bims-numges 2021-08-22 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–08–22
thirty papers selected by
Sean Rudd, Karolinska Institutet

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair.
A catalytic-independent function of human DNA polymerase Kappa controls the stability and abundance of the Checkpoint Kinase 1.
Heterogeneity of DNA damage incidence and repair in different chromatin contexts.
Toxic R-loops: Cause or consequence of replication stress?
Histone lysine modifying enzymes and their critical roles in DNA double-strand break repair.
Homologous Recombination Subpathways: A Tangle to Resolve.
Transcription coupled base excision repair in mammalian cells: So little is known and so much to uncover.
Role of histone acetyltransferases MOF and Tip60 in genome stability.
Local DNA synthesis is critical for DNA repair during oocyte maturation.
RNF168-mediated localization of BARD1 recruits the BRCA1-PALB2 complex to DNA damage.
A role for Rad5 in ribonucleoside monophosphate (rNMP) tolerance.
Photosensitivity and cGAS-dependent type I IFN activation in lupus patients with TREX1 deficiency.
Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion.
CX-5461 sensitises DNA damage repair proficient castrate-resistant prostate cancer to PARP inhibition.
Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5'-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives.
Transcription-coupled repair and the transcriptional response to UV-Irradiation.
Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair.
Targeting Wee1 kinase as a therapeutic approach in Hematological Malignancies.
Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.
ATR inhibitor M6620 (VX-970) enhances the effect of radiation in non-small cell lung cancer brain metastasis patient derived xenografts.
PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation.
Assessing AML susceptibility in rearrangement-driven patients by DNA breakage at topoisomerase II and CTCF/cohesin binding sites.
A laser-mediated photo-manipulative toolbox for generation and real-time monitoring of DNA lesions.
Molecular basis for DarT ADP-ribosylation of a DNA base.
A novel view on an old drug, 5-fluorouracil: an unexpected RNA modifier with intriguing impact on cancer cell fate.
PIKE-A promotes glioblastoma growth by driving PPP flux through increasing G6PD expression mediated by phosphorylation of STAT3.
Integrated multi-omics analysis of RB-loss identifies widespread cellular programming and synthetic weaknesses.
The frequency and clinical significance of DNA polymerase beta (POLβ) expression in breast ductal carcinoma in situ (DCIS).
SLFN11 is widely expressed in pediatric sarcoma and induces variable sensitization to replicative stress caused by DNA damaging agents.
Divergent synthesis and elaboration of structure activity relationship for quinoline derivatives as highly selective NTPDase inhibitor.

Nat Commun. 2021 Aug 20. 12(1): 5055

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair.

Joonas A Jamsen, Akira Sassa, Lalith Perera, David D Shock, William A Beard, Samuel H Wilson.

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

DOI: https://doi.org/10.1038/s41467-021-24486-x
Mol Cell Biol. 2021 Aug 16. MCB0009021

A catalytic-independent function of human DNA polymerase Kappa controls the stability and abundance of the Checkpoint Kinase 1.

Marina Dall'Osto, Laura Pierini, Nicolas Valery, Jean-Sébastien Hoffmann, Marie-Jeanne Pillaire.

DNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate, restart of stalled forks and replication checkpoint efficiency. Pol κ is also required for the stabilization of stalled forks although the mechanisms are poorly understood. Here we unveiled an unexpected role for Pol κ in controlling the stability and abundance of Chk1, an important actor for the replication checkpoint and fork stabilization. We found that loss of Pol κ decreased the Chk1 protein level in the nucleus of four human cell lines. Pol κ and not the other Y-family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation. Importantly, we also observed that the fork restart defects observed in Pol κ-depleted cells could be overcome by the re-expression of Chk1. Strikingly, this new function of Pol κ does not require its catalytic activity. We propose that Pol κ could contribute to the protection of stalled forks through Chk1 stability.

DOI: https://doi.org/10.1128/MCB.00090-21
DNA Repair (Amst). 2021 Aug 13. pii: S1568-7864(21)00166-X. [Epub ahead of print]107 103210

Heterogeneity of DNA damage incidence and repair in different chromatin contexts.

Pedro Ortega, Belén Gómez-González, Andrés Aguilera.

  It has been long known that some regions of the genome are more susceptible to damage and mutagenicity than others. Recent advances have determined a critical role of chromatin both in the incidence of damage and in its repair. Thus, chromatin arises as a guardian of the stability of the genome, which is altered in cancer cells. In this review, we focus into the mechanisms by which chromatin influences the occurrence and repair of the most cytotoxic DNA lesions, double-strand breaks, in particular at actively transcribed chromatin or related to DNA replication.

Keywords:  Chromatin; DNA damage; DNA repair; Double-strand break; Genome instability

DOI:  https://doi.org/10.1016/j.dnarep.2021.103210
DNA Repair (Amst). 2021 Aug 03. pii: S1568-7864(21)00155-5. [Epub ahead of print]107 103199

Toxic R-loops: Cause or consequence of replication stress?

Samira Kemiha, Jérôme Poli, Yea-Lih Lin, Armelle Lengronne, Philippe Pasero.

  Transcription-replication conflicts (TRCs) represent a potential source of endogenous replication stress (RS) and genomic instability in eukaryotic cells but the mechanisms that underlie this instability remain poorly understood. Part of the problem could come from non-B DNA structures called R-loops, which are formed of a RNA:DNA hybrid and a displaced ssDNA loop. In this review, we discuss different scenarios in which R-loops directly or indirectly interfere with DNA replication. We also present other types of TRCs that may not depend on R-loops to impede fork progression. Finally, we discuss alternative models in which toxic RNA:DNA hybrids form at stalled forks as a consequence - but not a cause - of replication stress and interfere with replication resumption.

Keywords:  Genomic instability; R-loops; RNA:DNA hybrids; Replication; Transcription

DOI:  https://doi.org/10.1016/j.dnarep.2021.103199
DNA Repair (Amst). 2021 Aug 12. pii: S1568-7864(21)00162-2. [Epub ahead of print]107 103206

Histone lysine modifying enzymes and their critical roles in DNA double-strand break repair.

Jun Zhang, Xiaopeng Lu, Sara MoghaddamKohi, Lei Shi, Xingzhi Xu, Wei-Guo Zhu.

  Cells protect the integrity of the genome against DNA double-strand breaks through several well-characterized mechanisms including nonhomologous end-joining repair, homologous recombination repair, microhomology-mediated end-joining and single-strand annealing. However, aberrant DNA damage responses (DDRs) lead to genome instability and tumorigenesis. Clarification of the mechanisms underlying the DDR following lethal damage will facilitate the identification of therapeutic targets for cancer. Histones are small proteins that play a major role in condensing DNA into chromatin and regulating gene function. Histone modifications commonly occur in several residues including lysine, arginine, serine, threonine and tyrosine, which can be acetylated, methylated, ubiquitinated and phosphorylated. Of these, lysine modifications have been extensively explored during DDRs. Here, we focus on discussing the roles of lysine modifying enzymes involved in acetylation, methylation, and ubiquitination during the DDR. We provide a comprehensive understanding of the basis of potential epigenetic therapies driven by histone lysine modifications.

Keywords:  Acetylation; DNA damage response; Enzymes; Histone lysine modification; Methylation; Ubiquitination

DOI:  https://doi.org/10.1016/j.dnarep.2021.103206
Front Genet. 2021 ;12 723847

Homologous Recombination Subpathways: A Tangle to Resolve.

Amira Elbakry, Markus Löbrich.

  Homologous recombination (HR) is an essential pathway for DNA double-strand break (DSB) repair, which can proceed through various subpathways that have distinct elements and genetic outcomes. In this mini-review, we highlight the main features known about HR subpathways operating at DSBs in human cells and the factors regulating subpathway choice. We examine new developments that provide alternative models of subpathway usage in different cell types revise the nature of HR intermediates involved and reassess the frequency of repair outcomes. We discuss the impact of expanding our understanding of HR subpathways and how it can be clinically exploited.

Keywords:  ATRX; RECQ5; crossover; double-strand break; holliday junction; homologous recombination; pathway choice; synthesis-dependent strand annealing

DOI:  https://doi.org/10.3389/fgene.2021.723847
DNA Repair (Amst). 2021 Aug 06. pii: S1568-7864(21)00160-9. [Epub ahead of print]107 103204

Transcription coupled base excision repair in mammalian cells: So little is known and so much to uncover.

Anirban Chakraborty, Nisha Tapryal, Azharul Islam, Sankar Mitra, Tapas Hazra.

  Oxidized bases in the genome has been implicated in various human pathologies, including cancer, aging and neurological diseases. Their repair is initiated with excision by DNA glycosylases (DGs) in the base excision repair (BER) pathway. Among the five oxidized base-specific human DGs, OGG1 and NTH1 preferentially excise oxidized purines and pyrimidines, respectively, while NEILs remove both oxidized purines and pyrimidines. However, little is known about why cells possess multiple DGs with overlapping substrate specificities. Studies of the past decades revealed that some DGs are involved in repair of oxidized DNA base lesions in the actively transcribed regions. Preferential removal of lesions from the transcribed strands of active genes, called transcription-coupled repair (TCR), was discovered as a distinct sub-pathway of nucleotide excision repair; however, such repair of oxidized DNA bases had not been established until our recent demonstration of NEIL2's role in TC-BER of the nuclear genome. We have shown that NEIL2 forms a distinct transcriptionally active, repair proficient complex. More importantly, we for the first time reconstituted TC-BER using purified components. These studies are important for characterizing critical requirement for the process. However, because NEIL2 cannot remove all types of oxidized bases, it is unlikely to be the only DNA glycosylase involved in TC-BER. Hence, we postulate TC-BER process to be universally involved in maintaining the functional integrity of active genes, especially in post-mitotic, non-growing cells. We further postulate that abnormal bases (e.g., uracil), and alkylated and other small DNA base adducts are also repaired via TC-BER. In this review, we have provided an overview of the various aspects of TC-BER in mammalian cells with the hope of generating significant interest of many researchers in the field. Further studies aimed at better understanding the mechanistic aspects of TC-BER could help elucidate the linkage of TC-BER deficiency to various human pathologies.

Keywords:  Base excision repair; DNA glycosylase; Human Nei like proteins (NEILs); Oxidative DNA damage; Transcription coupled repair

DOI:  https://doi.org/10.1016/j.dnarep.2021.103204
DNA Repair (Amst). 2021 Aug 08. pii: S1568-7864(21)00161-0. [Epub ahead of print]107 103205

Role of histone acetyltransferases MOF and Tip60 in genome stability.

Ulfat Syed Mir, Audesh Bhat, Arjamand Mushtaq, Shruti Pandita, Mohammad Altaf, Tej K Pandita.

  The accurate repair of DNA damage specifically the chromosomal double-strand breaks (DSBs) arising from exposure to physical or chemical agents, such as ionizing radiation (IR) and radiomimetic drugs is critical in maintaining genomic integrity. The DNA DSB response and repair is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes specifically histone modifications which impact cell-cycle checkpoints through enzymatic activities to repair the broken DNA ends. Various histone posttranslational modifications such as phosphorylation, acetylation, methylation and ubiquitylation have been shown to play a role in DNA damage repair. Recent studies have provided important insights into the role of histone-specific modifications in sensing DNA damage and facilitating the DNA repair. Histone modifications have been shown to determine the pathway choice for repair of DNA DSBs. This review will summarize the role of important histone acetyltransferases MOF and Tip60 mediated acetylation in repair of DNA DSBs in eukaryotic cells.

Keywords:  DNA DSB repair; Histone acetylation; Histone acetyltransferases; MOF; TIP60

DOI:  https://doi.org/10.1016/j.dnarep.2021.103205
J Cell Sci. 2021 Aug 20. pii: jcs.257774. [Epub ahead of print]

Local DNA synthesis is critical for DNA repair during oocyte maturation.

Ajay K Singh, S Lava Kumar, Rohit Beniwal, Aradhana Mohanty, Bhawna Kushwaha, H B D Prasada Rao.

  Mammalian oocytes can be very long-lived cells and thereby very likely to encounter DNA damage during their lifetime. Defective DNA repair may result in oocytes that are developmentally incompetent or give rise to progeny with congenital disorders. During oocyte maturation, damaged DNA is repaired primarily by non-homologous end joining (NHEJ) or homologous recombination (HR). Although these repair pathways have been studied extensively, the associated DNA synthesis is poorly characterized. Using porcine oocytes, we demonstrate that the DNA synthesis machinery is present during oocyte maturation and dynamically recruited to sites of DNA damage. DNA polymerase δ is identified as being crucial for oocyte DNA synthesis. Further, inhibiting synthesis causes DNA damage to accumulate and delays the progression of oocyte maturation. Importantly, inhibition of the spindle assembly checkpoint (SAC) bypassed the delay of oocyte maturation caused by DNA synthesis inhibition. Finally, we found that ∼20% of unperturbed oocytes experienced spontaneously-arising damage during maturation. Cumulatively, our findings indicate that oocyte maturation requires damage-associated DNA synthesis that is monitored by the SAC.

Keywords:  Break induced DNA repair in oocytes; Oocyte DNA damage and repair in higher order vertebrates; Oocyte maturation; SAC in higher order vertebrates

DOI:  https://doi.org/10.1242/jcs.257774
Nat Commun. 2021 08 18. 12(1): 5016

RNF168-mediated localization of BARD1 recruits the BRCA1-PALB2 complex to DNA damage.

John J Krais, Yifan Wang, Pooja Patel, Jayati Basu, Andrea J Bernhardy, Neil Johnson.

DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.

DOI: https://doi.org/10.1038/s41467-021-25346-4
Life Sci Alliance. 2021 Oct;pii: e202000966. [Epub ahead of print]4(10):

A role for Rad5 in ribonucleoside monophosphate (rNMP) tolerance.

Menattallah Elserafy, Iman El-Shiekh, Dalia Fleifel, Reham Atteya, Abdelrahman AlOkda, Mohamed M Abdrabbou, Mostafa Nasr, Sherif F El-Khamisy.

Ribonucleoside monophosphate (rNMP) incorporation in genomic DNA poses a significant threat to genomic integrity. In addition to repair, DNA damage tolerance mechanisms ensure replication progression upon encountering unrepaired lesions. One player in the tolerance mechanism is Rad5, which is an E3 ubiquitin ligase and helicase. Here, we report a new role for yeast Rad5 in tolerating rNMP incorporation, in the absence of the bona fide ribonucleotide excision repair pathway via RNase H2. This role of Rad5 is further highlighted after replication stress induced by hydroxyurea or by increasing rNMP genomic burden using a mutant DNA polymerase (Pol ε - Pol2-M644G). We further demonstrate the importance of the ATPase and ubiquitin ligase domains of Rad5 in rNMP tolerance. These findings suggest a similar role for the human Rad5 homologues helicase-like transcription factor (HLTF) and SNF2 Histone Linker PHD RING Helicase (SHPRH) in rNMP tolerance, which may impact the response of cancer cells to replication stress-inducing therapeutics.

DOI: https://doi.org/10.26508/lsa.202000966
J Invest Dermatol. 2021 Aug 13. pii: S0022-202X(21)01686-9. [Epub ahead of print]

Photosensitivity and cGAS-dependent type I IFN activation in lupus patients with TREX1 deficiency.

Nicole Berndt, Christine Wolf, Kristina Fischer, Emanuel Cura Costa, Peter Knuschke, Nick Zimmermann, Franziska Schmidt, Martin Merkel, Osvaldo Chara, Min Ae Lee-Kirsch, Claudia Günther.

  The exonuclease three prime repair exonuclease 1 (TREX1) safeguards the cell against DNA accumulation in the cytosol and thereby prevents innate immune activation and autoimmunity. TREX1 mutations lead to chronic DNA damage and cell-intrinsic type I interferon (IFN) response. Associated disease phenotypes include Aicardi-Goutières syndrome, familial chilblain lupus and systemic lupus erythematosus. Given the role of ultraviolet (UV) light in lupus pathogenesis, we assessed sensitivity to UV light in lupus patients with TREX1 mutation by phototesting which revealed an enhanced photosensitivity. TREX1-deficient fibroblasts and keratinocytes generated increased levels of reactive oxygen species in response to UV irradiation as well as increased levels of 8-oxo-guanine lesions after oxidative stress. Likewise, the primary UV-induced DNA lesions cyclobutane pyrimidine dimers (CPD) were induced more strongly in TREX1-deficient cells. Further analysis revealed that single-stranded DNA regions, frequently formed during DNA replication and repair, promote CPD formation. Together, this resulted in a strong UV-induced DNA damage response that was associated with a cyclic GMP-AMP synthase (cGAS)-dependent type I IFN activation. In conclusion, these findings link chronic DNA damage to photosensitivity and type I IFN production in TREX1 deficiency and explain the induction of disease flares upon UV exposure in lupus patients with TREX1 mutation.

Keywords:  DNA damage; UV irradiation; cutaneous lupus; interferon stimulated genes; pyrimidine dimers

DOI:  https://doi.org/10.1016/j.jid.2021.04.037
J Vis Exp. 2021 Jul 27.

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion.

Jing Zhang, Jing Huang, Ryan C James, Julia Gichimu, Manikandan Paramasivam, Durga Pokharel, Himabindu Gali, Marina A Bellani, Michael M Seidman.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins. Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

DOI: https://doi.org/10.3791/61689
Mol Cancer Ther. 2021 Aug 19. pii: molcanther.0932.2020. [Epub ahead of print]

CX-5461 sensitises DNA damage repair proficient castrate-resistant prostate cancer to PARP inhibition.

Mitchell G Lawrence, Laura H Porter, Nicholas Choo, David Pook, Jeremy P Grummet, Carmel J Pezaro, Shahneen Sandhu, Susanne Ramm, Jennii Luu, Andrew Bakshi, David L Goode, Elaine Sanij, Richard B Pearson, Ross D Hannan, Kaylene J Simpson, Renea A Taylor, Gail P Risbridger, Luc Furic.

Monotherapy with poly (ADP-ribose) polymerase (PARP) inhibitors is effective for the subset of castrate-resistant prostate cancer (CRPC) with defects in homologous recombination (HR) DNA repair. New treatments are required for the remaining tumours, and an emerging strategy is to combine PARP inhibitors with other therapies that induce DNA damage. Here we tested whether PARP inhibitors are effective for HR-proficient CRPC, including AR-null tumours, when used in combination with CX-5461, a small molecule that inhibits RNA polymerase I transcription and activates the DNA damage response, and has anti-tumour activity in early Phase I trials. The combination of CX-5461 and talazoparib significantly decreased in vivo growth of patient-derived xenografts of HR-proficient CRPC, including AR-positive, AR-null and neuroendocrine tumours. CX-5461 and talazoparib synergistically inhibited the growth of organoids and cell lines, and significantly increased the levels of DNA damage. Decreased tumour growth after combination therapy was maintained for two weeks without treatment, significantly increasing host survival. Therefore, combination treatment with CX-5461 and talazoparib is effective for HR-proficient tumours that are not suitable for monotherapy with PARP inhibitors, including AR-null CRPC. This expands the spectrum of CRPC that is sensitive to PARP inhibition.

DOI: https://doi.org/10.1158/1535-7163.MCT-20-0932
Purinergic Signal. 2021 Aug 17.

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5'-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives.

Emma Scaletti, Franziska U Huschmann, Uwe Mueller, Manfred S Weiss, Norbert Sträter.

  Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.

Keywords:  Crystal structure; Drug development; E5NT; Fragment screening; Purinergic signalling; eN

DOI:  https://doi.org/10.1007/s11302-021-09802-w
DNA Repair (Amst). 2021 Aug 10. pii: S1568-7864(21)00164-6. [Epub ahead of print]107 103208

Transcription-coupled repair and the transcriptional response to UV-Irradiation.

Liam Gaul, Jesper Q Svejstrup.

  Lesions in genes that result in RNA polymerase II (RNAPII) stalling or arrest are particularly toxic as they are a focal point of genome instability and potently block further transcription of the affected gene. Thus, cells have evolved the transcription-coupled nucleotide excision repair (TC-NER) pathway to identify damage-stalled RNAPIIs, so that the lesion can be rapidly repaired and transcription can continue. However, despite the identification of several factors required for TC-NER, how RNAPII is remodelled, modified, removed, or whether this is even necessary for repair remains enigmatic, and theories are intensely contested. Recent studies have further detailed the cellular response to UV-induced ubiquitylation and degradation of RNAPII and its consequences for transcription and repair. These advances make it pertinent to revisit the TC-NER process in general and with specific discussion of the fate of RNAPII stalled at DNA lesions.

Keywords:  Cockayne syndrome; Nucleotide excision repair; Transcription-coupled nucleotide excision repair; UVSSA; Ubiquitin; Xeroderma pigmentosum

DOI:  https://doi.org/10.1016/j.dnarep.2021.103208
Cell Discov. 2021 Aug 17. 7(1): 66

Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair.

Yu Jia, Zixiu Cheng, Sakshibeedu R Bharath, Qiangzu Sun, Nannan Su, Jun Huang, Haiwei Song.

SOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1, with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3, but how INTS3 interacts with INTS6, thereby impacting DSB repair, is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structural analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction impair the DSB repair process. Altogether, these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.

DOI: https://doi.org/10.1038/s41421-021-00283-0
DNA Repair (Amst). 2021 Aug 08. pii: S1568-7864(21)00159-2. [Epub ahead of print]107 103203

Targeting Wee1 kinase as a therapeutic approach in Hematological Malignancies.

Sajjad Vakili-Samiani, Abduladheem Turki Jalil, Walid Kamal Abdelbasset, Alexei Valerievich Yumashev, Vahid Karpisheh, Pooya Jalali, Sara Adibfar, Majid Ahmadi, Abbas Ali Hosseinpour Feizi, Farhad Jadidi-Niaragh.

  Hematologic malignancies include various diseases that develop from hematopoietic stem cells of bone marrow or lymphatic organs. Currently, conventional DNA-damage-based chemotherapy drugs are approved as standard therapeutic regimens for these malignancies. Although many improvements have been made, patients with relapsed or refractory hematological malignancies have a poor prognosis. Therefore, novel and practical therapeutic approaches are required for the treatment of these diseases. Interestingly several studies have shown that targeting Wee1 kinase in the Hematological malignancies, including AML, ALL, CML, CLL, DLBCL, BL, MCL, etc., can be an effective therapeutic strategy. It plays an essential role in regulating the cell cycle process by abrogating the G2-M cell-cycle checkpoint, which provides time for DNA damage repair before mitotic entry. Consistently, Wee1 overexpression is observed in various Hematological malignancies. Also, in healthy normal cells, repairing DNA damages occurs due to G1-S checkpoint function; however, in the cancer cells, which have an impaired G1-S checkpoint, the damaged DNA repair process depends on the G2-M checkpoint function. Thus, Wee1 inhibition could be a promising target in the presence of DNA damage in order to potentiate multiple therapeutic drugs. This review summarized the potentials and challenges of Wee1 inhibition combined with other therapies as a novel effective therapeutic strategy in Hematological malignancies.

Keywords:  Cancer; Hematologic malignancies; Leukemia; Lymphoma; Wee1 kinase

DOI:  https://doi.org/10.1016/j.dnarep.2021.103203
J Biol Chem. 2021 Aug 14. pii: S0021-9258(21)00883-8. [Epub ahead of print] 101080

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

Hai Pan, Parminder Kaur, Ryan Barnes, Ariana C Detwiler, Samantha Lynn Sanford, Ming Liu, Pengning Xu, Chelsea Mahn, Qingyu Tang, Pengyu Hao, Dhruv Bhattaram, Changjiang You, Xinyun Gu, Warren Lu, Jacob Piehler, Guozhou Xu, Keith Weninger, Robert Riehn, Patricia L Opresko, Hong Wang.

  TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ∼1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Pre-incubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

Keywords:  DNA binding protein; atomic force microscopy (AFM); single-molecule biophysics; telomere; telomeric repeat-binding factor 1

DOI:  https://doi.org/10.1016/j.jbc.2021.101080
Mol Cancer Ther. 2021 Aug 19. pii: molcanther.0305.2021. [Epub ahead of print]

ATR inhibitor M6620 (VX-970) enhances the effect of radiation in non-small cell lung cancer brain metastasis patient derived xenografts.

Andrew M Baschnagel, Jacob H Elnaggar, Haley J VanBeek, Ashley C Kromke, Justin H Skiba, Saakshi Kaushik, Lindsey Abel, Paul A Clark, Colin A Longhurst, Kwangok P Nickel, Ticiana A Leal, Shuang G Zhao, Randall J Kimple.

M6620, a selective ATP-competitive inhibitor of the ATM and RAD3-related (ATR) kinase, is currently under investigation with radiation in patients with non-small cell lung cancer (NSCLC) brain metastases. We evaluated the DNA damage response (DDR) pathway profile of NSCLC and assessed the radiosensitizing effects of M6620 in a preclinical NSCLC brain metastasis model. Mutation analysis and transcriptome profiling of DDR genes and pathways was performed on NSCLC patient samples. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, cell cycle, and DNA damage signaling and repair assays. NSCLC brain metastasis patient-derived xenograft models were used to assess intracranial response and overall survival. In vivo immunohistochemistry was performed to confirm in vitro results. A significant portion of NSCLC patient tumors demonstrated enrichment of DDR pathways. DDR pathways correlated with lung squamous cell histology; and mutations in ATR, ATM, BRCA1, BRCA2, CHEK1, and CHEK2 correlated with enrichment of DDR pathways in lung adenocarcinomas. M6620 reduced colony formation after radiotherapy and resulted in inhibition of DNA DSB repair, abrogation of the radiation-induced G2 cell checkpoint, and formation of dysfunctional micronuclei, leading to enhanced radiation-induced mitotic death. The combination of M6620 and radiation resulted in improved overall survival in mice compared to radiation alone. In vivo immunohistochemistry revealed inhibition of pChk1 in the radiation plus M6620 group. M6620 enhances the effect of radiation in our preclinical NSCLC brain metastasis models, supporting the ongoing clinical trial (NCT02589522) evaluating M6620 in combination with whole brain irradiation in patients with NSCLC brain metastases.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0305
Nat Commun. 2021 08 18. 12(1): 5010

PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation.

Yilun Sun, Jiji Chen, Shar-Yin N Huang, Yijun P Su, Wenjie Wang, Keli Agama, Sourav Saha, Lisa M Jenkins, John M Pascal, Yves Pommier.

Poly(ADP)-ribosylation (PARylation) regulates chromatin structure and recruits DNA repair proteins. Using single-molecule fluorescence microscopy to track topoisomerase I (TOP1) in live cells, we found that sustained PARylation blocked the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) in a similar fashion as inhibition of the ubiquitin-proteasome system (UPS). PARylation of TOP1-DPC was readily revealed by inhibiting poly(ADP-ribose) glycohydrolase (PARG), indicating the otherwise transient and reversible PARylation of the DPCs. As the UPS is a key repair mechanism for TOP1-DPCs, we investigated the impact of TOP1-DPC PARylation on the proteasome and found that the proteasome is unable to associate with and digest PARylated TOP1-DPCs. In addition, PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Our work identifies PARG as repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs. It also suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs.

DOI: https://doi.org/10.1038/s41467-021-25252-9
Genes Chromosomes Cancer. 2021 Aug 18.

Assessing AML susceptibility in rearrangement-driven patients by DNA breakage at topoisomerase II and CTCF/cohesin binding sites.

Naomi D Atkin, Heather M Raimer, Zhenjia Wang, Chongzhi Zang, Yuh-Hwa Wang.

  An initiating DNA double strand break (DSB) event precedes the formation of cancer-driven chromosomal abnormalities, such as gene rearrangements. Therefore, measuring DNA breaks at rearrangement-participating regions can provide a unique tool to identify and characterize susceptible individuals. Here, we developed a highly sensitive and low-input DNA break mapping method, the first of its kind for patient samples. We then measured genome-wide DNA breakage in normal cells of acute myeloid leukemia (AML) patients with KMT2A (previously MLL) rearrangements, compared to that of non-fusion AML individuals, as a means to evaluate individual susceptibility to gene rearrangements. DNA breakage at the KMT2A gene region was significantly greater in fusion-driven remission individuals, as compared to non-fusion individuals. Moreover, we identified select topoisomerase II (TOP2)-sensitive and CCCTC-binding factor (CTCF)/cohesin binding sites with preferential DNA breakage in fusion-driven patients. Importantly, measuring DSBs at these sites, in addition to the KMT2A gene region, provided greater predictive power when assessing individual break susceptibility. We also demonstrated that low-dose etoposide exposure further elevated DNA breakage at these regions in fusion-driven AML patients, but not in non-fusion patients, indicating that these sites are preferentially sensitive to TOP2 activity in fusion-driven AML patients. These results support that mapping of DSBs in patients enables discovery of novel break-prone regions and monitoring of individuals susceptible to chromosomal abnormalities, and thus cancer. This will build the foundation for early detection of cancer-susceptible individuals, as well as those preferentially susceptible to therapy-related malignancies caused by treatment with TOP2 poisons. This article is protected by copyright. All rights reserved.

Keywords:  CTCF; DNA fragility; acute myeloid leukemia; cohesin; topoisomerase

DOI:  https://doi.org/10.1002/gcc.22993
STAR Protoc. 2021 Sep 17. 2(3): 100700

A laser-mediated photo-manipulative toolbox for generation and real-time monitoring of DNA lesions.

Bindhu K Madhavan, Zhe Han, Albert Sickmann, Rainer Pepperkok, Peter P Nawroth, Varun Kumar.

  With the advancement of laser-based microscopy tools, it is now possible to explore mechano-kinetic processes occurring inside the cell. Here, we describe the advanced protocol for studying the DNA repair kinetics in real time using the laser to induce the DNA damage. This protocol can be used for inducing, testing, and studying the repair mechanisms associated with DNA double-strand breaks, interstrand cross-link repair, and single-strand break repair. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2017, 2020).

Keywords:  Cancer; Cell Biology; Microscopy; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2021.100700
Nature. 2021 Aug 18.

Molecular basis for DarT ADP-ribosylation of a DNA base.

Marion Schuller, Rachel E Butler, Antonio Ariza, Callum Tromans-Coia, Gytis Jankevicius, Tim D W Claridge, Sharon L Kendall, Shan Goh, Graham R Stewart, Ivan Ahel.

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.

DOI: https://doi.org/10.1038/s41586-021-03825-4
NAR Cancer. 2021 Sep;3(3): zcab032

A novel view on an old drug, 5-fluorouracil: an unexpected RNA modifier with intriguing impact on cancer cell fate.

Mounira Chalabi-Dchar, Tanguy Fenouil, Christelle Machon, Anne Vincent, Frédéric Catez, Virginie Marcel, Hichem C Mertani, Jean-Christophe Saurin, Philippe Bouvet, Jérôme Guitton, Nicole Dalla Venezia, Jean-Jacques Diaz.

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat patients with solid tumours, such as colorectal and pancreatic cancers. Colorectal cancer (CRC) is the second leading cause of cancer-related death and half of patients experience tumour recurrence. Used for over 60 years, 5-FU was long thought to exert its cytotoxic effects by altering DNA metabolism. However, 5-FU mode of action is more complex than previously anticipated since 5-FU is an extrinsic source of RNA modifications through its ability to be incorporated into most classes of RNA. In particular, a recent report highlighted that, by its integration into the most abundant RNA, namely ribosomal RNA (rRNA), 5-FU creates fluorinated active ribosomes and induces translational reprogramming. Here, we review the historical knowledge of 5-FU mode of action and discuss progress in the field of 5-FU-induced RNA modifications. The case of rRNA, the essential component of ribosome and translational activity, and the plasticity of which was recently associated with cancer, is highlighted. We propose that translational reprogramming, induced by 5-FU integration in ribosomes, contributes to 5-FU-driven cell plasticity and ultimately to relapse.

DOI: https://doi.org/10.1093/narcan/zcab032
Biochem Pharmacol. 2021 Aug 16. pii: S0006-2952(21)00352-X. [Epub ahead of print] 114736

PIKE-A promotes glioblastoma growth by driving PPP flux through increasing G6PD expression mediated by phosphorylation of STAT3.

Mingming Sun, Hao Sheng, Tingfeng Wu, Jiaqi Song, Huanran Sun, Yingzhi Wang, Jiyan Wang, Zhen Li, Huifang Zhao, Junzhen Tan, Yanping Li, Guo Chen, Qingrong Huang, Yuan Zhang, Bei Lan, Shuangping Liu, Changliang Shan, Shuai Zhang.

  Reprogramming of energy metabolism is a hallmarkofcancer, and the pentose phosphate pathway (PPP) is a major glucose metabolic pathway important for meeting the cellular demands of biosynthesis and anti-oxidant defense. Our previous study showed that phosphoinositide 3-kinase enhancer-activating Akt (PIKE-A) plays an important role in glioblastoma cell survival and growth under cellular energy stress condition. However, the crucial functions of PIKE-A in cancer energy metabolism are poorly understood.In the present study, we show that PIKE-A promotes DNA biosynthesis, NADPH production and inhibits reactive oxygen species (ROS) production, leading to increasing proliferation and growth of glioblastoma cell and suppressing cellular senescence. Mechanistically, PIKE-A binds to STAT3 and stimulates its phosphorylation mediated by tyrosine kinase Fyn, which enhances transcription of the rate-limitting enzyme glucose-6-phosphate dehydrogenase (G6PD) in the PPP. Finally, targeting PIKE-A-G6PD axis sensitizes glioblastoma to temozolomide (TMZ)treatment. This study reveals that STAT3 is a novel binding partner of PIKE-A which recruits Fyn to phosphorylate STAT3, contributing to the expression of G6PD, leading to promoting tumor growth and suppressing cellular senescence. Thus, the PIKE-A/STAT3/G6PD axis strongly links the PPP to carcinogenesis and may become a promising cancer therapeutic target.

Keywords:  Fyn; G6PD; PIKE-A; STAT3; glioblastoma; phosphorylation

DOI:  https://doi.org/10.1016/j.bcp.2021.114736
Commun Biol. 2021 Aug 17. 4(1): 977

Integrated multi-omics analysis of RB-loss identifies widespread cellular programming and synthetic weaknesses.

Swetha Rajasekaran, Jalal Siddiqui, Jessica Rakijas, Brandon Nicolay, Chenyu Lin, Eshan Khan, Rahi Patel, Robert Morris, Emanuel Wyler, Myriam Boukhali, Jayashree Balasubramanyam, R Ranjith Kumar, Capucine Van Rechem, Christine Vogel, Sailaja V Elchuri, Markus Landthaler, Benedikt Obermayer, Wilhelm Haas, Nicholas Dyson, Wayne Miles.

Inactivation of RB is one of the hallmarks of cancer, however gaps remain in our understanding of how RB-loss changes human cells. Here we show that pRB-depletion results in cellular reprogramming, we quantitatively measured how RB-depletion altered the transcriptional, proteomic and metabolic output of non-tumorigenic RPE1 human cells. These profiles identified widespread changes in metabolic and cell stress response factors previously linked to E2F function. In addition, we find a number of additional pathways that are sensitive to RB-depletion that are not E2F-regulated that may represent compensatory mechanisms to support the growth of RB-depleted cells. To determine whether these molecular changes are also present in RB1-/- tumors, we compared these results to Retinoblastoma and Small Cell Lung Cancer data, and identified widespread conservation of alterations found in RPE1 cells. To define which of these changes contribute to the growth of cells with de-regulated E2F activity, we assayed how inhibiting or depleting these proteins affected the growth of RB1-/- cells and of Drosophila E2f1-RNAi models in vivo. From this analysis, we identify key metabolic pathways that are essential for the growth of pRB-deleted human cells.

DOI: https://doi.org/10.1038/s42003-021-02495-2
Breast Cancer Res Treat. 2021 Aug 18.

The frequency and clinical significance of DNA polymerase beta (POLβ) expression in breast ductal carcinoma in situ (DCIS).

Abdulbaqi Al-Kawaz, Reem Ali, Michael S Toss, Islam M Miligy, Omar J Mohammed, Andrew R Green, Srinivasan Madhusudan, Emad A Rakha.

   BACKGROUND: The prediction of clinical behaviour of breast ductal carcinoma in situ (DCIS) and its progression to invasive disease remains a challenge. Alterations of DNA damage repair mechanisms are associated with invasive breast cancer (BC). This study aims to assess the role of base excision repair (BER) DNA Polymerase Beta (POLβ) in DCIS.
METHODS: A cohort of DCIS comprising pure DCIS (n = 776) and DCIS coexisting with invasive BC (n = 239) were prepared as tissue microarrays. POLβ protein expression was assessed using immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Preclinically, we investigated the impact of POLβ depletion on stem cell markers in representative DCIS cell line models.
RESULTS: Reduced POLβ expression was associated with aggressive DCIS features including high nuclear grade, comedo necrosis, larger tumour size, hormonal receptor negativity, HER2 overexpression and high Ki67 index. Combined low nuclear/low cytoplasmic POLβ expression showed the strongest association with the features' characteristics of aggressive behaviour. There was a gradual reduction in the POLβ expression from normal breast tissue, to DCIS, with the lowest expression observed in the invasive BC. Low POLβ expression was an independent predictor of recurrence in DCIS patients treated with breast conserving surgery (BCS). POLβ knockdown was associated with a significant increase in cell stemness markers including SOX2, NANOG and OCT4 levels in MCF10-DCIS cell lines.
CONCLUSION: Loss of POLβ in DCIS is associated with aggressive behaviour and it can predict recurrence. POLβ expression in DCIS provides an additional feature for patients' risk stratification for personalised therapy.

Keywords:  Breast cancer; DCIS; DNA damage response; POLβ; Prognosis

DOI:  https://doi.org/10.1007/s10549-021-06357-7
Mol Cancer Ther. 2021 Aug 19. pii: molcanther.MCT-21-0089-A.2021. [Epub ahead of print]

SLFN11 is widely expressed in pediatric sarcoma and induces variable sensitization to replicative stress caused by DNA damaging agents.

Jessica Gartrell, Marcia Mellado-Lagarde, Michael R Clay, Armita Bahrami, Natasha A Sahr, April Sykes, Kaley Blankenship, Lauren Hoffman, Jia Xie, Hyekyung P Cho, Nathaniel Twarog, Michele Connelly, Koon-Kiu Yan, Jiyang Yu, Shaina N Porter, Shondra M Pruett-Miller, Geoffrey Neale, Christopher L Tinkle, Sara M Federico, Elizabeth A Stewart, Anang A Shelat.

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated as a potential biomarker to predict sensitivity to DNA damaging agents (DDA). SLFN11 expression was quantified in 220 children with solid tumors using immunohistochemistry. Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out and over-expression models, and a patient-derived orthotopic xenograft model (PDOX). SLFN11 was expressed in 69% of pediatric sarcoma sampled, including 90% and 100% of Ewing sarcoma (ES) and desmoplastic small round cell tumors, respectively, although the magnitude of expression varied widely. In sarcoma cell lines, protein expression strongly correlated with response to TAL and IRN, with SLFN11 knockout resulting in significant loss of sensitivity in vitro and in vivo. Surprisingly, retrospective analysis of children with sarcoma found no association between SLFN11 levels and favorable outcome. Subsequently, high SLFN11 expression was confirmed in a PDOX model derived from a recurrent ES patient who failed to respond to treatment with TAL + IRN. Selective inhibition of BCL-xL increased sensitivity to TAL + IRN in SLFN11-positive resistant tumor cells. Although SLFN11 appears to drive sensitivity to replicative stress in pediatric sarcomas, its potential to act as a biomarker may be limited to certain tumor backgrounds or contexts. Impaired apoptotic response may be one mechanism of resistance to DDA-induced replicative stress.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0089
Bioorg Chem. 2021 Aug 04. pii: S0045-2068(21)00617-9. [Epub ahead of print]115 105240

Divergent synthesis and elaboration of structure activity relationship for quinoline derivatives as highly selective NTPDase inhibitor.

Amna Murtaza, Saira Afzal, Gohar Zaman, Aamer Saeed, Julie Pelletier, Jean Sévigny, Jamshed Iqbal, Abbas Hassan.

  Quinoline derivatives have interesting biological profile. In continuation for the comprehensive evaluations of substituted quinoline derivatives against human nucleoside triphosphate diphosphohydrolases (h-NTPDases) a series of substituted quinoline derivatives (2a-g, 3a-f, 4, 5a-c, 6) was synthesized. The inhibitory activities of the synthesized compounds were evaluated against four isoenzymes of human nucleoside triphosphate diphosphohydrolases (h-NTPDases). These quinoline derivatives had IC50 (µM) values in the range of 0.20-1.75, 0.77-2.20, 0.36-5.50 and 0.90-1.82 for NTPDase1, NTPDase2, NTPDase3 and NTPDase8, respectively. The derivative 3f was the most active compound against NTPDase1 (IC50, 0.20 ± 0.02 µM) that also possessed selectivity towards NTPDase1. Similarly, derivative 3b (IC50, 0.77 ± 0.06), 2h (IC50, 0.36 ± 0.01) and 2c (IC50, 0.90 ± 0.08) displayed excellent activity corresponding to NTPDase2, NTPDase3 and NTPdase8. The compound 5c emerged as a selective inhibitor of NTPDase8. The most active compounds were then investigated to determine their mode of inhibition and finally binding interactions of the active compounds were analyzed through molecular docking studies. The obtained results strongly support the quinoline scaffold's potential as potent and selective NTPDase inhibitor.

Keywords:  Human nucleoside triphosphate diphosphohydrolases (h-NTPDases); Iodine catalysis; Molecular docking studies; Quinoline derivatives; Structure activity relationship

DOI:  https://doi.org/10.1016/j.bioorg.2021.105240