression of the R-loop resolving enzyme RNaseH. Altogether, these findings reveal MED12-mutant UFs to be uniquely characterized by aberrant R-loop induced replication stress, suggesting a possible basis for genomic instability and new avenues for therapeutic intervention that involve the replication stress phenotype in this dominant UF subtype.

DOI: https://doi.org/10.1038/s41598-022-10188-x
NAR Cancer. 2022 Jun;4(2): zcac012

High replication stress and limited Rad51-mediated DNA repair capacity, but not oxidative stress, underlie oligodendrocyte precursor cell radiosensitivity.

N Daniel Berger, Peter M Brownlee, Myra J Chen, Hali Morrison, Katalin Osz, Nicolas P Ploquin, Jennifer A Chan, Aaron A Goodarzi.

Cranial irradiation is part of the standard of care for treating pediatric brain tumors. However, ionizing radiation can trigger serious long-term neurologic sequelae, including oligodendrocyte and brain white matter loss enabling neurocognitive decline in children surviving brain cancer. Oxidative stress-mediated oligodendrocyte precursor cell (OPC) radiosensitivity has been proposed as a possible explanation for this. Here, however, we demonstrate that antioxidants fail to improve OPC viability after irradiation, despite suppressing oxidative stress, suggesting an alternative etiology for OPC radiosensitivity. Using systematic approaches, we find that OPCs have higher irradiation-induced and endogenous γH2AX foci compared to neural stem cells, neurons, astrocytes and mature oligodendrocytes, and these correlate with replication-associated DNA double strand breakage. Furthermore, OPCs are reliant upon ATR kinase and Mre11 nuclease-dependent processes for viability, are more sensitive to drugs increasing replication fork collapse, and display synthetic lethality with PARP inhibitors after irradiation. This suggests an insufficiency for homology-mediated DNA repair in OPCs-a model that is supported by evidence of normal RPA but reduced RAD51 filament formation at resected lesions in irradiated OPCs. We therefore propose a DNA repair-centric mechanism of OPC radiosensitivity, involving chronically-elevated replication stress combined with 'bottlenecks' in RAD51-dependent DNA repair that together reduce radiation resilience.

DOI: https://doi.org/10.1093/narcan/zcac012
Chem Biol Interact. 2022 Apr 12. pii: S0009-2797(22)00143-0. [Epub ahead of print] 109938

Cinobufagin restrains the growth and triggers DNA damage of human hepatocellular carcinoma cells via proteasome-dependent degradation of thymidylate synthase.

Ailin Yang, Qi Wu, Qimei Chen, Jingyi Yang, Haoran Li, Yufan Tao, Anmei Wang, Yaxue Sun, Jiayu Zhang.

  Anti-tumor candidate drugs from natural products have gained increasing attention. Cinobufagin is a natural product isolated from the traditional chinese medicine Chansu. Herein, we find that cinobufagin inhibits the proliferation and colony-forming ability of human hepatoma HepG2 and SK-HEP-1 cells. Furthermore, cinobufagin induces G2-phase cell cycle arrest and DNA damage in cancer cells. Thymidylate synthase (TYMS), the major target of chemotherapeutic drugs 5-FU or other fluoropyrimidines, which catalyzes the conversion of dUMP to dTMP and provides the sole de novo source of thymidylate for DNA synthesis. We demonstrate that cinobufagin suppresses TYMS expression via proteasome-dependent degradation in human hepatoma cells, moreover, depletion of TYMS restrains the proliferation and colony formation of tumor cells, and the results of western blotting and immunofluorescence assay indicate DNA damage is induced in tumor cells transfected with TYMS-targeting siRNA (siTYMS), additionally, knockdown of TYMS enhances the inhibitory eﬀect of cinobufagin on the proliferative potential of HepG2 and SK-HEP-1 cells. It is worth noting that cinobufagin in combination with 5-FU exhibits antagonism or synergism combined effects on the proliferation of human hepatoma cells, indicating that Chansu-related preparations such as cinobufacini injection and Huachansu capsules applied to clinical practice should be used with caution in combination with 5-FU for the treatment of liver cancer. Collectively, cinobufagin exerts good anti-hepatoma activity through inhibition of growth and induction of DNA damage by promoting the degradation of TYMS. Our results provide evidence that cinobufagin might be a potential agent for the treatment of cancers such as hepatocellular carcinoma. It can also promote the scientific development of Chansu, and has great significance for enriching the application of TCM in the development of new anti-tumor drugs.

Keywords:  Cinobufagin; DNA damage; Hepatocellular carcinoma; Thymidylate synthase

DOI:  https://doi.org/10.1016/j.cbi.2022.109938
PLoS Genet. 2022 Apr 13. 18(4): e1010079

The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase.

Laura E Knighton, Nitika, Siddhi Omkar, Andrew W Truman.

The Hsp70 family of molecular chaperones is well-conserved and expressed in all organisms. In budding yeast, cells express four highly similar cytosolic Hsp70s Ssa1, 2, 3 and 4 which arose from gene duplication. Ssa1 and 2 are constitutively expressed while Ssa3 and 4 are induced upon heat shock. Recent evidence suggests that despite their amino acid similarity, these Ssas have unique roles in the cell. Here we examine the relative importance of Ssa1-4 in the regulation of the enzyme ribonucleotide reductase (RNR). We demonstrate that cells expressing either Ssa3 or Ssa4 as their sole Ssa are compromised for their resistance to DNA damaging agents and activation of DNA damage response (DDR)-regulated transcription. In addition, we show that the steady state levels and stability of RNR small subunits Rnr2 and Rnr4 are reduced in Ssa3 or Ssa4-expressing cells, a result of decreased Ssa-RNR interaction. Interaction between the Hsp70 co-chaperone Ydj1 and RNR is correspondingly decreased in cells only expressing Ssa3 and 4. Through studies of Ssa2/4 domain swap chimeras, we determined that the C-terminal domain of Ssas are the source of this functional specificity. Taking together, our work suggests a distinct role for Ssa paralogs in regulating DNA replication mediated by C-terminus sequence variation.

DOI: https://doi.org/10.1371/journal.pgen.1010079
Cell Rep. 2022 Apr 12. pii: S2211-1247(22)00449-1. [Epub ahead of print]39(2): 110691

TLR8 is activated by 5'-methylthioinosine, a Plasmodium falciparum-derived intermediate of the purine salvage pathway.

Gabriele Köllisch, Francisco Venegas Solis, Hannah-Lena Obermann, Jeannine Eckert, Thomas Müller, Tim Vierbuchen, Thomas Rickmeyer, Simon Muche, Jude M Przyborski, Holger Heine, Andreas Kaufmann, Stefan Baumeister, Klaus Lingelbach, Stefan Bauer.

  The innate immune recognition of the malaria-causing pathogen Plasmodium falciparum (P. falciparum) is not fully explored. Here, we identify the nucleoside 5'-methylthioinosine (MTI), a Plasmodium-specific intermediate of the purine salvage pathway, as a pathogen-derived Toll-like receptor 8 (TLR8) agonist. Co-incubation of MTI with the TLR8 enhancer poly(dT) as well as synthetic or P. falciparum-derived RNA strongly increase its stimulatory activity. Of note, MTI generated from methylthioadenosine (MTA) by P. falciparum lysates activates TLR8 when MTI metabolism is inhibited by immucillin targeting the purine nucleoside phosphorylase (PfPNP). Importantly, P. falciparum-infected red blood cells incubated with MTI or cultivated with MTA and immucillin lead to TLR8-dependent interleukin-6 (IL-6) production in human monocytes. Our data demonstrate that the nucleoside MTI is a natural human TLR8 ligand with possible in vivo relevance for innate sensing of P. falciparum.

Keywords:  5ʹ-methylthioadenosine; 5ʹ-methylthioinosine; CP: Immunology; CU-CPT9a; MTA; MTI; PBMC; Plasmodium falciparum; TLR8; immucillin; monocytes

DOI:  https://doi.org/10.1016/j.celrep.2022.110691
Cancer Gene Ther. 2022 Apr 14.

SHMT2 promotes cell viability and inhibits ROS-dependent, mitochondrial-mediated apoptosis via the intrinsic signaling pathway in bladder cancer cells.

Yun Zhang, Zhe Liu, Xueliang Wang, Hui Jian, Haihan Xiao, Tingyi Wen.

Mitochondrial serine hydroxymethyltransferase (SHMT2) catalyzes the conversion of serine to glycine and concomitantly produces one-carbon units to support cell growth and is upregulated in various cancer cells. SHMT2 knockdown triggers cell apoptosis; however, the detailed mechanism of apoptosis induced by SHMT2 inactivation remains unknown. Here, we demonstrate that SHMT2 supports the proliferation of bladder cancer (BC) cells by maintaining redox homeostasis. SHMT2 knockout decreased the pools of purine and one-carbon units and delayed cell cycle progression in a manner that was rescued by formate, demonstrating that SHMT2-mediated one-carbon units are essential for BC cell proliferation. SHMT2 deficiency promoted the accumulation of intracellular reactive oxygen species (ROS) by decreasing the NADH/NAD+, NADPH/NADP+, and GSH/GSSG ratios, leading to a loss in mitochondrial membrane potential, release of cytochrome c, translocation of Bcl-2 family protein and activation of caspase-3. Notably, blocking ROS production with the one-carbon donor formate and the ROS scavenger N-acetyl-cysteine (NAC) effectively rescued SHMT2 deficiency-induced cell apoptosis via the intrinsic signaling pathway. Treatment with the SHMT inhibitor SHIN1 resulted in a significant inhibitory effect on cell proliferation and induced cell apoptosis. Formate and NAC rescued SHIN1-induced cell apoptosis. Our findings reveal an important mechanism by which the loss of SHMT2 triggers ROS-dependent, mitochondrial-mediated apoptosis, which gives insight into the link between serine metabolism and cell apoptosis and provides a promising target for BC treatment and drug discovery.

DOI: https://doi.org/10.1038/s41417-022-00470-5
Chem Commun (Camb). 2022 Apr 13.

Multiple-turnover single nucleotide primer extension reactions to detect 8-oxo-2'-deoxyguanosine in DNA.

Yoshiya Kikukawa, Ryoji Kawazoe, Ryo Miyahara, Takato Sakurada, Yusuke Nagata, Shigeki Sasaki, Yosuke Taniguchi.

The identification of the position of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA is important to clarify the pathogenesis of many diseases. We herein developed a purine-1,3-diazaphenoxazine triphosphate (dPdapTP) and described the first example of detecting the presence of 8-oxo-dG by amplifying it several hundred times after the multiple-turnover single nucleotide primer extension reactions.

DOI: https://doi.org/10.1039/d2cc01372j
J Clin Lab Anal. 2022 Apr 11. e24416

Inosine monophosphate dehydrogenase type1 sustains tumor growth in hepatocellular carcinoma.

Xiaoyuan Jia, Yao Liu, Yan Cheng, Yin Wang, Hui Kang, Zhongren Ma, Kan Chen.

   BACKGROUND: Inosine monophosphate dehydrogenase (IMPDH) is the key enzyme in the biosynthesis of purine nucleotides. IMPDH1 and IMPDH2 are the two isoforms of IMPDH and they share 84% amino acid similarity and virtually indistinguishable catalytic activity. Although high expression of IMPDH2 has been reported in various cancers, the roles of IMPDH1 in hepatocellular carcinoma (HCC) are largely unknown.
METHODS: The expression and the clinical relevance of IMPDH1 in 154 HCC patients were detected by immunohistochemistry analysis. The stable IMPDH1 knockdown HuH7 cells were established by lentiviral RNAi approach. The single cell proliferation was detected by colony-forming unit assay. The tumor initiation and growth ability were measured by using xenograft tumor model in immunodeficient mice. The effect of IMPDH1 on cellular signaling pathways was analyzed by genome-wide transcriptomic profiling.
RESULTS: The expression of IMPDH1 is upregulated in tumor tissue compared with adjacent liver tissue, and higher expression of IMPDH1 is associated with better patient cumulative survival. In experimental models, loss of IMPDH1 in HCC cells inhibits the ability of single cell colony formation in vitro, and reduces the efficiency of tumor initiation and growth in immunodeficient mice. Consistently, loss of IMPDH1 results in distinct alterations of signaling pathways revealed by genome-wide transcriptomic profiling.
CONCLUSION: IMPDH1 sustains HCC growth and progression.

Keywords:  animal model; hepatocellular carcinoma; inosine monophosphate dehydrogenase; transcriptomic profiling

DOI:  https://doi.org/10.1002/jcla.24416
Mol Syndromol. 2022 Feb;13(2): 85-87

Deep White Matter Cysts in a Patient with Aicardi-Goutières Syndrome and SAMHD1 Variants.

Martin Poot.

DOI: https://doi.org/10.1159/000522096