bims-numges 2021-06-27 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–06–27
38 papers selected by
Sean Rudd, Karolinska Institutet

A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage.
RPA shields inherited DNA lesions for post-mitotic DNA synthesis.
The structure of ORC-Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6.
Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells.
Progerin impairs 3D genome organization and induces fragile telomeres by limiting the dNTP pools.
The human nucleoporin Tpr protects cells from RNA-mediated replication stress.
REV7 directs DNA repair pathway choice.
DNAPK inhibition preferentially compromises the repair of radiation-induced DNA double-strand breaks in chronically hypoxic tumor cells in xenograft models.
UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair.
Inhibition of MRN activity by a telomere protein motif.
PARP inhibitors in gastric cancer: beacon of hope.
Targeting FEN1 Suppresses the Proliferation of Chronic Myeloid Leukemia Cells Through Regulating Alternative End-Joining Pathways.
Impact of the interplay between stemness features, p53 and pol iota on replication pathway choices.
Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast.
Treacle TOPBP1 into nuclei to handle ribosomal-DNA replication stress.
Development of poly(ADP-ribose) polymerase inhibitor and immunotherapy combinations: progress, pitfalls, and promises.
Dissecting regulatory pathways for transcription recovery following DNA damage reveals a non-canonical function of the histone chaperone HIRA.
ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress.
Genes affecting ionizing radiation survival identified through combined exome sequencing and functional screening.
Activation of CDC25A phosphatase is limited by CDK2/cyclin A-mediated feedback inhibition.
ATR inhibitor-induced CDK1-SPOP axis destabilizes PD-L1 and enhances cytotoxicity in prostate cancer.
Radiopotentiation Profiling of Multiple Inhibitors of the DNA Damage Response for Early Clinical Development.
Novel insights into the mechanism of cell cycle kinases Mec1(ATR) and Tel1(ATM).
Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase.
Intracellular H2S production is an autophagy-dependent adaptive response to DNA damage.
Strand discrimination in DNA mismatch repair.
Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.
Combined epigenetic and metabolic treatments overcome differentiation blockade in acute myeloid leukemia.
From Bioorganic Models to Cells.
Strategies in Overcoming Homologous Recombination (HR) Proficiency and Poly (ADP-Ribose) Polymerase Inhibitor (PARPi) Resistance.
Multiplexed bioluminescence-mediated tracking of DNA double-strand break repairs in vitro and in vivo.
Small Molecule Inhibitors of Activation-Induced Deaminase Decrease Class Switch Recombination in B Cells.
Damage-Net: A program for DNA repair meta-analysis identifies a network of novel repair genes that facilitate cancer evolution.
Comparative nucleosomal reactivity of 5-formyl-uridine and 5-formyl-cytidine.
Exosome component 1 cleaves single-stranded DNA and sensitizes kidney renal clear cell carcinoma cells to poly(ADP-ribose) polymerase inhibitor.
Xanthine oxidoreductase promotes the progression of colitis-associated colorectal cancer by causing DNA damage and mediating macrophage M1 polarization.
Electron microscopy reveals unexpected cytoplasm and envelope changes during thymineless death in Escherichia coli.
MCM2-7 complex is a novel druggable target for neuroendocrine prostate cancer.

Nat Cancer. 2021 Jun;2(6): 643-657

A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage.

Genomics England Research Consortium

  Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. ΔOGG1, ΔUNG, ΔEXO1, ΔRNF168, ΔMLH1, ΔMSH2, ΔMSH6, ΔPMS1, and ΔPMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG elimination is sequence-context-specific while uracil clearance is sequence-context-independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C>A transversions), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a 'reverse template slippage' model for T>A transversions. ΔMLH1, ΔMSH6, and ΔMSH2 signatures were similar to each other but distinct from ΔPMS2. Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies.

Keywords:  CRISPR-Cas9 systems; Genomic instability; cancer; cancer genomics

DOI:  https://doi.org/10.1038/s43018-021-00200-0
Nat Commun. 2021 06 22. 12(1): 3827

RPA shields inherited DNA lesions for post-mitotic DNA synthesis.

Aleksandra Lezaja, Andreas Panagopoulos, Yanlin Wen, Edison Carvalho, Ralph Imhof, Matthias Altmeyer.

The paradigm that checkpoints halt cell cycle progression for genome repair has been challenged by the recent discovery of heritable DNA lesions escaping checkpoint control. How such inherited lesions affect genome function and integrity is not well understood. Here, we identify a new class of heritable DNA lesions, which is marked by replication protein A (RPA), a protein primarily known for shielding single-stranded DNA in S/G2. We demonstrate that post-mitotic RPA foci occur at low frequency during unperturbed cell cycle progression, originate from the previous cell cycle, and are exacerbated upon replication stress. RPA-marked inherited ssDNA lesions are found at telomeres, particularly of ALT-positive cancer cells. We reveal that RPA protects these replication remnants in G1 to allow for post-mitotic DNA synthesis (post-MiDAS). Given that ALT-positive cancer cells exhibit high levels of replication stress and telomere fragility, targeting post-MiDAS might be a new therapeutic opportunity.

DOI: https://doi.org/10.1038/s41467-021-23806-5
Nat Commun. 2021 06 23. 12(1): 3883

The structure of ORC-Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6.

Xiang Feng, Yasunori Noguchi, Marta Barbon, Bruce Stillman, Christian Speck, Huilin Li.

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.

DOI: https://doi.org/10.1038/s41467-021-24199-1
Nat Metab. 2021 Jun 21.

Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells.

Andrew J Schwartz, Joshua W Goyert, Sumeet Solanki, Samuel A Kerk, Brandon Chen, Cristina Castillo, Peggy P Hsu, Brian T Do, Rashi Singhal, Michael K Dame, Ho-Joon Lee, Jason R Spence, Samira Lakhal-Littleton, Matthew G Vander Heiden, Costas A Lyssiotis, Xiang Xue, Yatrik M Shah.

Colorectal cancer (CRC) requires massive iron stores, but the complete mechanisms by which CRC modulates local iron handling are poorly understood. Here, we demonstrate that hepcidin is activated ectopically in CRC. Mice deficient in hepcidin specifically in the colon tumour epithelium, compared with wild-type littermates, exhibit significantly diminished tumour number, burden and size in a sporadic model of CRC, whereas accumulation of intracellular iron by deletion of the iron exporter ferroportin exacerbates these tumour parameters. Metabolomic analysis of three-dimensional patient-derived CRC tumour enteroids indicates a prioritization of iron in CRC for the production of nucleotides, which is recapitulated in our hepcidin/ferroportin mouse CRC models. Mechanistically, our data suggest that iron chelation decreases mitochondrial function, thereby altering nucleotide synthesis, whereas exogenous supplementation of nucleosides or aspartate partially rescues tumour growth in patient-derived enteroids and CRC cell lines in the presence of an iron chelator. Collectively, these data suggest that ectopic hepcidin in the tumour epithelium establishes an axis to sequester iron in order to maintain the nucleotide pool and sustain proliferation in colorectal tumours.

DOI: https://doi.org/10.1038/s42255-021-00406-7
Sci Rep. 2021 Jun 23. 11(1): 13195

Progerin impairs 3D genome organization and induces fragile telomeres by limiting the dNTP pools.

Anna Kychygina, Marina Dall'Osto, Joshua A M Allen, Jean-Charles Cadoret, Vincent Piras, Hilda A Pickett, Laure Crabbe.

Chromatin organization within the nuclear volume is essential to regulate many aspects of its function and to safeguard its integrity. A key player in this spatial scattering of chromosomes is the nuclear envelope (NE). The NE tethers large chromatin domains through interaction with the nuclear lamina and other associated proteins. This organization is perturbed in cells from Hutchinson-Gilford progeria syndrome (HGPS), a genetic disorder characterized by premature aging features. Here, we show that HGPS-related lamina defects trigger an altered 3D telomere organization with increased contact sites between telomeres and the nuclear lamina, and an altered telomeric chromatin state. The genome-wide replication timing signature of these cells is perturbed, with a shift to earlier replication for regions that normally replicate late. As a consequence, we detected a higher density of replication forks traveling simultaneously on DNA fibers, which relies on limiting cellular dNTP pools to support processive DNA synthesis. Remarkably, increasing dNTP levels in HGPS cells rescued fragile telomeres, and improved the replicative capacity of the cells. Our work highlights a functional connection between NE dysfunction and telomere homeostasis in the context of premature aging.

DOI: https://doi.org/10.1038/s41598-021-92631-z
Nat Commun. 2021 06 24. 12(1): 3937

The human nucleoporin Tpr protects cells from RNA-mediated replication stress.

Martin Kosar, Michele Giannattasio, Daniele Piccini, Apolinar Maya-Mendoza, Francisco García-Benítez, Jirina Bartkova, Sonia I Barroso, Hélène Gaillard, Emanuele Martini, Umberto Restuccia, Miguel Angel Ramirez-Otero, Massimiliano Garre, Eleonora Verga, Miguel Andújar-Sánchez, Scott Maynard, Zdenek Hodny, Vincenzo Costanzo, Amit Kumar, Angela Bachi, Andrés Aguilera, Jiri Bartek, Marco Foiani.

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

DOI: https://doi.org/10.1038/s41467-021-24224-3
Trends Cell Biol. 2021 Jun 16. pii: S0962-8924(21)00114-8. [Epub ahead of print]

REV7 directs DNA repair pathway choice.

Connor S Clairmont, Alan D D'Andrea.

  REV7 is a small multifunctional protein that participates in multiple DNA repair pathways, most notably translesion DNA synthesis and double-strand break (DSB) repair. While the role of REV7 in translesion synthesis has been known for several decades, its function in DSB repair is a recent discovery. Investigations into the DSB repair function of REV7 have led to the discovery of a new DNA repair complex known as Shieldin. Recent studies have also highlighted the importance of REV7's HORMA domain, an ancient structural motif, in REV7 function and have identified the HORMA regulators, TRIP13 and p31, as novel DNA repair factors. In this review, we discuss these recent findings and their implications for repair pathway choice, at both DSBs and replication forks. We suggest that REV7, in particular the activation state of its HORMA domain, can act as a critical determinant of mutagenic versus error-free repair in multiple contexts.

Keywords:  DNA repair; Fanconi anemia; REV7; Shieldin; TRIP13; double-strand break repair; translesion synthesis

DOI:  https://doi.org/10.1016/j.tcb.2021.05.009
Mol Cancer Ther. 2021 Jun 22. pii: molcanther.0857.2020. [Epub ahead of print]

DNAPK inhibition preferentially compromises the repair of radiation-induced DNA double-strand breaks in chronically hypoxic tumor cells in xenograft models.

Yanyan Jiang, Elaine Willmore, Stephen R Wedge, Anderson J Ryan.

Radiation-induced DNA double strand breaks (DSBs) can be repaired by homologous recombination (HR) and non-homologous end joining (NHEJ). Recently, it has been found that chronic tumor hypoxia compromises HR repair of DNA DSBs, but activates the NHEJ protein DNAPK. We therefore hypothesized that inhibition of DNAPK can preferentially potentiate the sensitivity of chronically hypoxic cancer cells to radiation through contextual synthetic lethality in vivo. In this study, we investigated the impact of DNAPK inhibition by a novel selective DNAPK inhibitor, NU5455, on the repair of radiation-induced DNA DSBs in chronically hypoxic and nonhypoxic cells across a range of xenograft models. We found that NU5455 inhibited DSB repair following radiation in both chronically hypoxic and nonhypoxic tumor cells. Most importantly, the inhibitory effect was more pronounced in chronically hypoxic tumor cells than in nonhypoxic tumor cells. This is the first in vivo study to indicate that DNAPK inhibition may preferentially sensitizes chronically hypoxic tumor cells to radiotherapy, suggesting a broader therapeutic window for transient DNAPK inhibition combined with radiotherapy.

DOI: https://doi.org/10.1158/1535-7163.MCT-20-0857
Nat Commun. 2021 06 22. 12(1): 3849

UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair.

Greg H P Ngo, Julia W Grimstead, Duncan M Baird.

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.

DOI: https://doi.org/10.1038/s41467-021-24201-w
Nat Commun. 2021 06 22. 12(1): 3856

Inhibition of MRN activity by a telomere protein motif.

Freddy Khayat, Elda Cannavo, Majedh Alshmery, William R Foster, Charly Chahwan, Martino Maddalena, Christopher Smith, Antony W Oliver, Adam T Watson, Antony M Carr, Petr Cejka, Alessandro Bianchi.

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.

DOI: https://doi.org/10.1038/s41467-021-24047-2
J Exp Clin Cancer Res. 2021 Jun 24. 40(1): 211

PARP inhibitors in gastric cancer: beacon of hope.

Yali Wang, Kun Zheng, Yongbiao Huang, Hua Xiong, Jinfang Su, Rui Chen, Yanmei Zou.

  Defects in the DNA damage response (DDR) can lead to genome instability, producing mutations or aberrations that promote the development and progression of cancer. But it also confers such cells vulnerable to cell death when they inhibit DNA damage repair. Poly (ADP-ribose) polymerase (PARP) plays a central role in many cellular processes, including DNA repair, replication, and transcription. PARP induces the occurrence of poly (ADP-ribosylation) (PARylation) when DNA single strand breaks (SSB) occur. PARP and various proteins can interact directly or indirectly through PARylation to regulate DNA repair. Inhibitors that directly target PARP have been found to block the SSB repair pathway, triggering homologous recombination deficiency (HRD) cancers to form synthetic lethal concepts that represent an anticancer strategy. It has therefore been investigated in many cancer types for more effective anti-cancer strategies, including gastric cancer (GC). This review describes the antitumor mechanisms of PARP inhibitors (PARPis), and the preclinical and clinical progress of PARPis as monotherapy and combination therapy in GC.

Keywords:  Cancer treatment; DNA damage response; Gastric cancer; PARP inhibitor

DOI:  https://doi.org/10.1186/s13046-021-02005-6
DNA Cell Biol. 2021 Jun 22.

Targeting FEN1 Suppresses the Proliferation of Chronic Myeloid Leukemia Cells Through Regulating Alternative End-Joining Pathways.

Yalin Zhu, Hongdan Dai, Yonghong Wang, Yang Liang, Wenli Feng, Ying Yuan.

  Chronic myeloid leukemia (CML) is characterized by the formation of the BCR-ABL fusion gene. The BCR-ABL protein leads to an increased level of reactive oxygen species, which is a major cause of endogenous DNA double-strand breaks (DSBs). CML cells are prone to rely on a highly mutagenic alternative end-joining (Alt-EJ) pathway to cope with enhanced DSBs, which aggravates chromosomal instability. Hence, targeting dysregulated DNA repair proteins provides new insights into cancer treatment. In this study, we discovered the abnormal upregulation of Flap endonuclease 1 (FEN1) in CML, as well as FEN1's participation in the error-prone Alt-EJ repair pathway and its interplay with DNA Ligase1 and proliferating cell nuclear antigen in DSB repair. Knockdown of FEN1 by shRNA not only inhibited the proliferation and induced apoptosis but also enhanced the efficacy of imatinib (IM) in drug-resistant CML cell K562/G01. Moreover, excessive DSB accumulation was detected after FEN1 inhibition. In summary, our results demonstrated that FEN1 is a promising therapeutic target in CML treatment. This work extends the understanding of regulating abnormal DSB repair for cancer treatment.

Keywords:  Alt-EJ; BCR-ABL; DNA double-strand breaks; FEN1; chronic myeloid leukemia

DOI:  https://doi.org/10.1089/dna.2021.0239
Nucleic Acids Res. 2021 Jun 24. pii: gkab526. [Epub ahead of print]

Impact of the interplay between stemness features, p53 and pol iota on replication pathway choices.

Michaela Ihle, Stephanie Biber, Insa S Schroeder, Christine Blattner, Miriam Deniz, Giovanna Damia, Vanesa Gottifredi, Lisa Wiesmüller.

Using human embryonic, adult and cancer stem cells/stem cell-like cells (SCs), we demonstrate that DNA replication speed differs in SCs and their differentiated counterparts. While SCs decelerate DNA replication, differentiated cells synthesize DNA faster and accumulate DNA damage. Notably, both replication phenotypes depend on p53 and polymerase iota (POLι). By exploring protein interactions and newly synthesized DNA, we show that SCs promote complex formation of p53 and POLι at replication sites. Intriguingly, in SCs the translocase ZRANB3 is recruited to POLι and required for slow-down of DNA replication. The known role of ZRANB3 in fork reversal suggests that the p53-POLι complex mediates slow but safe bypass of replication barriers in SCs. In differentiated cells, POLι localizes more transiently to sites of DNA synthesis and no longer interacts with p53 facilitating fast POLι-dependent DNA replication. In this alternative scenario, POLι associates with the p53 target p21, which antagonizes PCNA poly-ubiquitination and, thereby potentially disfavors the recruitment of translocases. Altogether, we provide evidence for diametrically opposed DNA replication phenotypes in SCs and their differentiated counterparts putting DNA replication-based strategies in the spotlight for the creation of therapeutic opportunities targeting SCs.

DOI: https://doi.org/10.1093/nar/gkab526
Nucleic Acids Res. 2021 Jun 22. pii: gkab511. [Epub ahead of print]

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast.

Jakub Muraszko, Karol Kramarz, Bilge Argunhan, Kentaro Ito, Gabriela Baranowska, Yumiko Kurokawa, Yasuto Murayama, Hideo Tsubouchi, Sarah Lambert, Hiroshi Iwasaki, Dorota Dziadkowiec.

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

DOI: https://doi.org/10.1093/nar/gkab511
Nat Rev Mol Cell Biol. 2021 Jun 25.

Treacle TOPBP1 into nuclei to handle ribosomal-DNA replication stress.

Eytan Zlotorynski.

DOI: https://doi.org/10.1038/s41580-021-00393-3
Trends Cancer. 2021 Jun 19. pii: S2405-8033(21)00105-9. [Epub ahead of print]

Development of poly(ADP-ribose) polymerase inhibitor and immunotherapy combinations: progress, pitfalls, and promises.

Melissa M Pham, Natalie Y L Ngoi, Guang Peng, David S P Tan, Timothy A Yap.

  The efficacy of poly(ADP-ribose) polymerase inhibitors (PARPi) is restricted by inevitable drug resistance, while their use in combination with chemotherapy and targeted agents is commonly associated with dose-limiting toxicities. Immune checkpoint blockade (ICB) has demonstrated durable responses in different solid tumors and is well-established across multiple cancers. Despite this, single agent activity is limited to a minority of patients and drug resistance remains an issue. Building on the monotherapy success of both drug classes, combining PARPi with ICB may be a safe and well-tolerated strategy with the potential to improve survival outcomes. In this review, we present the preclinical, translational, and clinical data supporting the combination of DNA damage response (DDR) and ICB as well as consider important questions to be addressed with future research.

Keywords:  DNA damage response; PARP inhibitor; immune checkpoint inhibitor; immunotherapy

DOI:  https://doi.org/10.1016/j.trecan.2021.05.004
Nat Commun. 2021 06 22. 12(1): 3835

Dissecting regulatory pathways for transcription recovery following DNA damage reveals a non-canonical function of the histone chaperone HIRA.

Déborah Bouvier, Juliette Ferrand, Odile Chevallier, Michelle T Paulsen, Mats Ljungman, Sophie E Polo.

Transcription restart after a genotoxic challenge is a fundamental yet poorly understood process. Here, we dissect the interplay between transcription and chromatin restoration after DNA damage by focusing on the human histone chaperone complex HIRA, which is required for transcription recovery post UV. We demonstrate that HIRA is recruited to UV-damaged chromatin via the ubiquitin-dependent segregase VCP to deposit new H3.3 histones. However, this local activity of HIRA is dispensable for transcription recovery. Instead, we reveal a genome-wide function of HIRA in transcription restart that is independent of new H3.3 and not restricted to UV-damaged loci. HIRA coordinates with ASF1B to control transcription restart by two independent pathways: by stabilising the associated subunit UBN2 and by reducing the expression of the transcription repressor ATF3. Thus, HIRA primes UV-damaged chromatin for transcription restart at least in part by relieving transcription inhibition rather than by depositing new H3.3 as an activating bookmark.

DOI: https://doi.org/10.1038/s41467-021-24153-1
Nat Commun. 2021 06 23. 12(1): 3887

ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress.

Yu-Ching Teng, Aishwarya Sundaresan, Ryan O'Hara, Vincent U Gant, Minhua Li, Sara Martire, Jane N Warshaw, Amrita Basu, Laura A Banaszynski.

ATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.

DOI: https://doi.org/10.1038/s41467-021-24206-5
Hum Mutat. 2021 Jun 21.

Genes affecting ionizing radiation survival identified through combined exome sequencing and functional screening.

Meenal Gupta, Xiangfei Liu, Sharon N Teraoka, Jocyndra A Wright, Richard A Gatti, Aaron Quinlan, Patrick Concannon.

  The study of genetic syndromes characterized by sensitivity to DNA damaging agents has provided important insights into the mechanisms that maintain genome stability and identified novel targets for cancer therapies. Here we used exome sequencing to study 51 unrelated individuals with previously reported hypersensitivity to ionizing radiation as well as a range of neurologic, immunologic, and developmental features, but who did not clearly fit any previously defined genetic syndrome. Based on the combination of variant identification, computational evidence of deleteriousness, and functional screening, we identified three groups of subjects. Two subjects carried bi-allelic loss of function variants in causative genes for known DNA damage response syndromes. Eight subjects carried single loss of function variants in causative genes for DNA damage response syndromes, six of whom also carried predicted deleterious variants in other genes with DNA damage related functions. Three subjects carried deleterious mutations in genes without obvious roles in DNA damage responses. However, treatment of U2OS cells with siRNA targeting these genes resulted in significantly increased radiation sensitivity. Our results suggest that gene-gene interaction may contribute to ionizing radiation sensitivity as well as highlighting possible roles for several genes not obviously involved in the response to DNA damage. This article is protected by copyright. All rights reserved.

Keywords:  DNA damage disorders; cellular survival; genetic variants; radiation hypersensitivity; whole exome sequencing

DOI:  https://doi.org/10.1002/humu.24241
Cell Cycle. 2021 Jun 22. 1-12

Activation of CDC25A phosphatase is limited by CDK2/cyclin A-mediated feedback inhibition.

Jennifer P Ditano, Nandini Sakurikar, Alan Eastman.

  Cyclin-dependent kinase (CDK) 1 complexed with cyclin B is a driver of mitosis, while CDK2 drives S phase entry and replicon initiation. CDK2 activity increases as cells progress through S phase, and its cyclin partner switches from cyclin E to cyclin A. Activation of CDK2 requires dephosphorylation of tyrosine-15 by CDC25A. DNA damage activates the checkpoint protein CHK1, which phosphorylates and degrades CDC25A to prevent activation of CDK2 and protect from cell cycle progression before damage is repaired. CHK1 inhibitors were developed to circumvent this arrest and enhance the efficacy of many cancer chemotherapeutic agents. CHK1 inhibition results in the accumulation of CDC25A and activation of CDK2. We demonstrate that inhibition of CDK2 or suppression of cyclin A also results in accumulation of CDC25A suggesting a feedback loop that prevents over activation of this pathway. The feedback inhibition of CDC25A targets phosphorylation of S88-CDC25A, which resides within a CDK consensus sequence. In contrast, it appears that CDK complexes with cyclin B (and possibly cyclin E) stabilize CDC25A in a feed-forward activation loop. While CDK2/cyclin A would normally be active at late S/G2, we propose that this feedback inhibitory loop prevents over activation of CDK2 in early S phase, while still leaving CDK2/cyclin E to catalyze replicon initiation. One importance of this observation is that a subset of cancer cell lines are very sensitive to CHK1 inhibition, which is mediated by CDK2/cyclin A activity in S phase cells. Hence, dysregulation of this feedback loop might facilitate sensitivity of the cells.

Keywords:  CDC25A; CDK2; CHK1; WEE1; cell cycle regulation; cyclin A

DOI:  https://doi.org/10.1080/15384101.2021.1938813
Clin Cancer Res. 2021 Jun 24. pii: clincanres.1010.2021. [Epub ahead of print]

ATR inhibitor-induced CDK1-SPOP axis destabilizes PD-L1 and enhances cytotoxicity in prostate cancer.

Zhe Tang, Patrick G Pilie, Chuandong Geng, Ganiraju C Manyam, Guang Yang, Sanghee Park, Daoqi Wang, Shan Peng, Cheng Wu, Guang Peng, Timothy A Yap, Paul G Corn, Bradley M Broom, Timothy C Thompson.

PURPOSE: Despite significant benefit for other cancer subtypes, immune checkpoint blockade (ICB) therapy has not yet been shown to significantly improve outcomes for men with castration-resistant prostate cancer (CRPC). Prior data have shown that DNA damage response (DDR) deficiency, via genetic alteration and/or pharmacologic induction using DDR inhibitors (DDRi), may improve ICB response in solid tumors in part due to induction of mitotic catastrophe and innate immune activation. Discerning the underlying mechanisms of this DDRi-ICB interaction in a prostate-cancer specific manner is vital to guide novel clinical trials and provide durable clinical responses for men with CRPC.
EXPERIMENTAL DESIGN: We treated prostate cancer cell lines with potent, specific inhibitors of ATR kinase, as well as with PARP inhibitor, olaparib. We performed analyses of cGAS-STING and DDR signaling in treated cells, and treated a syngeneic androgen-indifferent, prostate cancer model with combined ATR inhibition and anti-PDL1, and performed scRNA-seq analysis in treated tumors.
RESULTS: ATRi (BAY1895433) directly repressed ATR-CHK1 signaling, activated CDK1-SPOP axis, leading to destabilization of PD-L1 protein. These effects of ATRi are distinct from those of olaparib, and resulted in a cGAS-STING-initiated, IFN-ß-mediated, autocrine, apoptotic response in CRPC. The combination of ATRi with anti-PDL1 therapy resulted in robust innate immune activation and a synergistic, T-cell dependent therapeutic response in our syngeneic mouse model.
CONCLUSIONS: This work provides a molecular mechanistic rationale for combining ATR-targeted agents with immune checkpoint blockade for patients with CRPC. Multiple early phase clinical trials of this combination are underway.

DOI: https://doi.org/10.1158/1078-0432.CCR-21-1010
Mol Cancer Ther. 2021 Jun 22. pii: molcanther.0502.2020. [Epub ahead of print]

Radiopotentiation Profiling of Multiple Inhibitors of the DNA Damage Response for Early Clinical Development.

Sonja J Gill, Paul W G Wijnhoven, Jacqueline H L Fok, Rebecca L Lloyd, Jonathan Cairns, Joshua Armenia, Jenni Nikkilä, Alan Lau, Christopher J Bakkenist, Susan M Galbraith, Conchita Vens, Mark J O'Connor.

Radiotherapy is an effective anti-cancer treatment, but combinations with targeted agents that maximise efficacy whilst sparing normal tissue are needed. Here, we assess the radiopotentiation profiles of DNA damage response inhibitors (DDRi) olaparib (PARP1/2), ceralasertib (ATR), adavosertib (WEE1), AZD0156 (ATM) and KU-60648 (DNA-PK). We performed a radiation combination screen and assessed how drug concentration and cellular DDR deficiencies influence the radiopotentiation ability of DDRi. We pre-selected six lung cancer cell lines with different genetic/signalling aberrations (including mutations in TP53 and ATM) and assessed multiple concentrations of DDRi in combination with a fixed radiation dose by clonogenic assay. The effective concentration of DDRi in radiation combinations is lower than that required for single-agent efficacy. This has the potential to be exploited further in the context of DDR deficiencies to increase therapeutic index and we demonstrate that low concentrations of AZD0156 preferentially sensitised p53-deficient cells. Moreover, testing multiple concentrations of DDRi in radiation combinations indicated that olaparib, ceralasertib and adavosertib have a desirable safety profile showing moderate increases in radiation dose enhancement with increasing inhibitor concentration. Small increases in concentration of AZD0156 and particularly KU-60648, however, result in steep increases in dose enhancement. Radiopotentiation profiling can inform on effective drug doses required for radiosensitisation in relation to biomarkers, providing an opportunity to increase therapeutic index. Moreover, multiple concentration testing demonstrates a relationship between drug concentration and radiation effect that provides valuable insights that, with future in vivo validation, can guide dose-escalation strategies in clinical trials.

DOI: https://doi.org/10.1158/1535-7163.MCT-20-0502
Crit Rev Biochem Mol Biol. 2021 Jun 20. 1-14

Novel insights into the mechanism of cell cycle kinases Mec1(ATR) and Tel1(ATM).

Elias A Tannous, Peter M Burgers.

  DNA replication is a highly precise process which usually functions in a perfect rhythm with cell cycle progression. However, cells are constantly faced with various kinds of obstacles such as blocks in DNA replication, lack of availability of precursors and improper chromosome alignment. When these problems are not addressed, they may lead to chromosome instability and the accumulation of mutations, and even cell death. Therefore, the cell has developed response mechanisms to keep most of these situations under control. Of the many factors that participate in this DNA damage response, members of the family of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), orchestrate the response landscape. Our understanding of two members of the PIKK family, human ATR (yeast Mec1) and ATM (yeast Tel1), and their associated partner proteins, has shown substantial progress through recent biochemical and structural studies. Emerging structural information of these unique kinases show common features that reveal the mechanism of kinase activity.

Keywords:  ATM; ATR; DNA damage response; DNA replication; Mec1; PIKK; Tel1; checkpoint

DOI:  https://doi.org/10.1080/10409238.2021.1925218
ACS Synth Biol. 2021 Jun 22.

Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase.

Erika Schaudy, Jory Lietard, Mark M Somoza.

  The untemplated activity of terminal deoxynucleotidyl transferase (TdT) represents its most appealing feature. Its use is well established in applications aiming for extension of a DNA initiator strand, but a more recent focus points to its potential in enzymatic de novo synthesis of DNA. Whereas its low substrate specificity for nucleoside triphosphates has been studied extensively, here we interrogate how the activity of TdT is modulated by the nature of the initiating strands, in particular their length, chemistry, and nucleotide composition. Investigation of full permutational libraries of mono- to pentamers of d-DNA, l-DNA, and 2'O-methyl-RNA of differing directionality immobilized to glass surfaces, and generated via photolithographic in situ synthesis, shows that the efficiency of extension strongly depends on the nucleobase sequence. We also show TdT being catalytically active on a non-nucleosidic substrate, hexaethylene glycol. These results offer new perspectives on constraints and strategies for de novo synthesis of DNA using TdT regarding the requirements for initiation of enzymatic generation of DNA.

Keywords:  TdT polymerase; enzymatic DNA synthesis; l-DNA; microarray; photolithographic synthesis; synthetic biology

DOI:  https://doi.org/10.1021/acssynbio.1c00142
Cell Chem Biol. 2021 Jun 12. pii: S2451-9456(21)00261-0. [Epub ahead of print]

Intracellular H2S production is an autophagy-dependent adaptive response to DNA damage.

Xiaofeng Jiang, Michael R MacArthur, J Humberto Treviño-Villarreal, Peter Kip, C Keith Ozaki, Sarah J Mitchell, James R Mitchell.

  Hydrogen sulfide (H2S) is a gasotransmitter with broad physiological activities, including protecting cells against stress, but little is known about the regulation of cellular H2S homeostasis. We have performed a high-content small-molecule screen and identified genotoxic agents, including cancer chemotherapy drugs, as activators of intracellular H2S levels. DNA damage-induced H2S in vitro and in vivo. Mechanistically, DNA damage elevated autophagy and upregulated H2S-generating enzyme CGL; chemical or genetic disruption of autophagy or CGL impaired H2S induction. Importantly, exogenous H2S partially rescued autophagy-deficient cells from genotoxic stress. Furthermore, stressors that are not primarily genotoxic (growth factor depletion and mitochondrial uncoupler FCCP) increased intracellular H2S in an autophagy-dependent manner. Our findings highlight the role of autophagy in H2S production and suggest that H2S generation may be a common adaptive response to DNA damage and other stressors.

Keywords:  CGL enzyme; DNA damage response; P3 probe; SF7-AM probe; autophagy; cancer chemotherapy; high-throughput screening; hydrogen sulfide; stress response; sulfide metabolism

DOI:  https://doi.org/10.1016/j.chembiol.2021.05.016
DNA Repair (Amst). 2021 Jun 19. pii: S1568-7864(21)00117-8. [Epub ahead of print]105 103161

Strand discrimination in DNA mismatch repair.

Christopher D Putnam.

  DNA mismatch repair (MMR) corrects non-Watson-Crick basepairs generated by replication errors, recombination intermediates, and some forms of chemical damage to DNA. In MutS and MutL homolog-dependent MMR, damaged bases do not identify the error-containing daughter strand that must be excised and resynthesized. In organisms like Escherichia coli that use methyl-directed MMR, transient undermethylation identifies the daughter strand. For other organisms, growing in vitro and in vivo evidence suggest that strand discrimination is mediated by DNA replication-associated daughter strand nicks that direct asymmetric loading of the replicative clamp (the β-clamp in bacteria and the proliferating cell nuclear antigen, PCNA, in eukaryotes). Structural modeling suggests that replicative clamps mediate strand specificity either through the ability of MutL homologs to recognize the fixed orientation of the daughter strand relative to one face of the replicative clamps or through parental strand-specific diffusion of replicative clamps on DNA, which places the daughter strand in the MutL homolog endonuclease active site. Finally, identification of bacteria that appear to lack strand discrimination mediated by a replicative clamp and a pre-existing nick suggest that other strand discrimination mechanisms exist or that these organisms perform MMR by generating a double-stranded DNA break intermediate, which may be analogous to NucS-mediated MMR.

Keywords:  DNA mismatch repair; DNA mispair; Strand discrimination

DOI:  https://doi.org/10.1016/j.dnarep.2021.103161
Mol Cell. 2021 Jun 17. pii: S1097-2765(21)00408-1. [Epub ahead of print]

Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

Weitao Wang, Kyle N Klein, Karel Proesmans, Hongbo Yang, Claire Marchal, Xiaopeng Zhu, Tyler Borrman, Alex Hastie, Zhiping Weng, John Bechhoefer, Chun-Long Chen, David M Gilbert, Nicholas Rhind.

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

DOI: https://doi.org/10.1016/j.molcel.2021.05.024
iScience. 2021 Jun 25. 24(6): 102651

Combined epigenetic and metabolic treatments overcome differentiation blockade in acute myeloid leukemia.

Barry M Zee, Kamrine E Poels, Cong-Hui Yao, Kimihito C Kawabata, Gongwei Wu, Cihangir Duy, William D Jacobus, Elizabeth Senior, Jennifer E Endress, Ashwini Jambhekar, Scott B Lovitch, Jiexian Ma, Abhinav Dhall, Isaac S Harris, M Andres Blanco, David B Sykes, Jonathan D Licht, David M Weinstock, Ari Melnick, Marcia C Haigis, Franziska Michor, Yang Shi.

  A hallmark of acute myeloid leukemia (AML) is the inability of self-renewing malignant cells to mature into a non-dividing terminally differentiated state. This differentiation block has been linked to dysregulation of multiple cellular processes, including transcriptional, chromatin, and metabolic regulation. The transcription factor HOXA9 and the histone demethylase LSD1 are examples of such regulators that promote differentiation blockade in AML. To identify metabolic targets that interact with LSD1 inhibition to promote myeloid maturation, we screened a small molecule library to identify druggable substrates. We found that differentiation caused by LSD1 inhibition is enhanced by combined perturbation of purine nucleotide salvage and de novo lipogenesis pathways, and identified multiple lines of evidence to support the specificity of these pathways and suggest a potential basis of how perturbation of these pathways may interact synergistically to promote myeloid differentiation. In sum, these findings suggest potential drug combination strategies in the treatment of AML.

Keywords:  molecular biology; stem cell research; systems biology

DOI:  https://doi.org/10.1016/j.isci.2021.102651
Annu Rev Biochem. 2021 Jun 20. 90 57-76

From Bioorganic Models to Cells.

Stephen J Benkovic.

  I endeavor to share how various choices-some deliberate, some unconscious-and the unmistakable influence of many others shaped my scientific pursuits. I am fascinated by how two long-term, major streams of my research, DNA replication and purine biosynthesis, have merged with unexpected interconnections. If I have imparted to many of the talented individuals who have passed through my lab a degree of my passion for uncloaking the mysteries hidden in scientific research and an understanding of the honesty and rigor it demands and its impact on the world community, then my mentorship has been successful.

Keywords:  DNA replication; autobiography; enzymatic catalysis; purine biosynthesis

DOI:  https://doi.org/10.1146/annurev-biochem-062320-062929
Mol Cancer Ther. 2021 Jun 25. pii: molcanther.0992.2020. [Epub ahead of print]

Strategies in Overcoming Homologous Recombination (HR) Proficiency and Poly (ADP-Ribose) Polymerase Inhibitor (PARPi) Resistance.

Nidhi Goel, McKenzie E Foxall, Carly Bess Scalise, Jaclyn A Wall, Rebecca C Arend.

Ovarian cancer is the second most common gynecologic malignancy in the United States (US) and the most common cause of gynecologic cancer-related death. The majority of ovarian cancers ultimately recur despite excellent response rates to upfront platinum and taxane-based chemotherapy. Maintenance therapy after frontline treatment has emerged in recent years as an effective tool for extending the platinum-free interval of these patients. Maintenance therapy with poly (ADP-ribose) polymerase inhibitors (PARPi) in particular has become part of standard of care in the upfront setting and in patients with platinum-sensitive disease. HR deficient (HRD) tumors have a nonfunctioning homologous recombination repair (HRR) pathway and respond well to PARPi, which takes advantage of synthetic lethality by concomitantly impairing DNA repair mechanisms. Conversely, patients with a functioning HRR pathway, i.e. HR proficient (HRP) tumors, can still elicit benefit from PARPi, but the efficacy is not as remarkable as what is seen in HRD tumors. PARPi are ineffective in some patients due to HR proficiency, which is either inherent to the tumor or potentially acquired as a method of therapeutic resistance. This review seeks to outline current strategies employed by clinicians and scientists to overcome PARPi resistance - either acquired or inherent to the tumor.

DOI: https://doi.org/10.1158/1535-7163.MCT-20-0992
Nat Protoc. 2021 Jun 23.

Multiplexed bioluminescence-mediated tracking of DNA double-strand break repairs in vitro and in vivo.

Jasper Che-Yung Chien, Christian E Badr, Charles Pin-Kuang Lai.

The dynamics of DNA double-strand break (DSB) repairs including homology-directed repair and nonhomologous end joining play an important role in diseases and therapies. However, investigating DSB repair is typically a low-throughput and cross-sectional process, requiring disruption of cells and organisms for subsequent nuclease-, sequencing- or reporter-based assays. In this protocol, we provide instructions for establishing a bioluminescent repair reporter system using engineered Gaussia and Vargula luciferases for noninvasive tracking of homology-directed repair and nonhomologous end joining, respectively, induced by SceI meganuclease, SpCas9 or SpCas9 D10A nickase-mediated editing. We also describe complementation with orthogonal DSB repair assays and omics analyses to validate the reporter readouts. The bioluminescent repair reporter system provides longitudinal and rapid readout (~seconds per sample) to accurately and efficiently measure the efficacy of genome-editing tools and small-molecule modulators on DSB repair. This protocol takes ~2-4 weeks to establish, and as little as 2 h to complete the assay. The entire bioluminescent repair reporter procedure can be performed by one person with standard molecular biology expertise and equipment. However, orthogonal DNA repair assays would require a specialized facility that performs Sanger sequencing or next-generation sequencing.

DOI: https://doi.org/10.1038/s41596-021-00564-8
ACS Pharmacol Transl Sci. 2021 Jun 11. 4(3): 1214-1226

Small Molecule Inhibitors of Activation-Induced Deaminase Decrease Class Switch Recombination in B Cells.

Juan Alvarez-Gonzalez, Adam Yasgar, Robert W Maul, Amanda E Rieffer, Daniel J Crawford, Daniel J Salamango, Dorjbal Dorjsuren, Alexey V Zakharov, Daniel J Jansen, Ganesha Rai, Juan Marugan, Anton Simeonov, Reuben S Harris, Rahul M Kohli, Patricia J Gearhart.

Activation-induced deaminase (AID) not only mutates DNA within the immunoglobulin loci to generate antibody diversity, but it also promotes development of B cell lymphomas. To tame this mutagen, we performed a quantitative high-throughput screen of over 90 000 compounds to see if AID activity could be mitigated. The enzymatic activity was assessed in biochemical assays to detect cytosine deamination and in cellular assays to measure class switch recombination. Three compounds showed promise via inhibition of switching in a transformed B cell line and in murine splenic B cells. These compounds have similar chemical structures, which suggests a shared mechanism of action. Importantly, the inhibitors blocked AID, but not a related cytosine DNA deaminase, APOBEC3B. We further determined that AID was continually expressed for several days after B cell activation to induce switching. This first report of small molecules that inhibit AID can be used to gain regulatory control over base editors.

DOI: https://doi.org/10.1021/acsptsci.1c00064
DNA Repair (Amst). 2021 Jun 10. pii: S1568-7864(21)00114-2. [Epub ahead of print]105 103158

Damage-Net: A program for DNA repair meta-analysis identifies a network of novel repair genes that facilitate cancer evolution.

Aldo S Bader, Martin Bushell.

  The advent of genome-wide methods for identifying novel components in biological processes including CRISPR screens and proteomic studies, has transformed the research landscape within the biological sciences. However, each study normally investigates a single aspect of a process without integration of other published datasets. Here, we present Damage-Net, a program with a curated database of published results from a broad range of studies investigating DNA repair, that facilitates simple and quick meta-analysis. Users can incorporate their own datasets for analysis, and query genes of interest in the database. Importantly, this program also allows users to examine the correlation of genes of interest with pan-cancer patient survival and mutational burden effects. Interrogating these datasets revealed a network of genes that associated with cancer progression in adrenocortical carcinoma via facilitating mutational burden, ultimately contributing substantially to adrenocortical carcinoma's poor prognosis. Download at www.damage-net.co.uk.

Keywords:  Adrenocortical carcinoma; Damage-Net; Genomics; Mass spectrometry; Mutational burden; Proteomics; TCGA

DOI:  https://doi.org/10.1016/j.dnarep.2021.103158
Chemistry. 2021 Jun 21.

Comparative nucleosomal reactivity of 5-formyl-uridine and 5-formyl-cytidine.

Leander S Runtsch, Michael Stadlmeier, Alexander Schön, Markus Müller, Thomas Carell.

  5-Formyl-deoxyuridine (fdU) and 5-formyl-deoxycytidine (fdC) are formyl-containing nucleosides that are created by oxidative stress in differentiated cells. While fdU is almost exclusively an oxidative stress lesion formed from deoxythymidine (T), the situation for fdC is more complex. Next to formation as an oxidative lesion, it is particularly abundant in stem cells, where it is more frequently formed in an epigenetically important oxidation reaction performed by α-ketoglutarate dependent TET enzymes from 5-methyl-deoxycytidine (mdC). Recently, it was shown that genomic fdC and fdU can react with the ε-aminogroups of nucleosomal lysines to give Schiff base adducts that covalently link nucleosomes to genomic DNA. Here we show that fdU features a significantly higher reactivity towards lysine side chains compared with fdC. This result shows that depending on the amounts of fdC and fdU, oxidative stress may have a bigger impact on nucleosome binding than epigenetics.

Keywords:  DNA damage; DNA histone adducts; DNA repair; Epigenetics; Nucleosomes

DOI:  https://doi.org/10.1002/chem.202102159
Elife. 2021 Jun 23. pii: e69454. [Epub ahead of print]10

Exosome component 1 cleaves single-stranded DNA and sensitizes kidney renal clear cell carcinoma cells to poly(ADP-ribose) polymerase inhibitor.

Qiaoling Liu, Qi Xiao, Zhen Sun, Bo Wang, Lina Wang, Na Wang, Kai Wang, Chengli Song, Qingkai Yang.

  Targeting DNA repair pathway offers an important therapeutic strategy for Homo sapiens (human) cancers. However, the failure of DNA repair inhibitors to markedly benefit patients necessitates the development of new strategies. Here, we show that exosome component 1 (EXOSC1) promotes DNA damages and sensitizes human kidney renal clear cell carcinoma (KIRC) cells to DNA repair inhibitor. Considering that endogenous source of mutation (ESM) constantly assaults genomic DNA and likely sensitize human cancer cells to the inhibitor, we first analyzed the statistical relationship between the expression of individual genes and the mutations for KIRC. Among the candidates, EXOSC1 most notably promoted DNA damages and subsequent mutations via preferentially cleaving C site(s) in single-stranded DNA. Consistently, EXOSC1 was more significantly correlated with C>A transversions in coding strands than these in template strands in human KIRC. Notably, KIRC patients with high EXOSC1 showed a poor prognosis, and EXOSC1 sensitized human cancer cells to poly(ADP-ribose) polymerase inhibitor. These results show that EXOSC1 acts as an ESM in KIRC, and targeting EXOSC1 might be a potential therapeutic strategy.

Keywords:  cancer biology; mouse

DOI:  https://doi.org/10.7554/eLife.69454
Eur J Pharmacol. 2021 Jun 22. pii: S0014-2999(21)00423-4. [Epub ahead of print] 174270

Xanthine oxidoreductase promotes the progression of colitis-associated colorectal cancer by causing DNA damage and mediating macrophage M1 polarization.

Hongling Li, Chengjuan Zhang, Hao Zhang, Haitao Li.

  In addition to its pivotal role in purine metabolism, xanthine oxidoreductase (XOR) is one of the key enzymes involved in superoxide radical generation. Oxidative stress has been implicated in the etiology of colorectal cancer, but the contribution of XOR remains unclear. Here we investigated the role of XOR in colitis-associated colorectal cancer (CAC) and the underlying mechanisms. Using clinical samples, we demonstrated that XOR up-regulation was an early event in colonic carcinogenesis. Pharmacological inhibition of XOR effectively delayed the progression of CAC. Moreover, XOR activity positively correlated with tumor necrosis factor-alpha (TNFα) protein levels. Mechanistically, TNFα may activate XOR transcription via activator protein-1 and, thus, promote endogenous hydrogen peroxide generation, resulting in oxidative DNA damage in colon cancer cells. On the other hand, XOR may regulate the TNFα mRNA transcripts by mediating LPS-induced macrophage M1 polarization. Collectively, XOR promotes tumor development by programming the tumor microenvironment and stimulates CAC progression via DNA damage-induced genetic instability.

Keywords:  Colitis-associated colorectal cancer; Macrophages; Oxidative stress; Xanthine oxidoreductase

DOI:  https://doi.org/10.1016/j.ejphar.2021.174270
J Bacteriol. 2021 Jun 21. JB0015021

Electron microscopy reveals unexpected cytoplasm and envelope changes during thymineless death in Escherichia coli.

T V Pritha Rao, Andrei Kuzminov.

Bacterial rod-shaped cells experiencing irreparable chromosome damage should filament without other morphological changes. Thymineless death (TLD) strikes thymidine auxotrophs denied external thymine/thymidine (T) supplementation. Such T-starved cells cannot produce the DNA precursor dTTP and therefore stop DNA replication. Stalled replication forks in T-starved cells were always assumed to experience mysterious chromosome lesions, but recently TLD was found to be independent of origin-dependent DNA replication, with the chromosome still remaining the main TLD target. T-starvation also induces morphological changes, as if thymidine prevents cell envelope or cytoplasm problems that otherwise translate into chromosome damage. We used transmission electron microscopy to examine cytoplasm and envelope changes in T-starved E. coli cells, using treatment with a DNA gyrase inhibitor as a control for "pure" chromosome death. Besides the expected cell filamentation in response to both treatments, we see the following morphological changes specific for T-starvation, that might lead to chromosome damage: 1) significant cell widening; 2) nucleoid diffusion; 3) cell pole damage; 4) formation of numerous cytoplasmic bubbles. We conclude that T-starvation does impact both the cytoplasm and the cell envelope in ways that could potentially affect the chromosome. Importance Thymineless death is a dramatic and medically-important phenomenon, whose mechanisms remain a mystery. Unlike most other auxotrophs in the absence of the required supplement, thymidine-requiring E. coli mutants not only go static in the absence of thymidine, but rapidly die of chromosomal damage of unclear nature. Since this chromosomal damage is independent of replication, we examined fine morphological changes in cells undergoing thymineless death in order to identify what could potentially affect the chromosome. We report several cytoplasm and cell envelope changes that develop in thymidine-starved cells, but not in gyrase-inhibitor-treated cells (negative control), that could be linked to subsequent irreparable chromosome damage. This is the first electron microscopy study of cell undergoing "genetic death" due to irreparable chromosome lesions.

DOI: https://doi.org/10.1128/JB.00150-21
Sci Rep. 2021 Jun 25. 11(1): 13305

MCM2-7 complex is a novel druggable target for neuroendocrine prostate cancer.

En-Chi Hsu, Michelle Shen, Merve Aslan, Shiqin Liu, Manoj Kumar, Fernando Garcia-Marques, Holly M Nguyen, Rosalie Nolley, Sharon J Pitteri, Eva Corey, James D Brooks, Tanya Stoyanova.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer that rarely develops de novo in primary tumors and is commonly acquired during the development of treatment resistance. NEPC is characterized by gain of neuroendocrine markers and loss of androgen receptor (AR), making it resistant to current therapeutic strategies targeting the AR signaling axis. Here, we report that MCM2, MCM3, MCM4, and MCM6 (MCM2/3/4/6) are elevated in human NEPC and high levels of MCM2/3/4/6 are associated with liver metastasis and poor survival in prostate cancer patients. MCM2/3/4/6 are four out of six proteins that form a core DNA helicase (MCM2-7) responsible for unwinding DNA forks during DNA replication. Inhibition of MCM2-7 by treatment with ciprofloxacin inhibits NEPC cell proliferation and migration in vitro, significantly delays NEPC tumor xenograft growth, and partially reverses the neuroendocrine phenotype in vivo. Our study reveals the clinical relevance of MCM2/3/4/6 proteins in NEPC and suggests that inhibition of MCM2-7 may represent a new therapeutic strategy for NEPC.

DOI: https://doi.org/10.1038/s41598-021-92552-x