bims-numges 2022-03-13 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–03–13
twenty-two papers selected by
Sean Rudd, Karolinska Institutet

Inhibition of oxidized nucleotide sanitation by TH1579 and conventional chemotherapy cooperatively enhance oxidative DNA-damage and survival in AML.
Replication stress induced by the ribonucleotide reductase inhibitor guanazole, triapine, and gemcitabine in fission yeast.
ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination.
Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.
Targeting of RecQ Helicases as a Novel Therapeutic Strategy for Ovarian Cancer.
Cyclin-dependent kinases in DNA damage response.
In Vivo Targeting Replication Protein A for Cancer Therapy.
Telomeric replication stress: the beginning and the end for alternative lengthening of telomeres cancers.
Active DNA damage eviction by HLTF stimulates nucleotide excision repair.
Cell cycle involvement in cancer therapy; WEE1 kinase, a potential target as therapeutic strategy.
The Dynamic Instability of the Aneuploid Genome.
Cell cycle checkpoints and beyond: Exploiting the ATR/CHK1/WEE1 pathway for the treatment of PARP inhibitor-resistant cancer.
Tumor-intrinsic PD-L1 promotes DNA repair in distinct cancers and suppresses PARP inhibitor-induced synthetic lethality.
Increased replication stress and R-loop accumulation in EGFRvIII-expressing glioblastoma present new therapeutic opportunities.
A distinctive requirement for p53 in the genome protective Topoisomerase 2a-dependent G2 arrest in hTERT positive cancer cells.
Cleavage of Abasic Sites in DNA by an Aminoquinoxaline Compound: Augmented Cytotoxicity and DNA Damage in Combination with an Anticancer Drug Chlorambucil in Human Colorectal Carcinoma Cells.
Investigating the Conformational Dynamics of a Y-Family DNA Polymerase during Its Folding and Binding to DNA and a Nucleotide.
Inhibiting nonhomologous end-joining repair would promote the antitumor activity of gemcitabine in nonsmall cell lung cancer cell lines.
The rate of formation and stability of abasic site interstrand crosslinks in the DNA duplex.
Inhibitors of PARP: Number crunching and structure gazing.
Four-Year Disease-Free Remission in a Patient With POLE Mutation-Associated Colorectal Cancer Treated Using Anti-PD-1 Therapy.
In vivo anti-tumor effect of PARP inhibition in IDH1/2 mutant MDS/AML resistant to targeted inhibitors of mutant IDH1/2.

Mol Cancer Ther. 2022 Feb 28. pii: molcanther.0185.2021. [Epub ahead of print]

Inhibition of oxidized nucleotide sanitation by TH1579 and conventional chemotherapy cooperatively enhance oxidative DNA-damage and survival in AML.

Anders Centio, Montserrat Estruch, Kristian Reckzeh, Kumar Sanjiv, Camilla Vittori, Sophia Engelhard, Ulrika Warpman Berglund, Thomas Helleday, Kim Theilgaard-Mönch.

Currently, the majority of AML patients still die of their disease due to primary resistance or relapse toward conventional ROS- and DNA damage-inducing chemotherapy regimens. Herein, we explored the therapeutic potential to enhance chemotherapy response in AML, by targeting the ROS scavenger enzyme MutT homolog 1 (MTH1, NUDT1), which protects cellular integrity through prevention of fatal chemotherapy-induced oxidative DNA damage. We demonstrate that MTH1 is a potential druggable target expressed by the majority of AML patients and the inv(16)/KITD816Y AML mouse model mimicking the genetics of AML patients exhibiting poor response to standard chemotherapy (i.e. anthracycline & cytarabine). Strikingly, combinatorial treatment of inv(16)/KITD816Y AML cells with the MTH1 inhibitor TH1579 and ROS- and DNA damage-inducing standard chemotherapy induced growth arrest and incorporated oxidized nucleotides into DNA leading to significantly increased DNA damage. Consistently, TH1579 and chemotherapy synergistically inhibited growth of clonogenic inv(16)/KITD816Y AML cells without substantially inhibiting normal clonogenic bone marrow cells. In addition, combinatorial treatment of inv(16)/KITD816Y AML mice with TH1579 and chemotherapy significantly reduced AML burden and prolonged survival compared to untreated or single treated mice. In conclusion, our study provides a rationale for future clinical studies combining standard AML chemotherapy with TH1579 to boost standard chemotherapy response in AML patients. Moreover, other cancer entities treated with ROS- and DNA damage-inducing chemo- or radiation therapies might benefit therapeutically from complementary treatment with TH1579.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0185
FEMS Yeast Res. 2022 Mar 09. pii: foac014. [Epub ahead of print]

Replication stress induced by the ribonucleotide reductase inhibitor guanazole, triapine, and gemcitabine in fission yeast.

Mashael Y Alyahya, Saman Khan, Sankhadip Bhadra, Rittu E Samuel, Yong-Jie Xu.

  Schizosaccharomyces pombe is an established yeast model for studying the cellular mechanisms conserved in humans, such as the DNA replication checkpoint. The replication checkpoint deals with replication stress caused by numerous endogenous and exogenous factors that perturb fork movement. If undealt with, perturbed forks collapse, causing chromosomal DNA damage or cell death. Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase (RNR) commonly used in checkpoint studies. It produces replication stress by depleting dNTPs, which slows the movement of ongoing forks and thus activates the replication checkpoint. However, HU also causes side effects such as oxidative stress, particularly under chronic exposure conditions, which complicates the studies. To find a drug that generates replication stress more specifically, we tested three other RNR inhibitors gemcitabine, guanazole, and triapine in S. pombe under various experimental conditions. Our results show that guanazole and triapine can produce replication stress more specifically than HU under chronic, not acute drug treatment conditions. Therefore, using the two drugs in spot assay, the method commonly used for testing drug sensitivity in yeasts, should benefit the checkpoint studies in S. pombe and likely the research in other model systems.

Keywords:   S. pombe ; Cds1; Chk1; Gemcitabine; Genome stability; Guanazole; Hydroxyurea; Oxidative Stress; Rad3; Ribonucleotide Reductase; The DNA Replication Checkpoint; Triapine

DOI:  https://doi.org/10.1093/femsyr/foac014
Nucleic Acids Res. 2022 Mar 07. pii: gkac160. [Epub ahead of print]

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination.

Doohyung Lee, Katja Apelt, Seong-Ok Lee, Hsin-Ru Chan, Martijn S Luijsterburg, Justin W C Leung, Kyle M Miller.

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.

DOI: https://doi.org/10.1093/nar/gkac160
Front Oncol. 2022 ;12 828684

Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.

Amirali B Bukhari, Gordon K Chan, Armin M Gamper.

  Cancer cells typically heavily rely on the G2/M checkpoint to survive endogenous and exogenous DNA damage, such as genotoxic stress due to genome instability or radiation and chemotherapy. The key regulator of the G2/M checkpoint, the cyclin-dependent kinase 1 (CDK1), is tightly controlled, including by its phosphorylation state. This posttranslational modification, which is determined by the opposing activities of the phosphatase cdc25 and the kinase Wee1, allows for a more rapid response to cellular stress than via the synthesis or degradation of modulatory interacting proteins, such as p21 or cyclin B. Reducing Wee1 activity results in ectopic activation of CDK1 activity and drives premature entry into mitosis with unrepaired or under-replicated DNA and causing mitotic catastrophe. Here, we review efforts to use small molecule inhibitors of Wee1 for therapeutic purposes, including strategies to combine Wee1 inhibition with genotoxic agents, such as radiation therapy or drugs inducing replication stress, or inhibitors of pathways that show synthetic lethality with Wee1. Furthermore, it become increasingly clear that Wee1 inhibition can also modulate therapeutic immune responses. We will discuss the mechanisms underlying combination treatments identifying both cell intrinsic and systemic anti-tumor activities.

Keywords:  DNA damage response (DDR); Wee1; cancer therapy; cell cycle; kinase; synthetic lethality

DOI:  https://doi.org/10.3389/fonc.2022.828684
Cancers (Basel). 2022 Feb 26. pii: 1219. [Epub ahead of print]14(5):

Targeting of RecQ Helicases as a Novel Therapeutic Strategy for Ovarian Cancer.

Jyotirindra Maity, Sachi Horibata, Grant Zurcher, Jung-Min Lee.

  RecQ helicases are essential for DNA replication, recombination, DNA damage repair, and other nucleic acid metabolic pathways required for normal cell growth, survival, and genome stability. More recently, RecQ helicases have been shown to be important for replication fork stabilization, one of the major mechanisms of PARP inhibitor resistance. Cancer cells often have upregulated helicases and depend on these enzymes to repair rapid growth-promoted DNA lesions. Several studies are now evaluating the use of RecQ helicases as potential biomarkers of breast and gynecologic cancers. Furthermore, RecQ helicases have attracted interest as possible targets for cancer treatment. In this review, we discuss the characteristics of RecQ helicases and their interacting partners that may be utilized for effective treatment strategies (as cancers depend on helicases for survival). We also discuss how targeting helicase in combination with DNA repair inhibitors (i.e., PARP and ATR inhibitors) can be used as novel approaches for cancer treatment to increase sensitivity to current treatment to prevent rise of treatment resistance.

Keywords:  BLM; RECQL4; RecQ helicases; WRN; novel treatment; ovarian cancer

DOI:  https://doi.org/10.3390/cancers14051219
Biochim Biophys Acta Rev Cancer. 2022 Mar 07. pii: S0304-419X(22)00041-5. [Epub ahead of print] 188716

Cyclin-dependent kinases in DNA damage response.

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

  The cyclin-dependent kinase (CDK) family plays a critical role in a variety of signaling pathways that regulate transcription and cell-cycle progression. Recently, the role of CDKs in DNA damage response (DDR) has emerged. CDKs affect both damage signaling and DNA repair, contributing to the fidelity of the cell division process as well as the maintenance of genomic integrity following DNA damage. This is due to the modulatory role of CDKs on double-strand break repair (DSBR) components, including their influence on enzymes involved in homologous recombination (HR) and non-homologous end-joining (NHEJ). In this review, the impact of CDKs on DDR and DNA repair is discussed.

Keywords:  Cell-cycle checkpoint; Cyclin-dependent kinase (CDK); DNA damage response (DDR); DNA repair

DOI:  https://doi.org/10.1016/j.bbcan.2022.188716
Front Oncol. 2022 ;12 826655

In Vivo Targeting Replication Protein A for Cancer Therapy.

Pamela S VanderVere-Carozza, Navnath S Gavande, Shadia I Jalal, Karen E Pollok, Elmira Ekinci, Joshua Heyza, Steve M Patrick, Andi Masters, John J Turchi, Katherine S Pawelczak.

  Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination, and the DNA damage response (DDR). Retrospective analysis of lung cancer patient data demonstrates high RPA expression as a negative prognostic biomarker for overall survival in smoking-related lung cancers. Similarly, relative expression of RPA is a predictive marker for response to chemotherapy. These observations are consistent with the increase in RPA expression serving as an adaptive mechanism that allows tolerance of the genotoxic stress resulting from carcinogen exposure. We have developed second-generation RPA inhibitors (RPAis) that block the RPA-DNA interaction and optimized formulation for in vivo analyses. Data demonstrate that unlike first-generation RPAis, second-generation molecules show increased cellular permeability and induce cell death via apoptosis. Second-generation RPAis elicit single-agent in vitro anticancer activity across a broad spectrum of cancers, and the cellular response suggests existence of a threshold before chemical RPA exhaustion induces cell death. Chemical RPA inhibition potentiates the anticancer activity of a series of DDR inhibitors and traditional DNA-damaging cancer therapeutics. Consistent with chemical RPA exhaustion, we demonstrate that the effects of RPAi on replication fork dynamics are similar to other known DDR inhibitors. An optimized formulation of RPAi NERx 329 was developed that resulted in single-agent anticancer activity in two non-small cell lung cancer models. These data demonstrate a unique mechanism of action of RPAis eliciting a state of chemical RPA exhaustion and suggest they will provide an effective therapeutic option for difficult-to-treat lung cancers.

Keywords:  DNA damage response; DNA repair and cancer; DNA repair inhibitors; Replication Protein A; Replication Stress Response

DOI:  https://doi.org/10.3389/fonc.2022.826655
Open Biol. 2022 Mar;12(3): 220011

Telomeric replication stress: the beginning and the end for alternative lengthening of telomeres cancers.

Robert Lu, Hilda A Pickett.

  Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Telomeric DNA comprises terminal tracts of G-rich tandem repeats, which are inherently difficult for the replication machinery to navigate. Structural aberrations that promote activation of the alternative lengthening of telomeres (ALT) pathway of telomere maintenance exacerbate replication stress at ALT telomeres, driving fork stalling and fork collapse. This form of telomeric DNA damage perpetuates recombination-mediated repair pathways and break-induced telomere synthesis. The relationship between replication stress and DNA repair is tightly coordinated for the purpose of regulating telomere length in ALT cells, but has been shown to be experimentally manipulatable. This raises the intriguing possibility that induction of replication stress can be used as a means to cause toxic levels of DNA damage at ALT telomeres, thereby selectively disrupting the viability of ALT cancers.

Keywords:  ALT; alternative lengthening of telomeres; replication stress; telomere maintenance

DOI:  https://doi.org/10.1098/rsob.220011
Mol Cell. 2022 Mar 04. pii: S1097-2765(22)00158-7. [Epub ahead of print]

Active DNA damage eviction by HLTF stimulates nucleotide excision repair.

Marvin van Toorn, Yasemin Turkyilmaz, Sueji Han, Di Zhou, Hyun-Suk Kim, Irene Salas-Armenteros, Mihyun Kim, Masaki Akita, Franziska Wienholz, Anja Raams, Eunjin Ryu, Sukhyun Kang, Arjan F Theil, Karel Bezstarosti, Maria Tresini, Giuseppina Giglia-Mari, Jeroen A Demmers, Orlando D Schärer, Jun-Hyuk Choi, Wim Vermeulen, Jurgen A Marteijn.

  Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a "cut-and-patch"-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3'-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity.

Keywords:  DNA damage; HLTF; TFIIH; UV damage response; damage eviction; genome stability; nucleotide excision repair; post-replication repair; repair synthesis

DOI:  https://doi.org/10.1016/j.molcel.2022.02.020
Mutat Res. 2022 Feb 19. pii: S0027-5107(22)00003-3. [Epub ahead of print]824 111776

Cell cycle involvement in cancer therapy; WEE1 kinase, a potential target as therapeutic strategy.

Sajjad Vakili-Samiani, Omid Joodi Khanghah, Elham Gholipour, Fatemeh Najafi, Elham Zeinalzadeh, Parisa Samadi, Parisa Sarvarian, Shiva Pourvahdani, Shohre Karimi Kelaye, Michael R Hamblin, Abbas Ali Hosseinpour Feizi.

  Mitosis is the process of cell division and is regulated by checkpoints in the cell cycle. G1-S, S, and G2-M are the three main checkpoints that prevent initiation of the next phase of the cell cycle phase until previous phase has completed. DNA damage leads to activation of the G2-M checkpoint, which can trigger a downstream DNA damage response (DDR) pathway to induce cell cycle arrest while the damage is repaired. If the DNA damage cannot be repaired, the replication stress response (RSR) pathway finally leads to cell death by apoptosis, in this case called mitotic catastrophe. Many cancer treatments (chemotherapy and radiotherapy) cause DNA damages based on SSBs (single strand breaks) or DSBs (double strand breaks), which cause cell death through mitotic catastrophe. However, damaged cells can activate WEE1 kinase (as a part of the DDR and RSR pathways), which prevents apoptosis and cell death by inducing cell cycle arrest at G2 phase. Therefore, inhibition of WEE1 kinase could sensitize cancer cells to chemotherapeutic drugs. This review focuses on the role of WEE1 kinase (as a biological macromolecule which has a molecular mass of 96 kDa) in the cell cycle, and its interactions with other regulatory pathways. In addition, we discuss the potential of WEE1 inhibition as a new therapeutic approach in the treatment of various cancers, such as melanoma, breast cancer, pancreatic cancer, cervical cancer, etc.

Keywords:  Cancer; Cell cycle; Checkpoint; DNA damage; WEE1 kinase

DOI:  https://doi.org/10.1016/j.mrfmmm.2022.111776
Front Cell Dev Biol. 2022 ;10 838928

The Dynamic Instability of the Aneuploid Genome.

Lorenza Garribba, Stefano Santaguida.

  Proper partitioning of replicated sister chromatids at each mitosis is crucial for maintaining cell homeostasis. Errors in this process lead to aneuploidy, a condition in which daughter cells harbor genome imbalances. Importantly, aneuploid cells often experience DNA damage, which in turn could drive genome instability. This might be the product of DNA damage accumulation in micronuclei and/or a consequence of aneuploidy-induced replication stress in S-phase. Although high levels of genome instability are associated with cell cycle arrest, they can also confer a proliferative advantage in some circumstances and fuel tumor growth. Here, we review the main consequences of chromosome segregation errors on genome stability, with a special focus on the bidirectional relationship between aneuploidy and DNA damage. Also, we discuss recent findings showing how increased genome instability can provide a proliferation improvement under specific conditions, including chemotherapeutic treatments.

Keywords:  aneuploidy; cancer; chromosomal instability; genome instability; mitotic errors

DOI:  https://doi.org/10.3389/fcell.2022.838928
Pharmacol Res. 2022 Mar 05. pii: S1043-6618(22)00107-4. [Epub ahead of print]178 106162

Cell cycle checkpoints and beyond: Exploiting the ATR/CHK1/WEE1 pathway for the treatment of PARP inhibitor-resistant cancer.

Nitasha Gupta, Tzu-Ting Huang, Sachi Horibata, Jung-Min Lee.

  Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have become a mainstay of therapy in ovarian cancer and other malignancies, including BRCA-mutant breast, prostate, and pancreatic cancers. However, a growing number of patients develop resistance to PARPis, highlighting the need to further understand the mechanisms of PARPi resistance and develop effective treatment strategies. Targeting cell cycle checkpoint protein kinases, e.g., ATR, CHK1, and WEE1, which are upregulated in response to replication stress, represents one such therapeutic approach for PARPi-resistant cancers. Mechanistically, activated cell cycle checkpoints promote cell cycle arrest, replication fork stabilization, and DNA repair, demonstrating the interplay of DNA repair proteins with replication stress in the development of PARPi resistance. Inhibitors of these cell cycle checkpoints are under investigation in PARPi-resistant ovarian and other cancers. In this review, we discuss the cell cycle checkpoints and their roles beyond mere cell cycle regulation as part of the arsenal to overcome PARPi-resistant cancers. We also address the current status and recent advancements as well as limitations of cell cycle checkpoint inhibitors in clinical trials.

Keywords:  ATR/CHK1/WEE1 pathway; Cell cycle checkpoint; DNA damage response; Ovarian cancer; PARP inhibitor resistance; Replication stress

DOI:  https://doi.org/10.1016/j.phrs.2022.106162
Cancer Res. 2022 Mar 03. pii: canres.2076.2021. [Epub ahead of print]

Tumor-intrinsic PD-L1 promotes DNA repair in distinct cancers and suppresses PARP inhibitor-induced synthetic lethality.

Anand V R Kornepati, Jacob T Boyd, Clare E Murray, Julia Saifetyarova, Bárbara de la Peña Avalos, Cody M Rogers, Haiyan Bai, Alvaro S Padron, Yiji Liao, Carlos Ontiveros, Robert S Svatek, Robert Hromas, Rong Li, Yanfen Hu, Jose R Conejo-Garcia, Ratna K Vadlamudi, Weixing Zhao, Eloise Dray, Patrick Sung, Tyler J Curiel.

BRCA1-mediated homologous recombination is an important DNA repair mechanism that is the target of FDA-approved PARP inhibitors, yet details of BRCA1-mediated functions remain to be fully elucidated. Similarly, immune checkpoint molecules are targets of FDA-approved cancer immunotherapies, but the biological and mechanistic consequences of their application are incompletely understood. We show here that the immune checkpoint molecule PD-L1 regulates homologous recombination in cancer cells by promoting BRCA1 nuclear foci formation and DNA end resection. Genetic depletion of tumor PD-L1 reduced homologous recombination, increased non-homologous end joining, and elicited synthetic lethality to PARP inhibitors olaparib and talazoparib in vitro in some, but not all, BRCA1 wild-type tumor cells. In vivo, genetic depletion of tumor PD-L1 rendered olaparib-resistant tumors senstive to olaparib. By contrast, anti-PD-L1 immune checkpoint blockade neither enhanced olaparib synthetic lethality nor improved its efficacy in vitro or in wild-type mice. Tumor PD-L1 did not alter expression of BRCA1 or its co-factor BARD1 but instead co-immunoprecipitated with BARD1 and increased BRCA1 nuclear accumulation. Tumor PD-L1 depletion enhanced tumor CCL5 expression and TBK1 activation in vitro, similar to known immune-potentiating effects of PARP inhibitors. Collectively, these data define immune-dependent and -independent effects of PARP inhibitor treatment and genetic tumor PD-L1 depletion. Moreover, they implicate a tumor cell-intrinsic, immune checkpoint-independent function of PD-L1 in cancer cell BRCA1-mediated DNA damage repair with translational potential, including as a treatment response biomarker.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2076
Neurooncol Adv. 2022 Jan-Dec;4(1):4(1): vdab180

Increased replication stress and R-loop accumulation in EGFRvIII-expressing glioblastoma present new therapeutic opportunities.

Nina Struve, Konstantin Hoffer, Anna-Sophie Weik, Britta Riepen, Leonie Krug, Meryem H Cetin, Jasmin Burmester, Leonie Ott, Jana Liebing, Fruzsina Gatzemeier, Justus Müller-Goebel, Mirja Gerbach, Lara Bußmann, Ann Christin Parplys, Kristian Unger, Wael Y Mansour, Ulrich Schüller, Thorsten Rieckmann, Cordula Petersen, Kai Rothkamm, Susan C Short, Malte Kriegs.

   Background: The oncogene epidermal growth factor receptor variant III (EGFRvIII) is expressed in approximately one-third of all glioblastomas (GBMs). So far it is not clear if EGFRvIII expression induces replication stress in GBM cells, which might serve as a therapeutical target.
Methods: Isogenetic EGFRvIII- and EGFRvIII+ cell lines with endogenous EGFRvIII expression were used. Markers of oncogenic and replication stress such as γH2AX, RPA, 53BP1, ATR, and CHK1 were analyzed using western blot, immunofluorescence, and flow cytometry. The DNA fiber assay was performed to analyze replication, transcription was measured by incorporation of EU, and genomic instability was investigated by micronuclei and CGH-Array analysis. Immunohistochemistry staining was used to detect replication stress markers and R-loops in human GBM samples.
Results: EGFRvIII+ cells exhibit an activated replication stress response, increased spontaneous DNA damage, elevated levels of single-stranded DNA, and reduced DNA replication velocity, which are all indicative characteristics of replication stress. Furthermore, we show here that EGFRvIII expression is linked to increased genomic instability. EGFRvIII-expressing cells display elevated RNA synthesis and R-loop formation, which could also be confirmed in EGFRvIII-positive GBM patient samples. Targeting replication stress by irinotecan resulted in increased sensitivity of EGFRvIII+ cells.
Conclusion: This study demonstrates that EGFRvIII expression is associated with increased replication stress, R-loop accumulation, and genomic instability. This might contribute to intratumoral heterogeneity but may also be exploited for individualized therapy approaches.

Keywords:  EGFRvIII; R-loops; genomic instability; glioblastoma; irinotecan sensitivity; replication stress

DOI:  https://doi.org/10.1093/noajnl/vdab180
Cancer Res. 2022 Mar 01. pii: canres.1785.2021. [Epub ahead of print]

A distinctive requirement for p53 in the genome protective Topoisomerase 2a-dependent G2 arrest in hTERT positive cancer cells.

Nicola Lockwood, Silvia Martini, Ainara López-Pardo, Katharina Deiss, Hendrika A Segeren, Robert K Semple, Ian Collins, Dimitra Repana, Mathias Cobbaut, Tanya N Soliman, Francesca Ciccarelli, Peter J Parker.

Topoisomerase 2a (Topo2a)-dependent G2 arrest engenders faithful segregation of sister chromatids, yet in certain tumor cell lines where this arrest is dysfunctional, a PKCε-dependent failsafe pathway can be triggered. Here we elaborate on recent advances in understanding the underlying mechanisms associated with this G2 arrest by determining that p53-p21 signaling is essential for efficient arrest in cell lines, in patient-derived cells, and in colorectal cancer organoids. Regulation of this p53 axis required the SMC5/6 complex, which is distinct from the p53 pathways observed in the DNA damage response. Topo2a inhibition specifically during S phase did not trigger G2 arrest despite affecting completion of DNA replication. Moreover, in cancer cells reliant upon the alternative lengthening of telomeres (ALT) mechanism, a distinct form of Topo2a-dependent, p53-independent G2 arrest was found to be mediated by BLM and Chk1. Importantly, the previously described PKCε-dependent mitotic failsafe was engaged in hTERT-positive cells when Topo2a-dependent G2 arrest was dysfunctional and where p53 was absent, but not in cells dependent on the ALT mechanism. In PKCε knockout mice, p53 deletion elicited tumors were less aggressive than in PKCε-replete animals and exhibited a distinct pattern of chromosomal rearrangements. This evidence suggests the potential of exploiting synthetic lethality in arrest-defective hTERT-positive tumors through PKCε-directed therapeutic intervention.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1785
ACS Omega. 2022 Mar 01. 7(8): 6488-6501

Cleavage of Abasic Sites in DNA by an Aminoquinoxaline Compound: Augmented Cytotoxicity and DNA Damage in Combination with an Anticancer Drug Chlorambucil in Human Colorectal Carcinoma Cells.

Chandra Sova Mandi, Tridib Mahata, Dipendu Patra, Jeet Chakraborty, Achyut Bora, Ritesh Pal, Sanjay Dutta.

The elevated level of endogenous oxidative DNA damage and spontaneous deamination of DNA bases in cancer cells substantially increase the abasic sites in DNA via base excision repairs (BERs). Thus, the predominant BER pathway is a favorable target for cancer therapy. Interestingly, elevated levels of glutathione (GSH) in certain cancer cells, such as colon cancer, are associated with acquired resistance to several chemotherapeutic agents, which increase the difficulty for the treatment of cancer. Here, we have reported an ideal nitro group-containing monoquinoxaline DNA intercalator (1d), which is reduced into a fluorescent quinoxaline amine (1e) in the presence of GSH; concurrently, 1e (∼100 nM concentration) selectively causes the in vitro cleavage of abasic sites in DNA. 1e also binds to the tetrahydrofuran analogue of the abasic site in the nanomolar to low micromolar range depending on the nucleotide sequence opposite to the abasic site and also induces a structural change in abasic DNA. Furthermore, the amine compound (1e) augments the response of the specific bifunctional alkylating drug chlorambucil at a much lower concentration in the human colorectal carcinoma cell (HCT-116), and their combination shows a potential strategy for targeted therapy. Alone or in combination, 1d and 1e lead to a cascade of cellular events such as induction of DNA double-stranded breaks and cell arrest at G0/G1 and G2/M phases, eventually leading to apoptotic cell death in HCT-116 cells. Hence, the outcome of this study provides a definitive approach that will help optimize the therapeutic applications for targeting the abasic site in cancer cells.

DOI: https://doi.org/10.1021/acsomega.1c04962
JACS Au. 2022 Feb 28. 2(2): 341-356

Investigating the Conformational Dynamics of a Y-Family DNA Polymerase during Its Folding and Binding to DNA and a Nucleotide.

Xiakun Chu, Zucai Suo, Jin Wang.

During DNA polymerization, the Y-family DNA polymerases are capable of bypassing various DNA damage, which can stall the replication fork progression. It has been well acknowledged that the structures of the Y-family DNA polymerases have been naturally evolved to undertake this vital task. However, the mechanisms of how these proteins utilize their unique structural and conformational dynamical features to perform the translesion DNA synthesis are less understood. Here, we developed structure-based models to study the precatalytic DNA polymerization process, including DNA and nucleotide binding to DPO4, a paradigmatic Y-family polymerase from Sulfolobus solfataricus. We studied the interplay between the folding and the conformational dynamics of DPO4 and found that DPO4 undergoes first unraveling (unfolding) and then folding for accomplishing the functional "open-to-closed" conformational transition. DNA binding dynamically modulates the conformational equilibrium in DPO4 during the stepwise binding through different types of interactions, leading to different conformational distributions of DPO4 at different DNA binding stages. We observed that nucleotide binding induces modulation of a few contacts surrounding the active site of the DPO4-DNA complex associated with a high free energy barrier. Our simulation results resonate with the experimental evidence that the conformational change at the active site led by nucleotide is the rate-limiting step of nucleotide incorporation. In combination with localized frustration analyses, we underlined the importance of DPO4 conformational dynamics and fluctuations in facilitating DNA and nucleotide binding. Our findings offer mechanistic insights into the processes of DPO4 conformational dynamics associated with the substrate binding and contribute to the understanding of the "structure-dynamics-function" relationship in the Y-family DNA polymerases.

DOI: https://doi.org/10.1021/jacsau.1c00368
Anticancer Drugs. 2022 Mar 08.

Inhibiting nonhomologous end-joining repair would promote the antitumor activity of gemcitabine in nonsmall cell lung cancer cell lines.

Nannan Guo, Shaojun Li, Bo Liu, Ping Chen, Jingbo Li, Yunlong Zhao, Yingnan Zhao, Jian Tang, Wen Zhang.

Nonsmall cell lung cancer (NSCLC) is a major type of lung cancer. In current study, we aim to evaluate whether the combination of Ku70/80 heterodimer protein inhibitor STL127705 and gemcitabine would be more favorable approach for the treatment of NSCLC compared with monotreatment with gemcitabine. Clongenic survival assay was used to determine the survival and sensitivity to irradiation. H1299 was stained with fluorescein isothiocyanate-Annexin V, and cell apoptosis was measured by flow cytometry. H1299 cells were transfected with nonhomologous end-joining (NHEJ) repair reporter, and stable cell line was selected by puromycin. NHEJ activity was determined based on the intensity of green fluorescent protein. DNA double-strand breaks (DSBs) were determined by the fluorescence intensity of γH2AX using flow cytometry. The mRNA expressions of Ku70 and Ku80 were determined using quantitative real-time PCR. Combination of STL127705 enhanced sensitivity of NSCLC cell lines to irradiation when compared with treatment with gemcitabine alone. However, small cell lung cancer cell line was not affected. H1299 cells treated with STL127705 in combination with gemcitabine showed a significantly increased apoptosis compared with H1299 cells treated with gemcitabine alone. Moreover, STL127705 treatment dramatically reduced NHEJ activity in H1299 cells when compared with gemcitabine single treatment. Increased DSBs were consistently observed in H1299 when treated with the combination of STL127705 and gemcitabine. However, the mRNA levels of Ku70 and Ku80 were upregulated by the combination treatment. It demonstrated that STL127705 enhanced antitumor activity of gemcitabine. Mechanistically, treatment with STL127705 enhanced DNA damage via inhibiting NHEJ pathway, blocking DNA-PK, and forming Ku70/80 heterodimer, eventually leading to tumor cells apoptosis.

DOI: https://doi.org/10.1097/CAD.0000000000001290
DNA Repair (Amst). 2022 Feb 11. pii: S1568-7864(22)00029-5. [Epub ahead of print]113 103300

The rate of formation and stability of abasic site interstrand crosslinks in the DNA duplex.

Andrea Huskova, Barbora Landova, Evzen Boura, Jan Silhan.

  DNA interstrand crosslinks (ICLs) strands pose an impenetrable barrier for DNA replication. Different ICLs are known to recruit distinct DNA repair pathways. NEIL3 glycosylase has been known to remove an abasic (Ap) site derived DNA crosslink (Ap-ICL). An Ap-ICL forms spontaneously from the Ap site with an adjacent adenine in the opposite strand. Lack of genetic models and a poor understanding of the fate of these lesions leads to many questions about the occurrence and the toxicity of Ap-ICL in cells. Here, we investigate the circumstances of Ap-ICL formation. With an array of different oligos, we have investigated the rates of formation, the yields, and the stability of Ap-ICL. Our findings point out how different bases in the vicinity of the Ap site change crosslink formation in vitro. We reveal that AT-rich rather than GC-rich regions in the surrounding Ap site lead to higher rates of Ap-ICL formation. Overall, our data reveal that Ap-ICL can be formed in virtually any DNA sequence context surrounding a hot spot of a 5'-Ap-dT pair, albeit with significantly different rates and yields. Based on Ap-ICL formation in vitro, we attempt to predict the number of Ap-ICLs in the cell.

Keywords:  Abasic site; DNA damage; DNA repair; Interstrand crosslink; Non-enzyme kinetics

DOI:  https://doi.org/10.1016/j.dnarep.2022.103300
Proc Natl Acad Sci U S A. 2022 03 15. 119(11): e2121979119

Inhibitors of PARP: Number crunching and structure gazing.

Johannes Rudolph, Karen Jung, Karolin Luger.

  SignificancePARP is an important target in the treatment of cancers, particularly in patients with breast, ovarian, or prostate cancer that have compromised homologous recombination repair (i.e., BRCA-/-). This review about inhibitors of PARP (PARPi) is for readers interested in the development of next-generation drugs for the treatment of cancer, providing insights into structure-activity relationships, in vitro vs. in vivo potency, PARP trapping, and synthetic lethality.

Keywords:  HPF1; cancer drugs; drug specificity; inhibitor of Parp; synthetic lethality

DOI:  https://doi.org/10.1073/pnas.2121979119
J Natl Compr Canc Netw. 2022 Mar;pii: jnccn21438. [Epub ahead of print]20(3): 218-223

Four-Year Disease-Free Remission in a Patient With POLE Mutation-Associated Colorectal Cancer Treated Using Anti-PD-1 Therapy.

Michael L Durando, Sanjay V Menghani, Jessica L Baumann, Danny G Robles, Tovah A Day, Cyrus Vaziri, Aaron J Scott.

The stability of the human genome depends upon a delicate balance between replication by high- and low-fidelity DNA polymerases. Aberrant replication by error-prone polymerases or loss of function of high-fidelity polymerases predisposes to genetic instability and, in turn, cancer. DNA polymerase epsilon (Pol ε) is a high-fidelity, processive polymerase that is responsible for the majority of leading strand synthesis, and mutations in Pol ε have been increasingly associated with various human malignancies. The clinical significance of Pol ε mutations, including how and whether they should influence management decisions, remains poorly understood. In this report, we describe a 24-year-old man with an aggressive stage IV high-grade, poorly differentiated colon carcinoma who experienced a dramatic response to single-agent checkpoint inhibitor immunotherapy after rapidly progressing on standard chemotherapy. His response was complete and durable and has been maintained for more than 48 months. Genetic testing revealed a P286R mutation in the endonuclease domain of POLE and an elevated tumor mutational burden of 126 mutations per megabase, both of which have been previously associated with response to immunotherapy. Interestingly, tumor staining for PD-L1 was negative. This case study highlights the importance of genetic profiling of both early and late-stage cancers, the clinical significance of POLE mutations, and how the interplay between genetic instability and immune-checkpoint blockade can impact clinical decision-making.

DOI: https://doi.org/10.6004/jnccn.2021.7115
Leukemia. 2022 Mar 10.

In vivo anti-tumor effect of PARP inhibition in IDH1/2 mutant MDS/AML resistant to targeted inhibitors of mutant IDH1/2.

Rana Gbyli, Yuanbin Song, Wei Liu, Yimeng Gao, Giulia Biancon, Namrata S Chandhok, Xiaman Wang, Xiaoying Fu, Amisha Patel, Ranjini Sundaram, Toma Tebaldi, Padmavathi Mamillapalli, Amer M Zeidan, Richard A Flavell, Thomas Prebet, Ranjit S Bindra, Stephanie Halene.

Treatment options for patients with relapsed/refractory acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are scarce. Recurring mutations, such as mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2) are found in subsets of AML and MDS, are therapeutically targeted by mutant enzyme-specific small molecule inhibitors (IDHmi). IDH mutations induce diverse metabolic and epigenetic changes that drive malignant transformation. IDHmi alone are not curative and resistance commonly develops, underscoring the importance of alternate therapeutic options. We were first to report that IDH1/2 mutations induce a homologous recombination (HR) defect, which confers sensitivity to poly (ADP)-ribose polymerase inhibitors (PARPi). Here, we show that the PARPi olaparib is effective against primary patient-derived IDH1/2-mutant AML/ MDS xeno-grafts (PDXs). Olaparib efficiently reduced overall engraftment and leukemia-initiating cell frequency as evident in serial transplantation assays in IDH1/2-mutant but not -wildtype AML/MDS PDXs. Importantly, we show that olaparib is effective in both IDHmi-naïve and -resistant AML PDXs, critical given the high relapse and refractoriness rates to IDHmi. Our pre-clinical studies provide a strong rationale for the translation of PARP inhibition to patients with IDH1/2-mutant AML/ MDS, providing an additional line of therapy for patients who do not respond to or relapse after targeted mutant IDH inhibition.

DOI: https://doi.org/10.1038/s41375-022-01536-x