bims-numges 2022-01-16 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–01–16
thirty papers selected by
Sean Rudd, Karolinska Institutet

Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells.
Chl1 helicase controls replication fork progression by regulating dNTP pools.
Combined inactivation of CTPS1 and ATR is synthetically lethal to MYC-overexpressing cancer cells.
Mechanism for inverted-repeat recombination induced by a replication fork barrier.
Linking DNA repair and cell cycle progression through serine ADP-ribosylation of histones.
Interaction of Wwox with Brca1 and associated complex proteins prevents premature resection at double-strand breaks and aberrant homologous recombination.
CANCER-ASSOCIATED SF3B1 MUTATIONS CONFER A BRCA-LIKE CELLULAR PHENOTYPE AND SYNTHETIC LETHALITY TO PARP INHIBITORS.
BRCA1 deficiency specific base substitution mutagenesis is dependent on translesion synthesis and regulated by 53BP1.
Metabolic remodeling maintains a reducing environment for rapid activation of the yeast DNA replication checkpoint.
Roles of the SUMO-related enzymes, PIAS1, PIAS4, and RNF4, in DNA double-strand break repair by homologous recombination.
Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη.
Disorders of purine biosynthesis metabolism.
Genome-wide sequencing analysis of Sgs1, Exo1, Rad51, and Srs2 in DNA repair by homologous recombination.
RECON syndrome is a genome instability disorder caused by mutations in the DNA helicase RECQL1.
Stimulation of adaptive gene amplification by origin firing under replication fork constraint.
The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin.
Discovery of small-molecule ATR inhibitors for potential cancer treatment: a patent review from 2014 to present.
BRD4 inhibition induces synthetic lethality in ARID2-deficient hepatocellular carcinoma by increasing DNA damage.
53BP1-shieldin-dependent DSB processing in BRCA1-deficient cells requires CST-Polα-primase fill-in synthesis.
SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia.
Shift in G1-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation.
SLFN11 biomarker status predicts response to lurbinectedin as a single agent and in combination with ATR inhibition in small cell lung cancer.
Checkpoint Kinase 1 (Chk1) inhibition fails to activate the Stimulator of Interferon Genes (STING) innate immune signalling in a human coculture cancer system.
Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.
IMPDH1 retinal variants control filament architecture to tune allosteric regulation.
RAD50 deficiency is a predictor of platinum sensitivity in sporadic epithelial ovarian cancers.
Genetic alterations of the SUMO isopeptidase SENP6 drive lymphomagenesis and genetic instability in diffuse large B-cell lymphoma.
Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression.
Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer.
Phosphoribosyl Pyrophosphate Amido Transferase: A New Prognostic Biomarker for Hepatocellular Carcinoma.

Cell Rep. 2022 Jan 11. pii: S2211-1247(21)01745-9. [Epub ahead of print]38(2): 110236

Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells.

Evan R Abt, Thuc M Le, Amanda M Dann, Joseph R Capri, Soumya Poddar, Vincent Lok, Luyi Li, Keke Liang, Amanda L Creech, Khalid Rashid, Woosuk Kim, Nanping Wu, Jing Cui, Arthur Cho, Hailey Rose Lee, Ethan W Rosser, Jason M Link, Johannes Czernin, Ting-Ting Wu, Robert Damoiseaux, David W Dawson, Timothy R Donahue, Caius G Radu.

  We determine that type I interferon (IFN) response biomarkers are enriched in a subset of pancreatic ductal adenocarcinoma (PDAC) tumors; however, actionable vulnerabilities associated with IFN signaling have not been systematically defined. Integration of a phosphoproteomic analysis and a chemical genomics synergy screen reveals that IFN activates the replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) in PDAC cells and sensitizes them to ATR inhibitors. IFN triggers cell-cycle arrest in S-phase, which is accompanied by nucleotide pool insufficiency and nucleoside efflux. In combination with IFN, ATR inhibitors induce lethal DNA damage and downregulate nucleotide biosynthesis. ATR inhibition limits the growth of PDAC tumors in which IFN signaling is driven by stimulator of interferon genes (STING). These results identify a cross talk between IFN, DNA replication stress response networks, and nucleotide metabolism while providing the rationale for targeted therapeutic interventions that leverage IFN signaling in tumors.

Keywords:  STING; interferon; nucleotide metabolism; pancreas cancer; replication stress

DOI:  https://doi.org/10.1016/j.celrep.2021.110236
Life Sci Alliance. 2022 Apr;pii: e202101153. [Epub ahead of print]5(4):

Chl1 helicase controls replication fork progression by regulating dNTP pools.

Amandine Batté, Sophie C van der Horst, Mireille Tittel-Elmer, Su Ming Sun, Sushma Sharma, Jolanda van Leeuwen, Andrei Chabes, Haico van Attikum.

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

DOI: https://doi.org/10.26508/lsa.202101153
Cancer Res. 2022 Jan 12. pii: canres.1707.2021. [Epub ahead of print]

Combined inactivation of CTPS1 and ATR is synthetically lethal to MYC-overexpressing cancer cells.

Zhe Sun, Ziheng Zhang, Qiao-Qi Wang, Ji-Long Liu.

The "undruggable" oncogene MYC supports cancer cell proliferation and survival through parallel induction of multiple anabolic processes. Here we find that inhibiting CTP synthase (CTPS) selectively decreases cell viability and induces DNA replication stress in MYC-overexpressing cells. MYC-driven rRNA synthesis caused the selective DNA replication stress upon CTPS inhibition. Combined inhibition of CTPS and ataxia telangiectasia and Rad3-related protein (ATR) was synthetically lethal in MYC-overexpressing cells, promoting cell death in vitro and decreasing tumor growth in vivo. Unexpectedly, interfering with CTPS1 but not CTPS2 was required to induce replication stress in MYC-deregulated cancer cells and consequent cell death in the presence of an ATR inhibitor. These results highlight a specific and key role of CTPS1 in MYC-driven cancer, suggesting that selectively inhibiting CTPS1 in combination with ATR could be a promising strategy to combat disease progression.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1707
Nat Commun. 2022 Jan 10. 13(1): 32

Mechanism for inverted-repeat recombination induced by a replication fork barrier.

Léa Marie, Lorraine S Symington.

Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. To gain insight into the mechanism of recombination between repeated sequences in the context of replication stress, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Our study reveals that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 nucleases. Physical analysis of the replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats that can robustly generate chromosome rearrangements.

DOI: https://doi.org/10.1038/s41467-021-27443-w
Nat Commun. 2022 Jan 13. 13(1): 185

Linking DNA repair and cell cycle progression through serine ADP-ribosylation of histones.

Julien Brustel, Tetsuya Muramoto, Kazuki Fumimoto, Jessica Ellins, Catherine J Pears, Nicholas D Lakin.

Although serine ADP-ribosylation (Ser-ADPr) by Poly(ADP-ribose)-polymerases is a cornerstone of the DNA damage response, how this regulates DNA repair and genome stability is unknown. Here, we exploit the ability to manipulate histone genes in Dictyostelium to identify that ADPr of the histone variant H3b at S10 and S28 maintains genome stability by integrating double strand break (DSB) repair with mitotic entry. Given the critical requirement for mitotic H3S10/28 phosphorylation, we develop separation of function mutations that maintain S10 phosphorylation whilst disrupting ADPr. Mechanistically, this reveals a requirement for H3bS10/28 ADPr in non-homologous end-joining by recruiting Ku to DSBs. Moreover, this also identifies H3bS10/S28 ADPr is critical to prevent premature mitotic entry with unresolved DNA damage, thus maintaining genome stability. Together, these data demonstrate how serine ADPr of histones coordinates DNA repair with cell cycle progression to maintain genome stability.

DOI: https://doi.org/10.1038/s41467-021-27867-4
DNA Repair (Amst). 2021 Dec 26. pii: S1568-7864(21)00220-2. [Epub ahead of print]110 103264

Interaction of Wwox with Brca1 and associated complex proteins prevents premature resection at double-strand breaks and aberrant homologous recombination.

Dongju Park, Mehdi Gharghabi, Morgan S Schrock, Rebecca Plow, Teresa Druck, Charles Yungvirt, C Marcelo Aldaz, Kay Huebner.

  Down regulation of Wwox protein expression occurs in many cancers, contributing to insensitivity to ionizing radiation (IR) and platinum drug treatments. Patients with reduced Wwox expression in their cancer tissue show decreased overall survival following these treatments, in accord with our earlier finding that reduced Wwox protein expression in cancers is associated with changes in choice of DNA double-strand break (DSB) repair pathway. Our current investigation of mechanisms underlying the initial choice of repair by homologous recombination/single-strand annealing (HR/SSA) in Wwox-deficient cells, showed immediate DNA end-resection at DSBs following IR, abrogating initial repair by the expected non-homologous end-joining (NHEJ) pathway. Mechanisms supporting the expected choice of DSB repair by NHEJ in Wwox-sufficient cells are: 1) direct recruitment of Wwox protein binding to Brca1 through the Brca1 981PPLF984 Wwox-binding motif; 2) possible Wwox blocking of Brca1-Rad50 interaction and of Brca1 activation by Chk2 phosphorylation of Brca1 S988; 3) Wwox suppression of Brca1 interaction with the B and C complex proteins, Brip1 and CtIP, thereby delaying the process of DSB end-resection post-IR. Wwox deficiency, instead, leads to early formation of the Brca1-CtIP/MRN complex at induced DSBs, stimulating immediate post-IR end-resection. This premature resection at DNA DSBs leads to inappropriate HR/SSA repair not restricted to late S/G2 cell cycle phases, and increases mutations in genomes of radiation or platinum-resistant colonies. Prevention of premature initiation of end-resection, by combining Chk2 inhibition with IR or carboplatin treatment, successfully sensitized IR and platinum-resistant Wwox-deficient cells by synthetic lethality, but did not alter response of Wwox-sufficient cells. Our results establish Wwox as a biomarker for treatment response and provide potential targets, such as Chk2, for reversal of treatment resistance.

Keywords:  Chk2 inhibition; IR and platinum treatment resistance; Premature end-resection at DSBs; Synthetic lethality; Wwox deficiency

DOI:  https://doi.org/10.1016/j.dnarep.2021.103264
Cancer Res. 2022 Jan 13. pii: canres.1843.2021. [Epub ahead of print]

CANCER-ASSOCIATED SF3B1 MUTATIONS CONFER A BRCA-LIKE CELLULAR PHENOTYPE AND SYNTHETIC LETHALITY TO PARP INHIBITORS.

Katrina M Lappin, Eliana M Barros, Satpal S Jhujh, Gareth W Irwin, Hayley McMillan, Fabio G Liberante, Cheryl Latimer, Melissa J LaBonte, Ken I Mills, D Paul Harkin, Grant S Stewart, Kienan I Savage.

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionising radiation and a variety of chemotherapeutic agents, including PARP inhibitors. Additionally, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation and defective replication fork restart. Taken together, these data suggest that tumour-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA damaging agents and PARP inhibitors, which can be exploited therapeutically.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1843
Nat Commun. 2022 Jan 11. 13(1): 226

BRCA1 deficiency specific base substitution mutagenesis is dependent on translesion synthesis and regulated by 53BP1.

Dan Chen, Judit Z Gervai, Ádám Póti, Eszter Németh, Zoltán Szeltner, Bernadett Szikriszt, Zsolt Gyüre, Judit Zámborszky, Marta Ceccon, Fabrizio d'Adda di Fagagna, Zoltan Szallasi, Andrea L Richardson, Dávid Szüts.

Defects in BRCA1, BRCA2 and other genes of the homology-dependent DNA repair (HR) pathway cause an elevated rate of mutagenesis, eliciting specific mutation patterns including COSMIC signature SBS3. Using genome sequencing of knock-out cell lines we show that Y family translesion synthesis (TLS) polymerases contribute to the spontaneous generation of base substitution and short insertion/deletion mutations in BRCA1 deficient cells, and that TLS on DNA adducts is increased in BRCA1 and BRCA2 mutants. The inactivation of 53BP1 in BRCA1 mutant cells markedly reduces TLS-specific mutagenesis, and rescues the deficiency of template switch-mediated gene conversions in the immunoglobulin V locus of BRCA1 mutant chicken DT40 cells. 53BP1 also promotes TLS in human cellular extracts in vitro. Our results show that HR deficiency-specific mutagenesis is largely caused by TLS, and suggest a function for 53BP1 in regulating the choice between TLS and error-free template switching in replicative DNA damage bypass.

DOI: https://doi.org/10.1038/s41467-021-27872-7
EMBO J. 2022 Jan 14. e108290

Metabolic remodeling maintains a reducing environment for rapid activation of the yeast DNA replication checkpoint.

Lili Li, Jie Wang, Zijia Yang, Yiling Zhao, Hui Jiang, Luguang Jiang, Wenya Hou, Risheng Ye, Qun He, Martin Kupiec, Brian Luke, Qinhong Cao, Zhi Qi, Zhen Li, Huiqiang Lou.

  Nucleotide metabolism fuels normal DNA replication and is also primarily targeted by the DNA replication checkpoint when replication stalls. To reveal a comprehensive interconnection between genome maintenance and metabolism, we analyzed the metabolomic changes upon replication stress in the budding yeast S. cerevisiae. We found that upon treatment of cells with hydroxyurea, glucose is rapidly diverted to the oxidative pentose phosphate pathway (PPP). This effect is mediated by the AMP-dependent kinase, SNF1, which phosphorylates the transcription factor Mig1, thereby relieving repression of the gene encoding the rate-limiting enzyme of the PPP. Surprisingly, NADPH produced by the PPP is required for efficient recruitment of replication protein A (RPA) to single-stranded DNA, providing the signal for the activation of the Mec1/ATR-Rad53/CHK1 checkpoint signaling kinase cascade. Thus, SNF1, best known as a central energy controller, determines a fast mode of replication checkpoint activation through a redox mechanism. These findings establish that SNF1 provides a hub with direct links to cellular metabolism, redox, and surveillance of DNA replication in eukaryotes.

Keywords:  DNA replication stress; carbon metabolism; cell cycle checkpoints; genome stability; reductive/oxidative (redox)

DOI:  https://doi.org/10.15252/embj.2021108290
Biochem Biophys Res Commun. 2021 Dec 30. pii: S0006-291X(21)01750-2. [Epub ahead of print]591 95-101

Roles of the SUMO-related enzymes, PIAS1, PIAS4, and RNF4, in DNA double-strand break repair by homologous recombination.

Moe Moe Han, Miyako Hirakawa, Motohiro Yamauchi, Naoki Matsuda.

  Post-translational modification of proteins by small ubiquitin-like modifier (SUMO) is known to be involved in a variety of cellular events. This modification, called SUMOylation, is carried out by the E1 activating enzyme, the E2 conjugating enzyme, and multiple E3 ligases. Previous studies have demonstrated that the SUMO E3 ligases, protein inhibitors of activated STAT 1 (PIAS1) and 4 (PIAS4), and the SUMO-targeted ubiquitin ligase, RING finger protein 4 (RNF4), play important roles in the repair of DNA double-strand breaks (DSBs). However, the mechanism by which these SUMO-related enzymes promote DSB repair is still poorly understood. In the present study, we focused on homologous recombination (HR), the most accurate DSB repair pathway, and aimed to elucidate the mechanism by which PIAS1, PIAS4, and RNF4 promote HR. In γ-ray-irradiated normal human fibroblasts, DSB end resection and RAD51 loading, the two essential steps of HR, were significantly impaired by small interfering RNA (siRNA)-mediated depletion of PIAS1, PIAS4, or RNF4. The recruitment of BRCA1, a major HR factor, to DSB sites was reduced in cells depleted of these SUMO-related enzymes. Consistent with the role of BRCA1 in counteracting the p53-binding protein 1 (53BP1)-mediated resection blockade, 53BP1 depletion rescued the reduced resection and RAD51 loading in the cells depleted of PIAS1, PIAS4, or RNF4. Moreover, Rap1-interacting factor 1 (RIF1), a resection inhibitor downstream of 53BP1, became more abundant at DSBs when PIAS1, PIAS4, RNF4, or BRCA1 was depleted. Importantly, the concomitant depletion of BRCA1 with either one of the SUMO-related enzymes did not further increase RIF1 at DSBs, when compared to single depletion of BRCA1. Collectively, these results suggest that PIAS1, PIAS4, RNF4, and BRCA1 work epistatically to counteract 53BP1/RIF1-mediated resection blockade, thereby promoting resection.

Keywords:  DNA double-strand break; Homologous recombination; Resection; Small ubiquitin-like modifier

DOI:  https://doi.org/10.1016/j.bbrc.2021.12.099
Int J Mol Sci. 2021 Dec 26. pii: 230. [Epub ahead of print]23(1):

Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη.

Eva Balint, Ildiko Unk.

  DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.

Keywords:  RNA extension; enzyme kinetics; human polymerase η; manganese; translesion synthesis

DOI:  https://doi.org/10.3390/ijms23010230
Mol Genet Metab. 2021 Dec 30. pii: S1096-7192(21)01201-4. [Epub ahead of print]

Disorders of purine biosynthesis metabolism.

Joseph P Dewulf, Sandrine Marie, Marie-Cécile Nassogne.

  Purines are essential molecules that are components of vital biomolecules, such as nucleic acids, coenzymes, signaling molecules, as well as energy transfer molecules. The de novo biosynthesis pathway starts from phosphoribosylpyrophosphate (PRPP) and eventually leads to the synthesis of inosine monophosphate (IMP) by means of 10 sequential steps catalyzed by six different enzymes, three of which are bi-or tri-functional in nature. IMP is then converted into guanosine monophosphate (GMP) or adenosine monophosphate (AMP), which are further phosphorylated into nucleoside di- or tri-phosphates, such as GDP, GTP, ADP and ATP. This review provides an overview of inborn errors of metabolism pertaining to purine synthesis in humans, including either phosphoribosylpyrophosphate synthetase (PRS) overactivity or deficiency, as well as adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), and adenylosuccinate synthetase (ADSS) deficiencies. ITPase deficiency is being described as well. The clinical spectrum of these disorders is broad, including neurological impairment, such as psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscle presentations or consequences of hyperuricemia, such as gouty arthritis or kidney stones. Clinical signs are often nonspecific and, thus, overlooked. It is to be hoped that this is likely to be gradually overcome by using sensitive biochemical investigations and next-generation sequencing technologies.

Keywords:  ADSL; ADSSL1; ATIC; ITPase; PAICS; PRPP synthase; PRPS1; Purine; Purine de novo

DOI:  https://doi.org/10.1016/j.ymgme.2021.12.016
Cell Rep. 2022 Jan 11. pii: S2211-1247(21)01705-8. [Epub ahead of print]38(2): 110201

Genome-wide sequencing analysis of Sgs1, Exo1, Rad51, and Srs2 in DNA repair by homologous recombination.

Facundo Ramos, Laura Durán, Mar Sánchez, Adrián Campos, David Hernández-Villamor, Francisco Antequera, Andrés Clemente-Blanco.

  Homologous recombination is essential to maintain genome stability in response to DNA damage. Here, we have used genome-wide sequencing to quantitatively analyze at nucleotide resolution the dynamics of DNA end resection, re-synthesis, and gene conversion at a double-strand break. Resection initiates asymmetrically in an MRX-independent manner before proceeding steadily in both directions. Sgs1, Exo1, Rad51, and Srs2 differently regulate the rate and symmetry of early and late resection. Exo1 also ensures the coexistence of resection and re-synthesis, while Srs2 guarantees a constant and symmetrical DNA re-polymerization. Gene conversion is MMR independent, spans only a minor fraction of the resected region, and its unidirectionality depends on Srs2. Finally, these repair factors prevent the development of alterations remote from the DNA lesion, such as subtelomeric instability, duplication of genomic regions, and over-replication of Ty elements. Altogether, this approach allows a quantitative analysis and a direct genome-wide visualization of DNA repair by homologous recombination.

Keywords:  DNA repair; DNA synthesis; double-strand break; gene conversion; homologous recombination; resection

DOI:  https://doi.org/10.1016/j.celrep.2021.110201
J Clin Invest. 2022 Jan 13. pii: e147301. [Epub ahead of print]

RECON syndrome is a genome instability disorder caused by mutations in the DNA helicase RECQL1.

Bassam Abu-Libdeh, Satpal S Jhujh, Srijita Dhar, Joshua A Sommers, Arindam Datta, Gabriel Mc Longo, Laura J Grange, John J Reynolds, Sophie L Cooke, Gavin S McNee, Robert Hollingworth, Beth L Woodward, Anil N Ganesh, Stephen J Smerdon, Claudia M Nicolae, Karina Durlacher-Betzer, Vered Molho-Pessach, Abdulsalam Abu-Libdeh, Vardiella Meiner, George-Lucian Moldovan, Vassilis Roukos, Tamar Harel, Robert M Brosh, Grant S Stewart.

  Despite being the first homolog of the bacterial RecQ helicase to be identified in humans the function of RECQL1 remains poorly characterised. Furthermore, unlike other members of the human RECQ family of helicases, mutations in RECQL1 have not been associated with a genetic disease. Here we identify two families with a novel genome instability disorder, named RECON (RECql ONe) Syndrome caused by biallelic mutations in the RECQL gene. The affected individuals exhibit short stature, progeroid facial features, a hypoplastic nose, xeroderma and skin photosensitivity. Affected individuals were homozygous for the same missense mutation in RECQL1 (p.Ala459Ser) located within its zinc binding domain. Biochemical analysis of the mutant RECQL1 protein revealed that the p.A459S missense mutation compromised its ATPase, helicase and fork restoration activity, whilst its capacity to promote single-strand DNA annealing was largely unaffected. At the cellular level, this mutation in RECQL1 gave rise to a defect in the ability to repair DNA damage induced by exposure to topoisomerase poisons and a failure of DNA replication to progress efficiently in the presence of abortive topoisomerase lesions. Taken together, RECQL1 is the fourth member of the RecQ family of helicases to be associated with a human genome instability disorder.

Keywords:  Cell Biology; DNA repair; Genetic diseases; Genetic instability; Genetics

DOI:  https://doi.org/10.1172/JCI147301
Nucleic Acids Res. 2022 Jan 08. pii: gkab1257. [Epub ahead of print]

Stimulation of adaptive gene amplification by origin firing under replication fork constraint.

Alex J Whale, Michelle King, Ryan M Hull, Felix Krueger, Jonathan Houseley.

Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 can cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32 and Pif1. CUP1 amplification is also critically dependent on late-firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after activation, leaving a tract of H3K56-acetylated chromatin that promotes secondary recombination events during elongation after replication fork re-start events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

DOI: https://doi.org/10.1093/nar/gkab1257
Nat Cell Biol. 2022 Jan;24(1): 62-73

The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin.

Dragomir B Krastev, Shudong Li, Yilun Sun, Andrew J Wicks, Gwendoline Hoslett, Daniel Weekes, Luned M Badder, Eleanor G Knight, Rebecca Marlow, Mercedes Calvo Pardo, Lu Yu, Tanaji T Talele, Jiri Bartek, Jyoti S Choudhary, Yves Pommier, Stephen J Pettitt, Andrew N J Tutt, Kristijan Ramadan, Christopher J Lord.

Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors.

DOI: https://doi.org/10.1038/s41556-021-00807-6
Expert Opin Ther Pat. 2022 Jan 10.

Discovery of small-molecule ATR inhibitors for potential cancer treatment: a patent review from 2014 to present.

Suwen Hu, Zi Hui, Jilong Duan, Carmen Garrido, Tian Xie, Xiang-Yang Ye.

   INTRODUCTION: Ataxia telangiectasia and RAD3-related kinase (ATR) is one of the key PIKKs family members important for DNA damage response and repair pathways. Targeting ATR kinase for potential cancer therapy has attracted a great deal of attention to both pharmaceutical industries and academic community.
AREA COVERED: This article surveys the patents published since 2014 aiming to analyze the structural features of scaffolds and the patent space. It also discusses the recent clinical developments and provides perspectives on the challenges and the future directions.
EXPERT OPINION: ATR kinase appears to be a viable drug target for anticancer therapy. Similar to DNA-PK inhibitors, the clinical investigation of an ATRi employs both monotherapy and combination strategy. In the combination strategy, an ATRi is typically combined with a radiation or a targeted drug such as chemotherapy agent poly (ADP-ribose) polymerase (PARP) inhibitor, etc. Diverse structures comprising different scaffolds from mono-heteroaryl to bicyclic heteroaryl to tricyclic heteroaryl to macrocycle are capable to achieve good ATR inhibitory activity and good ATR selectivity over other closely related enzymes. There are eight ATR inhibitors currently being evaluated in clinics, with the hope to get approval in the near future.

Keywords:  DNA damage response (DDR); ataxia telangiectasia and RAD3-related kinase (ATR); cancer therapy; inhibitor; patent review

DOI:  https://doi.org/10.1080/13543776.2022.2027911
Oncogene. 2022 Jan 11.

BRD4 inhibition induces synthetic lethality in ARID2-deficient hepatocellular carcinoma by increasing DNA damage.

Dan-Dan He, Xue-Ying Shang, Na Wang, Guang-Xing Wang, Kun-Yan He, Lan Wang, Ze-Guang Han.

Hepatocellular carcinoma (HCC) has emerged as the third cause of cancer-related death owing to lacking effective systemic therapies. Genomic DNA sequencing revealed the high frequency of loss-of-function mutations in ARID2, which encodes a subunit of SWI/SNF chromatin remodeling complex, however, the therapeutic strategy for the HCC patients with ARID2 mutations is still completely unclear. In this study, we first performed a high-throughput screening approach using a compound library consisting of 2 180 FDA-approved drugs and other compounds, to elicit the potential drugs for synthetic lethality to target ARID2-deficient HCC cells. Interestingly, JQ1, a selective inhibitor of bromodomain protein BRD4, uniquely suppressed the growth of ARID2- deficient HCC cells. Next JQ1 is further confirmed to predominantly induce cell lethality upon ARID2 depletion through exacerbating DNA damage, especially double strand breaks (DSBs). Functional assays demonstrated that both BRD4 inhibition and ARID2 deficiency synergistically impede two main DNA damage repair pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ), through attenuating the transcription of BRCA1, RAD51, and 53BP1, which encode the core molecules responsible for DSB repair. Mechanistically, both ARID2 and BRD4 exert a synergistic effect for maintaining transcriptional enhancer-promoter loops of these genes within chromatin conformation. However, as both ARID2 and BRD4 are disrupted, the expression of these DNA repair-related genes in response to DNA damage are hindered, resulting in DSB accumulation and cell apoptosis. Taken together, this study discloses that BRD4 inhibition may induce synthetic lethality in ARID2-deficient HCC cells, which might provide a potential therapeutic strategy for HCC patients with ARID2 mutations.

DOI: https://doi.org/10.1038/s41388-022-02176-2
Nat Cell Biol. 2022 Jan;24(1): 51-61

53BP1-shieldin-dependent DSB processing in BRCA1-deficient cells requires CST-Polα-primase fill-in synthesis.

Zachary Mirman, Nanda Kumar Sasi, Ashleigh King, J Ross Chapman, Titia de Lange.

The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit single-stranded DNA at double-strand breaks (DSBs) by blocking resection and/or through CST-Polα-primase-mediated fill-in. We show that primase (like 53BP1-shieldin and CST-Polα) promotes radial chromosome formation in PARPi-treated BRCA1-deficient cells and demonstrate shieldin-CST-Polα-primase-dependent incorporation of BrdU at DSBs. In the absence of 53BP1 or shieldin, radial formation in BRCA1-deficient cells was restored by the tethering of CST near DSBs, arguing that in this context, shieldin acts primarily by recruiting CST. Furthermore, a SHLD1 mutant defective in CST binding (SHLD1Δ) was non-functional in BRCA1-deficient cells and its function was restored after reconnecting SHLD1Δ to CST. Interestingly, at dysfunctional telomeres and at DNA breaks in class switch recombination where CST has been implicated, SHLD1Δ was fully functional, perhaps because these DNA ends carry CST recognition sites that afford SHLD1-independent binding of CST. These data establish that in BRCA1-deficient cells, CST-Polα-primase is the major effector of shieldin-dependent DSB processing.

DOI: https://doi.org/10.1038/s41556-021-00812-9
Cell Rep. 2022 Jan 11. pii: S2211-1247(21)01742-3. [Epub ahead of print]38(2): 110233

SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia.

Sofya A Polyanskaya, Rosamaria Y Moreno, Bin Lu, Ruopeng Feng, Yu Yao, Seema Irani, Olaf Klingbeil, Zhaolin Yang, Yiliang Wei, Osama E Demerdash, Lukas A Benjamin, Mitchell J Weiss, Yan Jessie Zhang, Christopher R Vakoc.

  Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we use domain-focused CRISPR screening and identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.

Keywords:  PDIK1L; SCP4; STK35; acute myeloid leukemia; complex; dependency; kinase; phosphatase

DOI:  https://doi.org/10.1016/j.celrep.2021.110233
Cells. 2021 Dec 27. pii: 63. [Epub ahead of print]11(1):

Shift in G1-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation.

Fanghua Li, Emil Mladenov, Rositsa Dueva, Martin Stuschke, Beate Timmermann, George Iliakis.

  The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions.

Keywords:  ATM; ATR; DNA double-strand breaks; DNA end resection; DNA-PKcs; cell cycle; checkpoints; ionizing radiation

DOI:  https://doi.org/10.3390/cells11010063
Transl Lung Cancer Res. 2021 Nov;10(11): 4095-4105

SLFN11 biomarker status predicts response to lurbinectedin as a single agent and in combination with ATR inhibition in small cell lung cancer.

Kiran Kundu, Robert J Cardnell, Bingnan Zhang, Li Shen, C Allison Stewart, Kavya Ramkumar, Kasey R Cargill, Jing Wang, Carl M Gay, Lauren A Byers.

   Background: Lurbinectedin recently received FDA accelerated approval as a second line treatment option for metastatic small cell lung cancer (SCLC). However, there are currently no established biomarkers to predict SCLC sensitivity or resistance to lurbinectedin or preclinical studies to guide rational combinations.
Methods: Drug sensitivity was assayed in proliferation assays and xenograft models. Baseline proteomic profiling was performed by reverse-phase protein array. Lurbinectedin-induced changes in intracellular signaling pathways were assayed by Western blot.
Results: Among 21 human SCLC cell lines, cytotoxicity was observed following lurbinectedin treatment at a low dose (median IC50 0.46 nM, range, 0.06-1.83 nM). Notably, cell lines with high expression of Schlafen-11 (SLFN11) protein, a promising biomarker of response to other DNA damaging agents (e.g., chemotherapy, PARP inhibitors), were more sensitive to single-agent lurbinectedin (FC =3.2, P=0.005). SLFN11 was validated as a biomarker of sensitivity to lurbinectedin using siRNA knockdown and in xenografts representing SLFN11 high and low SCLC. Replication stress and DNA damage markers (e.g., γH2AX, phosphorylated CHK1, phosphorylated RPA32) increased in SCLC cell lines following treatment with lurbinectedin. Lurbinectedin also induced PD-L1 expression via cGAS-STING pathway activation. Finally, the combination of lurbinectedin with the ataxia telangiectasia and Rad3-related protein (ATR) inhibitors ceralasertib and berzosertib showed a greater than additive effect in SLFN11-low models.
Conclusions: Together our data confirm the activity of lurbinectedin across a large cohort of SCLC models and identify SLFN11 as a top candidate biomarker for lurbinectedin sensitivity. In SLFN11-low SCLC cell lines which are relatively resistance to lurbinectedin, the addition of an ATR inhibitor to lurbinectedin re-sensitized otherwise resistant cells, confirming previous observations that SLFN11 is a master regulator of DNA damage response independent of ATR, and the absence of SLFN11 leads to synthetic lethality with ATR inhibition. This study provides a rationale for lurbinectedin in combination with ATR inhibitors to overcome resistance in SCLC with low SLFN11 expression.

Keywords:  SLFN11; Small cell lung cancer (SCLC); ataxia telangiectasia and Rad3-related protein (ATR); lurbinectedin; replication stress

DOI:  https://doi.org/10.21037/tlcr-21-437
Mol Biomed. 2021 Jun 20. 2(1): 19

Checkpoint Kinase 1 (Chk1) inhibition fails to activate the Stimulator of Interferon Genes (STING) innate immune signalling in a human coculture cancer system.

Teresa Brooks, Joanne Wayne, Andrew J Massey.

  Utilising Checkpoint Kinase 1 (Chk1) inhibitors to increase cytoplasmic DNA may be a potential strategy to increase the sensitivity of tumours to immune checkpoint modulators. The appearance of DNA in the cytoplasm can drive Cyclic GMP-AMP Synthase-2',3'-Cyclic Guanosine Monophosphate-Adenosine Monophosphate-Stimulator of Interferon Genes (cGAS-cGAMP-STING) inflammatory, anti-tumour T-cell activity via a type I interferon (IFN) and nuclear factor-κB response. In the THP1-Dual reporter cell line, the STING agonist cGAMP activated both reporters, and increased phosphorylation of the innate immune pathway signallers Tank Binding Kinase 1 (TBK1) and Interferon Regulatory Factor (IRF) 3. Inhibition of Chk1 increased TBK1 but not IRF3 phosphorylation and did not induce IRF or NF-κB reporter activation. cGAMP induced a Type I IFN response in THP1 cells whereas inhibition of Chk1 did not. HT29 or HCC1937 cell treatment with a Chk1 inhibitor increased cytoplasmic dsDNA in treated HCC1937 but not HT29 cells and increased IRF reporter activation in cocultured THP1-Dual cells. HT29 cells pre-treated with gemcitabine or camptothecin had elevated cytoplasmic dsDNA and IRF reporter activation in cocultured THP1-Dual cells. Camptothecin or gemcitabine plus a Chk1 inhibitor increased cytoplasmic dsDNA but Chk1 inhibition suppressed IRF reporter activation in cocultured THP1 cells. In THP1-Dual cells treated with cGAMP, Chk1 inhibition suppressed the activation of the IRF reporter compared to cGAMP alone. These results suggest that, in some cellular models, there is little evidence to support the combination of Chk1 inhibitors with immune checkpoint modulators and, in some combination regimes, may even prove deleterious.

Keywords:  Chk1; DNA damage; STING; TBK1; Type I interferon response

DOI:  https://doi.org/10.1186/s43556-021-00044-1
Proc Natl Acad Sci U S A. 2022 Jan 18. pii: e2105898119. [Epub ahead of print]119(3):

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Yiji Liao, Chen-Hao Chen, Tengfei Xiao, Bárbara de la Peña Avalos, Eloise V Dray, Changmeng Cai, Shuai Gao, Neel Shah, Zhao Zhang, Avery Feit, Pengya Xue, Zhijie Liu, Mei Yang, Ji Hoon Lee, Han Xu, Wei Li, Shenglin Mei, Roodolph S Pierre, Shaokun Shu, Teng Fei, Melissa Duarte, Jin Zhao, James E Bradner, Kornelia Polyak, Philip W Kantoff, Henry Long, Steven P Balk, X Shirley Liu, Myles Brown, Kexin Xu.

  Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Keywords:  DNA damage repair; EZH2 inhibitors; cancer therapy; mechanism of drug action

DOI:  https://doi.org/10.1073/pnas.2105898119
Nat Struct Mol Biol. 2022 Jan 10.

IMPDH1 retinal variants control filament architecture to tune allosteric regulation.

Anika L Burrell, Chuankai Nie, Meerit Said, Jacqueline C Simonet, David Fernández-Justel, Matthew C Johnson, Joel Quispe, Rubén M Buey, Jeffrey R Peterson, Justin M Kollman.

Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.

DOI: https://doi.org/10.1038/s41594-021-00706-2
Mol Biomed. 2020 Dec 30. 1(1): 19

RAD50 deficiency is a predictor of platinum sensitivity in sporadic epithelial ovarian cancers.

Adel Alblihy, Muslim L Alabdullah, Michael S Toss, Mashael Algethami, Nigel P Mongan, Emad A Rakha, Srinivasan Madhusudan.

  Intrinsic or acquired resistance seriously limits the use of platinating agents in advanced epithelial ovarian cancers. Increased DNA repair capacity is a key route to platinum resistance. RAD50 is a critical component of the MRN complex, a 'first responder' to DNA damage and essential for the repair of DSBs and stalled replication forks. We hypothesised a role for RAD50 in ovarian cancer pathogenesis and therapeutics. Clinicopathological significance of RAD50 expression was evaluated in clinical cohorts of ovarian cancer at the protein level (n = 331) and at the transcriptomic level (n = 1259). Sub-cellular localization of RAD50 at baseline and following cisplatin therapy was tested in platinum resistant (A2780cis, PEO4) and sensitive (A2780, PEO1) ovarian cancer cells. RAD50 was depleted and cisplatin sensitivity was investigated in A2780cis and PEO4 cells. RAD50 deficiency was associated with better progression free survival (PFS) at the protein (p = 0.006) and transcriptomic level (p < 0.001). Basal level of RAD50 was higher in platinum resistant cells. Following cisplatin treatment, increased nuclear localization of RAD50 was evident in A2780cis and PEO4 compared to A2780 and PEO1 cells. RAD50 depletion using siRNAs in A2780cis and PEO4 cells increased cisplatin cytotoxicity, which was associated with accumulation of DSBs, S-phase cell cycle arrest and increased apoptosis. We provide evidence that RAD50 deficiency is a predictor of platinum sensitivity. RAD50 expression-based stratification and personalization could be viable clinical strategy in ovarian cancers.

Keywords:  DNA repair; Ovarian cancer; Platinum therapy; Predictive biomarker; RAD50

DOI:  https://doi.org/10.1186/s43556-020-00023-y
Nat Commun. 2022 Jan 12. 13(1): 281

Genetic alterations of the SUMO isopeptidase SENP6 drive lymphomagenesis and genetic instability in diffuse large B-cell lymphoma.

Markus Schick, Le Zhang, Sabine Maurer, Hans Carlo Maurer, Konstandina Isaakaidis, Lara Schneider, Upayan Patra, Kathrin Schunck, Elena Rohleder, Julia Hofstetter, Apoorva Baluapuri, Anna Katharina Scherger, Julia Slotta-Huspenina, Franziska Hettler, Julia Weber, Thomas Engleitner, Roman Maresch, Jolanta Slawska, Richard Lewis, Rouzanna Istvanffy, Stefan Habringer, Katja Steiger, Armin Baiker, Robert A J Oostendorp, Cornelius Miething, Hans-Peter Lenhof, Florian Bassermann, Björn Chapuy, Matthias Wirth, Elmar Wolf, Roland Rad, Stefan Müller, Ulrich Keller.

SUMOylation is a post-translational modification of proteins that regulates these proteins' localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma.

DOI: https://doi.org/10.1038/s41467-021-27704-8
Nucleic Acids Res. 2022 Jan 08. pii: gkab1251. [Epub ahead of print]

Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression.

Meenakshi Singh, Viktor Posse, Bradley Peter, Maria Falkenberg, Claes M Gustafsson.

Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome. Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed. Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM. Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs.

DOI: https://doi.org/10.1093/nar/gkab1251
Cancers (Basel). 2021 Dec 31. pii: 191. [Epub ahead of print]14(1):

Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer.

Adrianne Wallace-Povirk, Zhanjun Hou, Md Junayed Nayeen, Aleem Gangjee, Larry H Matherly.

  New therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. To identify new approaches for targeting EOC, metabolic vulnerabilities must be discovered and strategies for the selective delivery of therapeutic agents must be established. Folate receptor (FR) α and the proton-coupled folate transporter (PCFT) are expressed in the majority of EOCs. FRβ is expressed on tumor-associated macrophages, a major infiltrating immune population in EOC. One-carbon (C1) metabolism is partitioned between the cytosol and mitochondria and is important for the synthesis of nucleotides, amino acids, glutathione, and other critical metabolites. Novel inhibitors are being developed with the potential for therapeutic targeting of tumors via FRs and the PCFT, as well as for inhibiting C1 metabolism. In this review, we summarize these exciting new developments in targeted therapies for both tumors and the tumor microenvironment in EOC.

Keywords:  epithelial ovarian cancer; folate; folate receptor; folate transport; one-carbon metabolism; proton-coupled folate transporter; tumor microenvironment

DOI:  https://doi.org/10.3390/cancers14010191
Int J Gen Med. 2022 ;15 353-358

Phosphoribosyl Pyrophosphate Amido Transferase: A New Prognostic Biomarker for Hepatocellular Carcinoma.

Qingfei Chu, Xinyu Gu, Qiuxian Zheng, Jing Wang, Haihong Zhu.

   Background: PPAT (phosphoribosyl pyrophosphate amido transferase) catalyzes the first committed step of de novo purine biosynthesis and is a key regulatory point in the biosynthesis of nascent purine nucleotides. However, the clinical significance and biologic role of PPAT in hepatocellular carcinoma (HCC) remain unknown.
Methods: We compared the expression of PPAT in carcinomatous and precancerous hepatocellular carcinoma tissues by immunohistochemistry in 90 cases of HCC. Correlation analysis was also made on clinical data, survival, classification, and staging.
Results: The expression of PPAT in HCC tumor tissues is significantly higher than that in adjacent normal tissues. The results of the Kaplan-Meier analysis showed that HCC patients with high PPAT expression survived shorter than those with low PPAT expression. Moreover, the expression of PPAT was significantly associated with the tumor grade (P=0.014), PD-L1 (P<0.001), and CTLA4 (P=0.003). The later grade of the tumor, the higher the expression of PPAT. In the PD-L1 high expression group, PPAT is also highly expressed.
Conclusion: Our study demonstrated that PPAT expression might be included in the process of carcinogenesis and prognosis. Hence, PPAT could be served as a new prognostic biomarker for patients of HCC.

Keywords:  HCC; PPAT; biomarker; metabolism; prognosis

DOI:  https://doi.org/10.2147/IJGM.S340758