bims-numges 2020-11-08 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2020–11–08
24 papers selected by
Sean Rudd, Karolinska Institutet

Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε.
Ongoing replication forks delay nuclear envelope breakdown upon mitotic entry.
PRDX1 Counteracts Catastrophic Telomeric Cleavage Events That Are Triggered by DNA Repair Activities Post Oxidative Damage.
dUTPase inhibition confers susceptibility to a thymidylate synthase inhibitor in DNA-repair-defective human cancer cells.
CRM1 inhibitor anti-tumor activity is enhanced with salicylates by S-phase arrest and impaired DNA-damage repair.
The POLD1R689W variant increases the sensitivity of colorectal cancer cells to ATR and CHK1 inhibitors.
Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.
The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal.
Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation.
DNA helicases and their roles in cancer.
RAD51-Mediated DNA Homologous Recombination Is Independent of PTEN Mutational Status.
Combining PARP and DNA-PK Inhibitors With Irradiation Inhibits HPV-Negative Head and Neck Cancer Squamous Carcinoma Growth.
Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase η from DNA Polymerization toward RNA Polymerization.
Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors.
Targeting DNA damage response kinases in cancer therapy.
Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1.
Targeting BCL-2 in B-cell malignancies and overcoming therapeutic resistance.
Fission yeast condensin contributes to interphase chromatin organization and prevents transcription-coupled DNA damage.
Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47phox.
The MYC Paralog-PARP1 Axis as a Potential Therapeutic Target in MYC Paralog-Activated Small Cell Lung Cancer.
Targeting DNA Damage Response in Prostate and Breast Cancer.
Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases.
Rev7 loss alters cisplatin response and increases drug efficacy in chemotherapy-resistant lung cancer.
Dimeric Hold States of Anti-HIV Protein SAMHD1 are Redox Tunable.

Mol Cell. 2020 Oct 28. pii: S1097-2765(20)30723-1. [Epub ahead of print]

Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε.

Vladislav O Sviderskiy, Lili Blumenberg, Elizabeth Gorodetsky, Triantafyllia R Karakousi, Nicole Hirsh, Samantha W Alvarez, Erdem M Terzi, Efiyenia Kaparos, Gabrielle C Whiten, Shakirah Ssebyala, Peter Tonzi, Hannan Mir, Benjamin G Neel, Tony T Huang, Sylvia Adams, Kelly V Ruggles, Richard Possemato.

  Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.

Keywords:  CDK2; Cancer Metabolism; DNA damage; DNA replication; Iron-sulfur clusters; NFS1; POLE1; breast cancer

DOI:  https://doi.org/10.1016/j.molcel.2020.10.016
J Biol Chem. 2020 Nov 04. pii: jbc.RA120.015142. [Epub ahead of print]

Ongoing replication forks delay nuclear envelope breakdown upon mitotic entry.

Yoshitami Hashimoto, Hirofumi Tanaka.

  DNA replication is a major contributor to genomic instability and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S-phase of the cell cycle. If these stalled forks persist into the M-phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5'-triphosphate (Ara-CTP)-induced stalled forks were able to restart with the addition of excess dCTPduring early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase (CDK) activities, owing to which DNA replication and mitosis occur in a mutually exclusive and sequential manner.

Keywords:  DNA replication; Wee1/Myt1; Xenopus; cell cycle; chromatin; cyclin-dependent kinase (CDK); nuclear envelope; replication fork; replisome; xenopus egg extract

DOI:  https://doi.org/10.1074/jbc.RA120.015142
Cell Rep. 2020 Nov 03. pii: S2211-1247(20)31336-X. [Epub ahead of print]33(5): 108347

PRDX1 Counteracts Catastrophic Telomeric Cleavage Events That Are Triggered by DNA Repair Activities Post Oxidative Damage.

Wareed Ahmed, Joachim Lingner.

  Telomeres are prone to damage inflicted by reactive oxygen species (ROS). Oxidized telomeric DNA and nucleotide substrates inhibit telomerase, causing telomere shortening. In addition, ROS can induce telomeric single-strand DNA breaks (SSBs). The peroxiredoxin-PRDX1 is enriched in telomeric chromatin and this counteracts ROS-induced telomere damage. Here, we identify DNA processing after oxidative stress as a main source of telomeric DNA cleavage events in the absence of PRDX1. In PRDX1-depleted cells, poly(ADP-ribose) polymerase (PARP)-dependent telomeric repair is often incomplete, giving persistent SSBs that are converted into telomeric double-strand breaks during replication, leading to rapid telomere shortening. Interestingly, PARP1 inhibition dampens telomere shortening, triggering stabilization of the homologous recombination (HR) factor BRCA1 and RAD51-mediated repair of telomeres. Overall, our results reveal that, in the absence PRDX1, incomplete PARP1-dependent DNA repair and competition between PARP1 and HR cause ROS-induced telomeric catastrophe.

Keywords:  DNA damage; PARP inhibitors; homologous recombination; oxidative stress; telomeres

DOI:  https://doi.org/10.1016/j.celrep.2020.108347
Cancer Sci. 2020 Nov 02.

dUTPase inhibition confers susceptibility to a thymidylate synthase inhibitor in DNA-repair-defective human cancer cells.

Tatsushi Yokogawa, Wakako Yano, Sayaka Tsukioka, Akiko Osada, Takeshi Wakasa, Hiroyuki Ueno, Takuya Hoshino, Keisuke Yamamura, Akio Fujioka, Masayoshi Fukuoka, Shuichi Ohkubo, Kenichi Matsuo.

  Deficiency in DNA repair proteins confers susceptibility to DNA damage, making cancer cells vulnerable to various cancer chemotherapies. 5-Fluorouracil (5-FU) is an anticancer nucleoside analog that both inhibits thymidylate synthase (TS) and causes DNA damage via the misincorporation of FdUTP and dUTP into DNA under the conditions of dTTP depletion. However, the role of the DNA damage response to its antitumor activity is still unclear. To determine which DNA repair pathway contributes to DNA damage caused by 5-FU and uracil misincorporation, we examined cancer cells treated with 2'-deoxy-5-fluorouridine (FdUrd) in the presence of TAS-114, a highly potent inhibitor of dUTPase that restricts aberrant base misincorporation. Addition of TAS-114 increased FdUTP and dUTP levels in HeLa cells and facilitated 5-FU and uracil misincorporation into DNA, but did not alter TS inhibition or 5-FU incorporation into RNA. TAS-114 showed synergistic potentiation of FdUrd cytotoxicity and caused aberrant base misincorporation, leading to DNA damage and induced cell death even after short-term exposure to FdUrd. Base excision repair (BER) and homologous recombination (HR) were found to be involved in the DNA repair of 5-FU and uracil misincorporation caused by dUTPase inhibition in genetically modified chicken DT40 cell lines and siRNA-treated HeLa cells. These results suggest that BER and HR are major pathways that protect cells from the antitumor effects of massive incorporation of 5-FU and uracil. Further, dUTPase inhibition has the potential to maximize the antitumor activity of fluoropyrimidines in cancers that are defective in BER or HR.

Keywords:  5-fluorouracil/uracil misincorporation; DNA repair; base excision repair; dUTPase; homologous recombination

DOI:  https://doi.org/10.1111/cas.14718
Blood. 2020 Nov 02. pii: blood.2020009013. [Epub ahead of print]

CRM1 inhibitor anti-tumor activity is enhanced with salicylates by S-phase arrest and impaired DNA-damage repair.

Jithma Abeykoon, Xiaosheng Wu, Kevin Edward Nowakowski, Surendra Dasari, Jonas Paludo, S John Weroha, Chunling Hu, Xiaonan Hou, Jann N Sarkaria, Ann C Mladek, Jessica Lynne Phillips, Andrew Feldman, Aishwarya Ravindran, Rebecca L King, Justin C Boysen, Mary Stenson, Ryan M Carr, Michelle K Manske, Julian R Molina, Prashant Kapoor, Sameer A Parikh, Shaji K Kumar, Steven I Robinson, Jia Yu, Judy C Boughey, Liewei Wang, Matthew P Goetz, Fergus J Couch, Mrinal M Patnaik, Thomas E Witzig.

Chromosome region maintenance protein1 (CRM1) mediates protein export from the nucleus and is a new target for anti-cancer therapeutics. Broader application of KPT-330 (selinexor), a first in class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the anti-tumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid organ cancers ex vivo and in vivo. The K+CS combination was not toxic to non-malignant cells as compared to malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared to KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51 and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of PARP inhibitors further potentiates the K+CS anti-tumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

DOI: https://doi.org/10.1182/blood.2020009013
Sci Rep. 2020 Nov 03. 10(1): 18924

The POLD1R689W variant increases the sensitivity of colorectal cancer cells to ATR and CHK1 inhibitors.

Albert Job, Marina Tatura, Cora Schäfer, Veronika Lutz, Hanna Schneider, Brigitte Lankat-Buttgereit, Alexandra Zielinski, Kerstin Borgmann, Christian Bauer, Thomas M Gress, Malte Buchholz, Eike Gallmeier.

Inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in certain tumors. As previously shown, this is at least partly attributable to synthetic lethal interactions between ATR and POLD1, the catalytic subunit of the polymerase δ. Various POLD1 variants have been found in colorectal cancer, but their significance as therapeutic targets for ATR pathway inhibition remains unknown. Using CRISPR/Cas9 in the colorectal cancer cell line DLD-1, which harbors four POLD1 variants, we established heterozygous POLD1-knockout clones with exclusive expression of distinct variants to determine the functional relevance of these variants individually by assessing their impact on ATR pathway activation, DNA replication, and cellular sensitivity to inhibition of ATR or its effector kinase CHK1. Of the four variants analyzed, only POLD1R689W affected POLD1 function, as demonstrated by compensatory ATR pathway activation and impaired DNA replication. Upon treatment with ATR or CHK1 inhibitors, POLD1R689W strongly decreased cell survival in vitro, which was attributable at least partly to S phase impairment and apoptosis. Similarly, treatment with the ATR inhibitor AZD6738 inhibited growth of murine xenograft tumors, harboring the POLD1R689W variant, in vivo. Our POLD1-knockout model thus complements algorithm-based models to predict the pathogenicity of tumor-specific variants of unknown significance and illustrates a novel and potentially clinically relevant therapeutic approach using ATR/CHK1 inhibitors in POLD1-deficient tumors.

DOI: https://doi.org/10.1038/s41598-020-76033-1
Mol Cell. 2020 Oct 28. pii: S1097-2765(20)30719-X. [Epub ahead of print]

Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

Felix A Dingler, Meng Wang, Anfeng Mu, Christopher L Millington, Nina Oberbeck, Sam Watcham, Lucas B Pontel, Ashley N Kamimae-Lanning, Frederic Langevin, Camille Nadler, Rebecca L Cordell, Paul S Monks, Rui Yu, Nicola K Wilson, Asuka Hira, Kenichi Yoshida, Minako Mori, Yusuke Okamoto, Yusuke Okuno, Hideki Muramatsu, Yuichi Shiraishi, Masayuki Kobayashi, Toshinori Moriguchi, Tomoo Osumi, Motohiro Kato, Satoru Miyano, Etsuro Ito, Seiji Kojima, Hiromasa Yabe, Miharu Yabe, Keitaro Matsuo, Seishi Ogawa, Berthold Göttgens, Michael R G Hodskinson, Minoru Takata, Ketan J Patel.

  Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

Keywords:  DNA damage; ageing; bone marrow failure; cancer; formaldehyde; hematopoiesis; hematopoietic stem cells; immunodeficiency; mutagenesis; oncometabolite

DOI:  https://doi.org/10.1016/j.molcel.2020.10.012
Nat Commun. 2020 Nov 06. 11(1): 5643

The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal.

Karol Kramarz, Kamila Schirmeisen, Virginie Boucherit, Anissia Ait Saada, Claire Lovo, Benoit Palancade, Catherine Freudenreich, Sarah A E Lambert.

Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences.

DOI: https://doi.org/10.1038/s41467-020-19516-z
J Mol Biol. 2020 Nov 03. pii: S0022-2836(20)30616-1. [Epub ahead of print]

Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation.

Yoshihiro Matsumoto, Rhys C Brooks, Aleksandr Sverzhinsky, John M Pascal, Alan E Tomkinson.

  More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5' end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.

Keywords:  DNA flap cleavage; DNA ligation; DNA polymerase δ; DNA replication; DNA synthesis; DNA-protein complex; primase; proliferating cell nuclear antigen; replication fork; sliding clamp

DOI:  https://doi.org/10.1016/j.jmb.2020.10.032
DNA Repair (Amst). 2020 Oct 17. pii: S1568-7864(20)30253-6. [Epub ahead of print]96 102994

DNA helicases and their roles in cancer.

Srijita Dhar, Arindam Datta, Robert M Brosh.

  DNA helicases, known for their fundamentally important roles in genomic stability, are high profile players in cancer. Not only are there monogenic helicase disorders with a strong disposition to cancer, it is well appreciated that helicase variants are associated with specific cancers (e.g., breast cancer). Flipping the coin, DNA helicases are frequently overexpressed in cancerous tissues and reduction in helicase gene expression results in reduced proliferation and growth capacity, as well as DNA damage induction and apoptosis of cancer cells. The seminal roles of helicases in the DNA damage and replication stress responses, as well as DNA repair pathways, validate their vital importance in cancer biology and suggest their potential values as targets in anti-cancer therapy. In recent years, many laboratories have characterized the specialized roles of helicase to resolve transcription-replication conflicts, maintain telomeres, mediate cell cycle checkpoints, remodel stalled replication forks, and regulate transcription. In vivo models, particularly mice, have been used to interrogate helicase function and serve as a bridge for preclinical studies that may lead to novel therapeutic approaches. In this review, we will summarize our current knowledge of DNA helicases and their roles in cancer, emphasizing the latest developments.

Keywords:  Aging; Cancer; DNA repair; Disease; Genomic instability; Helicase; Replication; Transcription

DOI:  https://doi.org/10.1016/j.dnarep.2020.102994
Cancers (Basel). 2020 Oct 29. pii: E3178. [Epub ahead of print]12(11):

RAD51-Mediated DNA Homologous Recombination Is Independent of PTEN Mutational Status.

Asha Sinha, Ali Saleh, Raelene Endersby, Shek H Yuan, Chirayu R Chokshi, Kevin R Brown, Bozena Kuzio, Tiina Kauppinen, Sheila K Singh, Suzanne J Baker, Peter J McKinnon, Sachin Katyal.

  PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors.
METHODS: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs.
RESULTS: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status.
CONCLUSIONS: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.

Keywords:  DNA damage; HRD; PARP inhibitor; PTEN; RAD51; RAD51 foci; RNA expression; alkaline comet assay; base excision repair; brain tumor initiating cells; combination therapy; glioblastoma multiforme; homologous recombination; olaparib; synthetic lethality; γH2AX foci

DOI:  https://doi.org/10.3390/cancers12113178
Front Genet. 2020 ;11 1036

Combining PARP and DNA-PK Inhibitors With Irradiation Inhibits HPV-Negative Head and Neck Cancer Squamous Carcinoma Growth.

Ling Zeng, Drexell Hunter Boggs, Chuan Xing, Zhuo Zhang, Joshua C Anderson, Narendra Wajapeyee, Chris Veale, Markus Bredel, Lewis Z Shi, James A Bonner, Christopher D Willey, Eddy S Yang.

  Novel targeted agents to inhibit DNA repair pathways to sensitize tumors to irradiation (IR) are being investigated as an alternative to chemoradiation for locally advanced human papilloma virus negative (HPV-negative) head and neck squamous cell carcinoma (HNSCC). Two well-characterized targets that, when inhibited, exhibit potent IR sensitization are PARP1 and DNA-PKcs. However, their cooperation in sensitizing HPV-negative HNSCC to IR remains to be explored given that PARP1 and DNA-Pk CS bind to unresected stalled DNA replication forks and cooperate to recruit XRCC1 to facilitate double-strand break repair. Here, we show that the combination of the DNA-PK inhibitor NU7441 and the PARP inhibitor olaparib significantly decrease proliferation (61-78%) compared to no reduction with either agent alone (p < 0.001) in both SCC1 and SCC6 cell lines. Adding IR to the combination further decreased cell proliferation (91-92%, p < 0.001) in SCC1 and SCC6. Similar results were observed using long-term colony formation assays [dose enhancement ratio (DER) 2.3-3.2 at 4Gy, p < 0.05]. Reduced cell survival was attributed to increased apoptosis and G2/M cell cycle arrest. Kinomic analysis using tyrosine (PTK) and serine/threonine (STK) arrays reveals that combination treatment results in the most potent inhibition of kinases involved in the CDK and ERK pathways compared to either agent alone. In vivo, a significant delay of tumor growth was observed in UM-SCC1 xenografts receiving IR with olaparib and/or NU7441, which was similar to the cisplatin-IR group. Both regimens were less toxic than cisplatin-IR as assessed by loss of mouse body weight. Taken together, these results demonstrate that the combination of NU7441 and olaparib with IR enhances HPV-negative HNSCC inhibition in both cell culture and in mice, suggesting a potential innovative combination for effectively treating patients with HPV-negative HNSCC.

Keywords:  DNA damage; DNA repair; DNA-PK inhibitors; PARP inhibitors; homologous recombination; non-homologous end-joining

DOI:  https://doi.org/10.3389/fgene.2020.01036
Int J Mol Sci. 2020 Nov 04. pii: E8248. [Epub ahead of print]21(21):

Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase η from DNA Polymerization toward RNA Polymerization.

Eva Balint, Ildiko Unk.

  Polymerase eta (Polη) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Polη in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Polη proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here, we show that manganese triggers drastic changes in the activity of Polη. Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by ~400-2000-fold opposite undamaged DNA, and ~3000 and ~6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination is almost lost during DNA synthesis by Polη with manganese. Moreover, Polη shows strong preference for manganese during RNA synthesis even at a 25-fold excess magnesium concentration. Based on this, we suggest that a new regulatory mechanism, selective metal cofactor utilization, modulates the specificity of Polη helping it to perform distinct activities needed for its separate functions during replication and transcription.

Keywords:  enzyme kinetics; manganese; polymerase η; yeast

DOI:  https://doi.org/10.3390/ijms21218248
PLoS Genet. 2020 Nov 02. 16(11): e1009176

Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors.

Emily M Schleicher, Ashna Dhoonmoon, Lindsey M Jackson, Kristen E Clements, Coryn L Stump, Claudia M Nicolae, George-Lucian Moldovan.

The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFβ signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.

DOI: https://doi.org/10.1371/journal.pgen.1009176
Mutat Res. 2020 Nov 01. pii: S0027-5107(20)30058-0. [Epub ahead of print]821 111725

Targeting DNA damage response kinases in cancer therapy.

Vanesa Gottifredi.

Cancer cells die when their decimated DNA damage response (DDR) unsuccessfully handles DNA damage. This notion has been successfully exploited when targeting PARP (poly ADP-ribose polymerase) in homologous recombination-deficient cells. With the greater understanding of DDR achieved in the last decade, new cancer therapy targets within the DDR network have been identified. Intriguingly, many of the molecules that have advanced into clinical trials are inhibitors of DDR kinases. This special issue is devoted to discussing the mechanism of cell killing and the level of success that such inhibitors have reached in pre-clinical and clinical settings.

DOI: https://doi.org/10.1016/j.mrfmmm.2020.111725
PLoS One. 2020 ;15(11): e0240932

Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1.

Guillaume Gaullier, Genevieve Roberts, Uma M Muthurajan, Samuel Bowerman, Johannes Rudolph, Jyothi Mahadevan, Asmita Jha, Purushka S Rae, Karolin Luger.

Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

DOI: https://doi.org/10.1371/journal.pone.0240932
Cell Death Dis. 2020 Nov 02. 11(11): 941

Targeting BCL-2 in B-cell malignancies and overcoming therapeutic resistance.

Isha Kapoor, Juraj Bodo, Brian T Hill, Eric D Hsi, Alexandru Almasan.

Defects in apoptosis can promote tumorigenesis and impair responses of malignant B cells to chemotherapeutics. Members of the B-cell leukemia/lymphoma-2 (BCL-2) family of proteins are key regulators of the intrinsic, mitochondrial apoptotic pathway. Overexpression of antiapoptotic BCL-2 family proteins is associated with treatment resistance and poor prognosis. Thus, inhibition of BCL-2 family proteins is a rational therapeutic option for malignancies that are dependent on antiapoptotic BCL-2 family proteins. Venetoclax (ABT-199, GDC-0199) is a highly selective BCL-2 inhibitor that represents the first approved agent of this class and is currently widely used in the treatment of chronic lymphocytic leukemia (CLL) as well as acute myeloid leukemia (AML). Despite impressive clinical activity, venetoclax monotherapy for a prolonged duration can lead to drug resistance or loss of dependence on the targeted protein. In this review, we provide an overview of the mechanism of action of BCL-2 inhibition and the role of this approach in the current treatment paradigm of B-cell malignancies. We summarize the drivers of de novo and acquired resistance to venetoclax that are closely associated with complex clonal shifts, interplay of expression and interactions of BCL-2 family members, transcriptional regulators, and metabolic modulators. We also examine how tumors initially resistant to venetoclax become responsive to it following prior therapies. Here, we summarize preclinical data providing a rationale for efficacious combination strategies of venetoclax to overcome therapeutic resistance by a targeted approach directed against alternative antiapoptotic BCL-2 family proteins (MCL-1, BCL-xL), compensatory prosurvival pathways, epigenetic modifiers, and dysregulated cellular metabolism/energetics for durable clinical remissions.

DOI: https://doi.org/10.1038/s41419-020-03144-y
Genome Biol. 2020 Nov 05. 21(1): 272

Fission yeast condensin contributes to interphase chromatin organization and prevents transcription-coupled DNA damage.

Yasutaka Kakui, Christopher Barrington, David J Barry, Tereza Gerguri, Xiao Fu, Paul A Bates, Bhavin S Khatri, Frank Uhlmann.

   BACKGROUND: Structural maintenance of chromosomes (SMC) complexes are central organizers of chromatin architecture throughout the cell cycle. The SMC family member condensin is best known for establishing long-range chromatin interactions in mitosis. These compact chromatin and create mechanically stable chromosomes. How condensin contributes to chromatin organization in interphase is less well understood.
RESULTS: Here, we use efficient conditional depletion of fission yeast condensin to determine its contribution to interphase chromatin organization. We deplete condensin in G2-arrested cells to preempt confounding effects from cell cycle progression without condensin. Genome-wide chromatin interaction mapping, using Hi-C, reveals condensin-mediated chromatin interactions in interphase that are qualitatively similar to those observed in mitosis, but quantitatively far less prevalent. Despite their low abundance, chromatin mobility tracking shows that condensin markedly confines interphase chromatin movements. Without condensin, chromatin behaves as an unconstrained Rouse polymer with excluded volume, while condensin constrains its mobility. Unexpectedly, we find that condensin is required during interphase to prevent ongoing transcription from eliciting a DNA damage response.
CONCLUSIONS: In addition to establishing mitotic chromosome architecture, condensin-mediated long-range chromatin interactions contribute to shaping chromatin organization in interphase. The resulting structure confines chromatin mobility and protects the genome from transcription-induced DNA damage. This adds to the important roles of condensin in maintaining chromosome stability.

Keywords:  Chromosome architecture; Condensin; DNA damage; Interphase chromatin; Polymer physics; S. pombe; Transcription

DOI:  https://doi.org/10.1186/s13059-020-02183-0
Proc Natl Acad Sci U S A. 2020 Nov 02. pii: 202017830. [Epub ahead of print]

Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47phox.

Christos Polytarchou, Maria Hatziapostolou, Tung On Yau, Niki Christodoulou, Philip W Hinds, Filippos Kottakis, Ioannis Sanidas, Philip N Tsichlis.

  Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidant N-acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression in p53 -/- and INK4a -/-/Arf -/- mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47phox, which results in NADPH oxidase activation. Expression of Akt3 in p47 phox-/- MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47phox, but not by the phosphorylation site mutant of p47phox In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important, Akt3 alterations correlate with a higher frequency of mutation of p53, suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.

Keywords:  Akt isoforms; DNA damage; NADPH oxidase; cancer; oxidative stress

DOI:  https://doi.org/10.1073/pnas.2017830117
Front Oncol. 2020 ;10 565820

The MYC Paralog-PARP1 Axis as a Potential Therapeutic Target in MYC Paralog-Activated Small Cell Lung Cancer.

Xing Bian, Xiaolin Wang, Qiuyan Zhang, Liying Ma, Guozhen Cao, Ao Xu, Jinhua Han, Jun Huang, Wenchu Lin.

  Poly (ADP-ribose) polymerase 1 (PARP1) is highly expressed in small cell lung cancer (SCLC) and has emerged as an attractive target for treatment of SCLC. However, the clinical significance of PARP1 expression in SCLC remains elusive. In this study, we showed that high PARP1 expression was associated with better overall survival (OS), and was positively correlated with the expression of MYC paralogs in patients with SCLC. We demonstrated that PARP1 was transcriptionally regulated by MYC paralogs. Integrative analysis of multiple RNA-seq data sets indicated that DNA damage response (DDR) genes involved in the replication stress response (RSR) and homologous recombination (HR) repair pathways were highly enriched in MYC paralog-addicted SCLC cell models and in human SCLC specimens. Targeting the MYC paralog-PARP1 axis with concomitant BET and PARP inhibition resulted in synergistic effects in MYC paralog-activated SCLC. Our study identified a critical PARP1 regulatory pathway, and provided evidence for a rational combination treatment strategy for MYC paralog-activated SCLC.

Keywords:  BET; DNA damage response; MYC paralog; PARP1; small cell lung cancer

DOI:  https://doi.org/10.3389/fonc.2020.565820
Int J Mol Sci. 2020 Nov 04. pii: E8273. [Epub ahead of print]21(21):

Targeting DNA Damage Response in Prostate and Breast Cancer.

Antje M Wengner, Arne Scholz, Bernard Haendler.

  Steroid hormone signaling induces vast gene expression programs which necessitate the local formation of transcription factories at regulatory regions and large-scale alterations of the genome architecture to allow communication among distantly related cis-acting regions. This involves major stress at the genomic DNA level. Transcriptionally active regions are generally instable and prone to breakage due to the torsional stress and local depletion of nucleosomes that make DNA more accessible to damaging agents. A dedicated DNA damage response (DDR) is therefore essential to maintain genome integrity at these exposed regions. The DDR is a complex network involving DNA damage sensor proteins, such as the poly(ADP-ribose) polymerase 1 (PARP-1), the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the ataxia-telangiectasia-mutated (ATM) kinase and the ATM and Rad3-related (ATR) kinase, as central regulators. The tight interplay between the DDR and steroid hormone receptors has been unraveled recently. Several DNA repair factors interact with the androgen and estrogen receptors and support their transcriptional functions. Conversely, both receptors directly control the expression of agents involved in the DDR. Impaired DDR is also exploited by tumors to acquire advantageous mutations. Cancer cells often harbor germline or somatic alterations in DDR genes, and their association with disease outcome and treatment response led to intensive efforts towards identifying selective inhibitors targeting the major players in this process. The PARP-1 inhibitors are now approved for ovarian, breast, and prostate cancer with specific genomic alterations. Additional DDR-targeting agents are being evaluated in clinical studies either as single agents or in combination with treatments eliciting DNA damage (e.g., radiation therapy, including targeted radiotherapy, and chemotherapy) or addressing targets involved in maintenance of genome integrity. Recent preclinical and clinical findings made in addressing DNA repair dysfunction in hormone-dependent and -independent prostate and breast tumors are presented. Importantly, the combination of anti-hormonal therapy with DDR inhibition or with radiation has the potential to enhance efficacy but still needs further investigation.

Keywords:  ATM; ATR; DNA damage response; DNA repair; DNA-PKcs; PARP-1; breast cancer; hormone-dependent; prostate cancer; radiation

DOI:  https://doi.org/10.3390/ijms21218273
Nucleic Acids Res. 2020 Nov 02. pii: gkaa948. [Epub ahead of print]

Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases.

Valentina Riva, Anna Garbelli, Federica Casiraghi, Francesca Arena, Claudia Immacolata Trivisani, Assunta Gagliardi, Luca Bini, Martina Schroeder, Antonio Maffia, Simone Sabbioneda, Giovanni Maga.

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5' of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols β and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.

DOI: https://doi.org/10.1093/nar/gkaa948
Proc Natl Acad Sci U S A. 2020 Nov 03. pii: 202016067. [Epub ahead of print]

Rev7 loss alters cisplatin response and increases drug efficacy in chemotherapy-resistant lung cancer.

Faye-Marie Vassel, Ke Bian, Graham C Walker, Michael T Hemann.

  Cisplatin is a standard of care for lung cancer, yet platinum therapy rarely results in substantial tumor regression or a dramatic extension in patient survival. Here, we examined whether targeting Rev7 (also referred to as Mad2B, Mad2L2, and FANCV), a component of the translesion synthesis (TLS) machinery, could potentiate the action of cisplatin in non-small cell lung cancer (NSCLC) treatment. Rev7 loss led to an enhanced tumor cell sensitivity to cisplatin and dramatically improved chemotherapeutic response in a highly drug-resistant mouse model of NSCLC. While cisplatin monotherapy resulted in tumor cell apoptosis, Rev7 deletion promoted a cisplatin-induced senescence phenotype. Moreover, Rev7 deficiency promoted greater cisplatin sensitivity than that previously shown following targeting of other Pol ζ-proteins, suggesting that Pol ζ-dependent and -independent roles of Rev7 are relevant to cisplatin response. Thus, targeting Rev7 may represent a unique strategy for altering and enhancing chemotherapeutic response.

Keywords:  Rev7; chemotherapy; cisplatin; lung cancer; translesion synthesis

DOI:  https://doi.org/10.1073/pnas.2016067117
J Chem Inf Model. 2020 Nov 02.

Dimeric Hold States of Anti-HIV Protein SAMHD1 are Redox Tunable.

Gauri Thapa, Akash Bhattacharya, Swati Bhattacharya.

The sterile α motif and histidine-aspartate domain-containing protein 1 (or SAMHD1) is a human protein that restricts HIV-1 in select terminally differentiated cells of the immune system by acting as a triphosphohydrolase, lowering dNTP pools. The functionally active form of the protein has been reported to be a tetramer where adjacent monomers are linked by GTP-Mg+2-dNTP cross-bridges, although some studies have also suggested the existence of a dimeric form of this protein. In this in silico study, we have investigated the stability of SAMHD1 dimeric "hold states" as well as the role of intrachain disulfide bonds. We have found that dimeric-GTP bound SAMHD1 can exist as a viable meso-stable hold state with extensive motion in the C-terminal domain, which is quenched upon tetramer assembly. The redox switch comprised of residues C341, C350, and C522 was found to be linked to changes in the allosteric site, suggesting a mechanism for initiating tetramer disassembly. The disulfide state of the protein dimer (C341-S-S-C350 vs C341-S-S-C522) also plays a role in driving affinities for the allosteric dATP molecules. In sum, our results suggest a model wherein dimeric SAMHD1 exists as a "hold state" in the cytosol, ready to be activated by dATP concentrations, where the "tunability" of this activation is further regulated by the redox state of the enzyme.

DOI: https://doi.org/10.1021/acs.jcim.0c00629