bims-numges 2021-12-26 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–12–26
thirty-one papers selected by
Sean Rudd, Karolinska Institutet

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51.
RPA phosphorylation facilitates RAD52 dependent homologous recombination in BRCA-deficient cells.
Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells.
SUMO-Based Regulation of Nuclear Positioning to Spatially Regulate Homologous Recombination Activities at Replication Stress Sites.
Homologous Recombination as a Fundamental Genome Surveillance Mechanism during DNA Replication.
The Interplay of Cohesin and the Replisome at Processive and Stressed DNA Replication Forks.
Role and Regulation of the RECQL4 Family during Genomic Integrity Maintenance.
Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea.
PARP Inhibitors and Myeloid Neoplasms: A Double-Edged Sword.
Biochemical analysis of DNA synthesis blockage by G-quadruplex structure and bypass facilitated by a G4-resolving helicase.
The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis.
UBE2O and USP7 co-regulate RECQL4 ubiquitinylation and homologous recombination-mediated DNA repair.
Cell-Cycle-Dependent Chromatin Dynamics at Replication Origins.
Tools for Decoding Ubiquitin Signaling in DNA Repair.
The Abscission Checkpoint: A Guardian of Chromosomal Stability.
Multiple DSB Resection Activities Redundantly Promote Alternative End Joining-Mediated Class Switch Recombination.
New Synthetic Lethality Re-Sensitizing Platinum-Refractory Cancer Cells to Cisplatin In Vitro: The Rationale to Co-Use PARP and ATM Inhibitors.
Structural Insight into the Mechanism of PALB2 Interaction with MRG15.
IFI16 inhibits DNA repair that potentiates type-I interferon-induced antitumor effects in triple negative breast cancer.
Maternal Smc3 protects the integrity of the zygotic genome through DNA replication and mitosis.
Mitochondrial glutamine metabolism regulates sensitivity of cancer cells after chemotherapy via amphiregulin.
Recent advances in biosensor for DNA glycosylase activity detection.
Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos.
Chemotherapeutic drugs induce oxidative stress associated with DNA repair and metabolism modulation.
The rate-limiting step of DNA synthesis by DNA polymerase occurs in the fingers-closed conformation.
Autophosphorylation transforms DNA-PK from protecting to processing DNA ends.
Protein phosphatase 2A-dependent mitotic hnRNPA1 dephosphorylation and TERRA formation facilitate telomere capping.
RB expression confers sensitivity to CDK4/6 inhibitor-mediated radiosensitization across breast cancer subtypes.
MLH1 mediates cytoprotective nucleophagy to resist 5-Fluorouracil-induced cell death in colorectal carcinoma.
On-flow magnetic particle activity assay for the screening of human purine nucleoside phosphorylase inhibitors.
Design, synthesis and biological evaluation studies of novel small molecule ENPP1 inhibitors for cancer immunotherapy.

Nature. 2021 Dec 22.

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51.

Roopesh Anand, Erika Buechelmaier, Ondrej Belan, Matthew Newton, Aleksandra Vancevska, Artur Kaczmarczyk, Tohru Takaki, David S Rueda, Simon N Powell, Simon J Boulton.

DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3' to 5' polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2-4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

DOI: https://doi.org/10.1038/s41586-021-04261-0
Mol Cell Biol. 2021 Dec 20. mcb0052421

RPA phosphorylation facilitates RAD52 dependent homologous recombination in BRCA-deficient cells.

Alison C Carley, Manisha Jalan, Shyamal Subramanyam, Rohini Roy, Gloria E O Borgstahl, Simon N Powell.

Loss of RAD52 is synthetically lethal in BRCA-deficient cells, owing to its role in backup homologous recombination (HR) repair of DNA double-strand breaks (DSBs). In HR in mammalian cells, DSBs are processed to single-stranded DNA (ssDNA) overhangs, which are then bound by Replication Protein A(RPA). RPA is exchanged for RAD51 by mediator proteins: in mammals BRCA2 is the primary mediator, however, RAD52 provides an alternative mediator pathway in BRCA-deficient cells. RAD51 stimulates strand exchange between homologous DNA duplexes, a critical step in HR. RPA phosphorylation and de-phosphorylation are important for HR, but its effect on RAD52 mediator function is unknown. Here, we show that RPA phosphorylation is required for RAD52 to salvage HR in BRCA-deficient cells. Using BRCA2-depleted human cells, in which the only available mediator pathway is RAD52-dependent, the expression of phosphorylation-deficient RPA mutant reduced HR. Furthermore, RPA-phospho-mutant cells showed reduced association of RAD52 with RAD51. Interestingly, there was no effect of RPA phosphorylation on RAD52 recruitment to repair foci. Finally, we show that RPA phosphorylation does not affect RAD52-dependent ssDNA annealing. Thus, although RAD52 can be recruited independently of RPA's phosphorylation status, RPA phosphorylation is required for RAD52's association with RAD51, and its subsequent promotion of RAD52-mediated HR.

DOI: https://doi.org/10.1128/mcb.00524-21
Cancers (Basel). 2021 Dec 10. pii: 6215. [Epub ahead of print]13(24):

Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells.

David King, Harriet E D Southgate, Saskia Roetschke, Polly Gravells, Leona Fields, Jessica B Watson, Lindi Chen, Devon Chapman, Daniel Harrison, Daniel Yeomanson, Nicola J Curtin, Deborah A Tweddle, Helen E Bryant.

  Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB.

Keywords:  ATR; MYCN; PARP; neuroblastoma; replication stress

DOI:  https://doi.org/10.3390/cancers13246215
Genes (Basel). 2021 Dec 17. pii: 2010. [Epub ahead of print]12(12):

SUMO-Based Regulation of Nuclear Positioning to Spatially Regulate Homologous Recombination Activities at Replication Stress Sites.

Kamila Schirmeisen, Sarah A E Lambert, Karol Kramarz.

  DNA lesions have properties that allow them to escape their nuclear compartment to achieve DNA repair in another one. Recent studies uncovered that the replication fork, when its progression is impaired, exhibits increased mobility when changing nuclear positioning and anchors to nuclear pore complexes, where specific types of homologous recombination pathways take place. In yeast models, increasing evidence points out that nuclear positioning is regulated by small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to maintaining genome integrity at sites of replication stress. Here, we review how SUMO-based pathways are instrumental to spatially segregate the subsequent steps of homologous recombination during replication fork restart. In particular, we discussed how routing towards nuclear pore complex anchorage allows distinct homologous recombination pathways to take place at halted replication forks.

Keywords:  DNA; SUMO; chromatin mobility; genome stability; homologous recombination; nuclear pore complex; replication stress; yeast

DOI:  https://doi.org/10.3390/genes12122010
Genes (Basel). 2021 Dec 09. pii: 1960. [Epub ahead of print]12(12):

Homologous Recombination as a Fundamental Genome Surveillance Mechanism during DNA Replication.

Julian Spies, Hana Polasek-Sedlackova, Jiri Lukas, Kumar Somyajit.

  Accurate and complete genome replication is a fundamental cellular process for the proper transfer of genetic material to cell progenies, normal cell growth, and genome stability. However, a plethora of extrinsic and intrinsic factors challenge individual DNA replication forks and cause replication stress (RS), a hallmark of cancer. When challenged by RS, cells deploy an extensive range of mechanisms to safeguard replicating genomes and limit the burden of DNA damage. Prominent among those is homologous recombination (HR). Although fundamental to cell division, evidence suggests that cancer cells exploit and manipulate these RS responses to fuel their evolution and gain resistance to therapeutic interventions. In this review, we focused on recent insights into HR-mediated protection of stress-induced DNA replication intermediates, particularly the repair and protection of daughter strand gaps (DSGs) that arise from discontinuous replication across a damaged DNA template. Besides mechanistic underpinnings of this process, which markedly differ depending on the extent and duration of RS, we highlight the pathophysiological scenarios where DSG repair is naturally silenced. Finally, we discuss how such pathophysiological events fuel rampant mutagenesis, promoting cancer evolution, but also manifest in adaptative responses that can be targeted for cancer therapy.

Keywords:  BRCA1/2; DNA polymerases; DNA replication; RAD51; adaptative mutagenesis; cancer evolution; cancer microenvironment; cancer therapy; chromosome stability; daughter strand gaps; homologous recombination; replication fork protection; replication stress

DOI:  https://doi.org/10.3390/genes12121960
Cells. 2021 Dec 08. pii: 3455. [Epub ahead of print]10(12):

The Interplay of Cohesin and the Replisome at Processive and Stressed DNA Replication Forks.

Janne J M van Schie, Job de Lange.

  The cohesin complex facilitates faithful chromosome segregation by pairing the sister chromatids after DNA replication until mitosis. In addition, cohesin contributes to proficient and error-free DNA replication. Replisome progression and establishment of sister chromatid cohesion are intimately intertwined processes. Here, we review how the key factors in DNA replication and cohesion establishment cooperate in unperturbed conditions and during DNA replication stress. We discuss the detailed molecular mechanisms of cohesin recruitment and the entrapment of replicated sister chromatids at the replisome, the subsequent stabilization of sister chromatid cohesion via SMC3 acetylation, as well as the role and regulation of cohesin in the response to DNA replication stress.

Keywords:  DNA entrapment; DNA replication; DNA replication stress; SMC3 acetylation; cohesin complex; cohesion establishment; fork stalling; replisome; sister chromatid cohesion

DOI:  https://doi.org/10.3390/cells10123455
Genes (Basel). 2021 Nov 29. pii: 1919. [Epub ahead of print]12(12):

Role and Regulation of the RECQL4 Family during Genomic Integrity Maintenance.

Thong T Luong, Kara A Bernstein.

  RECQL4 is a member of the evolutionarily conserved RecQ family of 3' to 5' DNA helicases. RECQL4 is critical for maintaining genomic stability through its functions in DNA repair, recombination, and replication. Unlike many DNA repair proteins, RECQL4 has unique functions in many of the central DNA repair pathways such as replication, telomere, double-strand break repair, base excision repair, mitochondrial maintenance, nucleotide excision repair, and crosslink repair. Consistent with these diverse roles, mutations in RECQL4 are associated with three distinct genetic diseases, which are characterized by developmental defects and/or cancer predisposition. In this review, we provide an overview of the roles and regulation of RECQL4 during maintenance of genome homeostasis.

Keywords:  DNA crosslink repair; RECQL4; base excision repair; double-strand break repair; genome stability; mitochondria; nucleotide excision repair; replication; telomere

DOI:  https://doi.org/10.3390/genes12121919
Int J Mol Sci. 2021 Dec 10. pii: 13296. [Epub ahead of print]22(24):

Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea.

Mariarosaria De Falco, Mariarita De Felice.

  All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.

Keywords:  DNA end resection; DNA repair; DSB; archaea; homologous recombination

DOI:  https://doi.org/10.3390/ijms222413296
Cancers (Basel). 2021 Dec 20. pii: 6385. [Epub ahead of print]13(24):

PARP Inhibitors and Myeloid Neoplasms: A Double-Edged Sword.

Clifford M Csizmar, Antoine N Saliba, Elizabeth M Swisher, Scott H Kaufmann.

  Despite recent discoveries and therapeutic advances in aggressive myeloid neoplasms, there remains a pressing need for improved therapies. For instance, in acute myeloid leukemia (AML), while most patients achieve a complete remission with conventional chemotherapy or the combination of a hypomethylating agent and venetoclax, de novo or acquired drug resistance often presents an insurmountable challenge, especially in older patients. Poly(ADP-ribose) polymerase (PARP) enzymes, PARP1 and PARP2, are involved in detecting DNA damage and repairing it through multiple pathways, including base excision repair, single-strand break repair, and double-strand break repair. In the context of AML, PARP inhibitors (PARPi) could potentially exploit the frequently dysfunctional DNA repair pathways that, similar to deficiencies in homologous recombination in BRCA-mutant disease, set the stage for cell killing. PARPi appear to be especially effective in AML with certain gene rearrangements and molecular characteristics (RUNX1-RUNX1T1 and PML-RARA fusions, FLT3- and IDH1-mutated). In addition, PARPi can enhance the efficacy of other agents, particularly alkylating agents, TOP1 poisons, and hypomethylating agents, that induce lesions ordinarily repaired via PARP1-dependent mechanisms. Conversely, emerging reports suggest that long-term treatment with PARPi for solid tumors is associated with an increased incidence of myelodysplastic syndrome (MDS) and AML. Here, we (i) review the pre-clinical and clinical data on the role of PARPi, specifically olaparib, talazoparib, and veliparib, in aggressive myeloid neoplasms and (ii) discuss the reported risk of MDS/AML with PARPi, especially as the indications for PARPi use expand to include patients with potentially curable cancer.

Keywords:  DNA damage repair; PARP inhibitors; acute myeloid leukemia; base excision repair; myelodysplastic syndrome; myeloid neoplasms; non-homologous end-joining; secondary malignancies; synthetic lethality

DOI:  https://doi.org/10.3390/cancers13246385
Methods. 2021 Dec 17. pii: S1046-2023(21)00282-6. [Epub ahead of print]

Biochemical analysis of DNA synthesis blockage by G-quadruplex structure and bypass facilitated by a G4-resolving helicase.

Joshua A Sommers, Katrina N Estep, Robert W Maul, Robert M Brosh.

  G-quadruplex (G4) DNA poses a unique obstacle to DNA synthesis during replication or DNA repair due to its unusual structure which deviates significantly from the conventional DNA double helix. A mechanism to overcome the G4 roadblock is provided by the action of a G4-resolving helicase that collaborates with the DNA polymerase to smoothly catalyze polynucleotide synthesis past the unwound G4. In this technique-focused paper, we describe the experimental approaches of the primer extension assay using a G4 DNA template to measure the extent and fidelity of DNA synthesis by a DNA polymerase acting in concert with a G4-resolving DNA helicase. Important parameters pertaining to reaction conditions and controls are discussed to aid in the design of experiments and interpretation of the data obtained. This methodology can be applied in multiple capacities that may depend on the DNA substrate, DNA polymerase, or DNA helicase under investigation.

Keywords:  DNA repair; DNA replication; G-quadruplex; Genomic instability; Helicase; Human disease; Mutagenesis; Nucleic acid metabolism; Polymerase

DOI:  https://doi.org/10.1016/j.ymeth.2021.12.005
J Biol Chem. 2021 Dec 17. pii: S0021-9258(21)01316-8. [Epub ahead of print] 101506

The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis.

Kodavati Manohar, Prashant Khandagale, Shraddheya Kumar Patel, Jugal Kishor Sahu, Narottam Acharya.

  DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be non-essential for Polη's function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses we show that the ubz domain of Polη which intrinsically lacks the pip motif directly binds to the inter-domain connecting loop (IDCL) of PCNA and regulates Polη's TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη's function.

Keywords:  Candida; DNA Replication; DNA polymerase; PCNA; Polη; Rad30; Ubiquitin; pip box motif; ubz

DOI:  https://doi.org/10.1016/j.jbc.2021.101506
FASEB J. 2022 Jan;36(1): e22112

UBE2O and USP7 co-regulate RECQL4 ubiquitinylation and homologous recombination-mediated DNA repair.

Qiuling Huang, Dajiang Qin, Duanqing Pei, Michiel Vermeulen, Xiaofei Zhang.

  The human RecQ DNA helicase, RECQL4, plays a pivotal role in maintaining genomic stability by regulating the DNA double-strand breaks (DSBs) repair pathway, and is, thus, involved in the regulation of aging and cancer onset. However, the regulatory mechanisms of RECQL4, especially its post-translational modifications, have not been fully illustrated. Here, we report that the E2/E3 hybrid ubiquitin-conjugating enzyme, UBE2O, physically interacts with RECQL4, and mediates the multi-monoubiquitinylation of RECQL4, subsequently leading to its proteasomal degradation. Functionally, we showed that UBE2O inhibits homologous recombination (HR)-mediated DSBs repair, and this inhibition depends on its E2 catalytic activity and RECQL4 expression. Mechanistically, we showed that UBE2O attenuates the interaction of RECQL4 and DNA damage repair proteins, the MRE11-RAD50-NBS1 complex and CtIP. Furthermore, we show that deubiquitinylase USP7 interacts with both UBE2O and RECQL4, and in that it antagonizes UBE2O-mediated regulation of RECQL4 stability and function. Collectively, we found a novel regulatory mechanism of ubiquitin-mediated regulation of RECQL4 in HR-mediated DSBs repair process.

Keywords:  DNA damage repair; RECQL4; UBE2O; USP7; multi-monoubiquitinylation

DOI:  https://doi.org/10.1096/fj.202100974RRR
Genes (Basel). 2021 Dec 16. pii: 1998. [Epub ahead of print]12(12):

Cell-Cycle-Dependent Chromatin Dynamics at Replication Origins.

Yulong Li, Alexander J Hartemink, David M MacAlpine.

  Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell-cycle-dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors, and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between chromatin occupancy at the ACS and origin efficiency occurred in early S phase, consistent with the rate-limiting formation of the Cdc45-Mcm2-7-GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell-cycle-regulated chromatin dynamics and how they relate to the regulation of origin activity.

Keywords:  DNA replication; cell cycle; chromatin; replication origins

DOI:  https://doi.org/10.3390/genes12121998
Front Cell Dev Biol. 2021 ;9 760226

Tools for Decoding Ubiquitin Signaling in DNA Repair.

Benjamin Foster, Martin Attwood, Ian Gibbs-Seymour.

  The maintenance of genome stability requires dedicated DNA repair processes and pathways that are essential for the faithful duplication and propagation of chromosomes. These DNA repair mechanisms counteract the potentially deleterious impact of the frequent genotoxic challenges faced by cells from both exogenous and endogenous agents. Intrinsic to these mechanisms, cells have an arsenal of protein factors that can be utilised to promote repair processes in response to DNA lesions. Orchestration of the protein factors within the various cellular DNA repair pathways is performed, in part, by post-translational modifications, such as phosphorylation, ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs). In this review, we firstly explore recent advances in the tools for identifying factors involved in both DNA repair and ubiquitin signaling pathways. We then expand on this by evaluating the growing repertoire of proteomic, biochemical and structural techniques available to further understand the mechanistic basis by which these complex modifications regulate DNA repair. Together, we provide a snapshot of the range of methods now available to investigate and decode how ubiquitin signaling can promote DNA repair and maintain genome stability in mammalian cells.

Keywords:  CRISPR-Cas9 screen; DNA damage; DNA repair; cross-linking mass spectrometry; cryo-EM; genome stability; proteomics; ubiquitin

DOI:  https://doi.org/10.3389/fcell.2021.760226
Cells. 2021 Nov 29. pii: 3350. [Epub ahead of print]10(12):

The Abscission Checkpoint: A Guardian of Chromosomal Stability.

Eleni Petsalaki, George Zachos.

  The abscission checkpoint contributes to the fidelity of chromosome segregation by delaying completion of cytokinesis (abscission) when there is chromatin lagging in the intercellular bridge between dividing cells. Although additional triggers of an abscission checkpoint-delay have been described, including nuclear pore defects, replication stress or high intercellular bridge tension, this review will focus only on chromatin bridges. In the presence of such abnormal chromosomal tethers in mammalian cells, the abscission checkpoint requires proper localization and optimal kinase activity of the Chromosomal Passenger Complex (CPC)-catalytic subunit Aurora B at the midbody and culminates in the inhibition of Endosomal Sorting Complex Required for Transport-III (ESCRT-III) components at the abscission site to delay the final cut. Furthermore, cells with an active checkpoint stabilize the narrow cytoplasmic canal that connects the two daughter cells until the chromatin bridges are resolved. Unsuccessful resolution of chromatin bridges in checkpoint-deficient cells or in cells with unstable intercellular canals can lead to chromatin bridge breakage or tetraploidization by regression of the cleavage furrow. In turn, these outcomes can lead to accumulation of DNA damage, chromothripsis, generation of hypermutation clusters and chromosomal instability, which are associated with cancer formation or progression. Recently, many important questions regarding the mechanisms of the abscission checkpoint have been investigated, such as how the presence of chromatin bridges is signaled to the CPC, how Aurora B localization and kinase activity is regulated in late midbodies, the signaling pathways by which Aurora B implements the abscission delay, and how the actin cytoskeleton is remodeled to stabilize intercellular canals with DNA bridges. Here, we review recent progress toward understanding the mechanisms of the abscission checkpoint and its role in guarding genome integrity at the chromosome level, and consider its potential implications for cancer therapy.

Keywords:  ATM; Aurora B; CPC; Chk2; Chmp4c; DNA damage; ESCRT; abscission checkpoint; actin patches; cancer; chromatin bridges; chromosomal instability; cytokinesis; midbody

DOI:  https://doi.org/10.3390/cells10123350
Front Cell Dev Biol. 2021 ;9 767624

Multiple DSB Resection Activities Redundantly Promote Alternative End Joining-Mediated Class Switch Recombination.

Xikui Sun, Jingning Bai, Jiejie Xu, Xiaoli Xi, Mingyu Gu, Chengming Zhu, Hongman Xue, Chun Chen, Junchao Dong.

  Alternative end joining (A-EJ) catalyzes substantial level of antibody class switch recombination (CSR) in B cells deficient for classical non-homologous end joining, featuring increased switch (S) region DSB resection and junctional microhomology (MH). While resection has been suggested to initiate A-EJ in model DSB repair systems using engineered endonucleases, the contribution of resection factors to A-EJ-mediated CSR remains unclear. In this study, we systematically dissected the requirement for individual DSB resection factors in A-EJ-mediated class switching with a cell-based assay system and high-throughput sequencing. We show that while CtIP and Mre11 both are mildly required for CSR in WT cells, they play more critical roles in mediating A-EJ CSR, which depend on the exonuclease activity of Mre11. While DNA2 and the helicase/HRDC domain of BLM are required for A-EJ by mediating long S region DSB resection, in contrast, Exo1's resection-related function does not play any obvious roles for class switching in either c-NHEJ or A-EJ cells, or mediated in an AID-independent manner by joining of Cas9 breaks. Furthermore, ATM and its kinase activity functions at least in part independent of CtIP/Mre11 to mediate A-EJ switching in Lig4-deficient cells. In stark contrast to Lig4 deficiency, 53BP1-deficient cells do not depend on ATM/Mre11/CtIP for residual joining. We discuss the roles for each resection factor in A-EJ-mediated CSR and suggest that the extent of requirements for resection is context dependent.

Keywords:  DNA double-strand breaks repair; DSB end resection; alternative end joining; class switch recombination; microhomology

DOI:  https://doi.org/10.3389/fcell.2021.767624
Int J Mol Sci. 2021 Dec 11. pii: 13324. [Epub ahead of print]22(24):

New Synthetic Lethality Re-Sensitizing Platinum-Refractory Cancer Cells to Cisplatin In Vitro: The Rationale to Co-Use PARP and ATM Inhibitors.

Watson P Folk, Alpana Kumari, Tetsushi Iwasaki, Erica K Cassimere, Slovénie Pyndiah, Elizabeth Martin, Kelly Homlar, Daitoku Sakamuro.

  The pro-apoptotic tumor suppressor BIN1 inhibits the activities of the neoplastic transcription factor MYC, poly (ADP-ribose) polymerase-1 (PARP1), and ATM Ser/Thr kinase (ATM) by separate mechanisms. Although BIN1 deficits increase cancer-cell resistance to DNA-damaging chemotherapeutics, such as cisplatin, it is not fully understood when BIN1 deficiency occurs and how it provokes cisplatin resistance. Here, we report that the coordinated actions of MYC, PARP1, and ATM assist cancer cells in acquiring cisplatin resistance by BIN1 deficits. Forced BIN1 depletion compromised cisplatin sensitivity irrespective of Ser15-phosphorylated, pro-apoptotic TP53 tumor suppressor. The BIN1 deficit facilitated ATM to phosphorylate the DNA-damage-response (DDR) effectors, including MDC1. Consequently, another DDR protein, RNF8, bound to ATM-phosphorylated MDC1 and protected MDC1 from caspase-3-dependent proteolytic cleavage to hinder cisplatin sensitivity. Of note, long-term and repeated exposure to cisplatin naturally recapitulated the BIN1 loss and accompanying RNF8-dependent cisplatin resistance. Simultaneously, endogenous MYC was remarkably activated by PARP1, thereby repressing the BIN1 promoter, whereas PARP inhibition abolished the hyperactivated MYC-dependent BIN1 suppression and restored cisplatin sensitivity. Since the BIN1 gene rarely mutates in human cancers, our results suggest that simultaneous inhibition of PARP1 and ATM provokes a new BRCAness-independent synthetic lethal effect and ultimately re-establishes cisplatin sensitivity even in platinum-refractory cancer cells.

Keywords:  ATM; BIN1; MDC1-RNF8 complex; MYC; PARP1; apoptosis; cisplatin resistance

DOI:  https://doi.org/10.3390/ijms222413324
Genes (Basel). 2021 Dec 17. pii: 2002. [Epub ahead of print]12(12):

Structural Insight into the Mechanism of PALB2 Interaction with MRG15.

Jennifer Redington, Jaigeeth Deveryshetty, Lakshmi Kanikkannan, Ian Miller, Sergey Korolev.

  The tumor suppressor protein partner and localizer of BRCA2 (PALB2) orchestrates the interactions between breast cancer susceptibility proteins 1 and 2 (BRCA1, -2) that are critical for genome stability, homologous recombination (HR) and DNA repair. PALB2 mutations predispose patients to a spectrum of cancers, including breast and ovarian cancers. PALB2 localizes HR machinery to chromatin and links it with transcription through multiple DNA and protein interactions. This includes its interaction with MRG15 (Morf-related gene on chromosome 15), which is part of many transcription complexes, including the HAT-associated and the HDAC-associated complexes. This interaction is critical for PALB2 localization in actively transcribed genes, where transcription/replication conflicts lead to frequent replication stress and DNA breaks. We solved the crystal structure of the MRG15 MRG domain bound to the PALB2 peptide and investigated the effect of several PALB2 mutations, including patient-derived variants. PALB2 interacts with an extended surface of the MRG that is known to interact with other proteins. This, together with a nanomolar affinity, suggests that the binding of MRG15 partners, including PALB2, to this region is mutually exclusive. Breast cancer-related mutations of PALB2 cause only minor attenuation of the binding affinity. New data reveal the mechanism of PALB2-MRG15 binding, advancing our understanding of PALB2 function in chromosome maintenance and tumorigenesis.

Keywords:  DNA repair; cancer mutations; crystal structure; genome maintenance; homologous recombination; protein-protein interaction; recombination mediator; transcription complex

DOI:  https://doi.org/10.3390/genes12122002
Cell Rep. 2021 Dec 21. pii: S2211-1247(21)01634-X. [Epub ahead of print]37(12): 110138

IFI16 inhibits DNA repair that potentiates type-I interferon-induced antitumor effects in triple negative breast cancer.

Na-Lee Ka, Ga Young Lim, Sewon Hwang, Seung-Su Kim, Mi-Ock Lee.

  Tumor DNA-damage response (DDR) has an important role in driving type-I interferon (IFN)-mediated host antitumor immunity, but it is not clear how tumor DNA damage is interconnected with the immune response. Here, we report the role of IFN-γ-inducible protein 16 (IFI16) in DNA repair, which amplifies the stimulator of IFN genes (STING)-type-I IFN signaling, particularly in triple-negative breast cancer (TNBC). IFI16 is rapidly induced and accumulated to the histone-evicted DNA at double-stranded breakage (DSB) sites, where it inhibits recruitment of DDR factors. Subsequently, IFI16 increases the release of DNA fragments to the cytoplasm and induces STING-mediated type-I IFN production. Synergistic cytotoxic and immunomodulatory effects of doxorubicin and type-I IFNs are decreased upon IFI16 depletion in vivo. Furthermore, IFI16 expression correlates with improved clinical outcome in patients with TNBC treated with chemotherapy. Together, our findings suggest that type-I IFNs and IFI16 could offer potential therapeutic strategies for TNBC.

Keywords:  DNA damage; DNA repair; IFI16; STING; antitumor immunity; cytosolic DNA; triple-negative breast cancer; type-I IFN

DOI:  https://doi.org/10.1016/j.celrep.2021.110138
Development. 2021 Dec 15. pii: dev199800. [Epub ahead of print]148(24):

Maternal Smc3 protects the integrity of the zygotic genome through DNA replication and mitosis.

Wei-Ting Yueh, Vijay Pratap Singh, Jennifer L Gerton.

  Aneuploidy is frequently observed in oocytes and early embryos, begging the question of how genome integrity is monitored and preserved during this crucial period. SMC3 is a subunit of the cohesin complex that supports genome integrity, but its role in maintaining the genome during this window of mammalian development is unknown. We discovered that, although depletion of Smc3 following meiotic S phase in mouse oocytes allowed accurate meiotic chromosome segregation, adult females were infertile. We provide evidence that DNA lesions accumulated following S phase in SMC3-deficient zygotes, followed by mitosis with lagging chromosomes, elongated spindles, micronuclei, and arrest at the two-cell stage. Remarkably, although centromeric cohesion was defective, the dosage of SMC3 was sufficient to enable embryogenesis in juvenile mutant females. Our findings suggest that, despite previous reports of aneuploidy in early embryos, chromosome missegregation in zygotes halts embryogenesis at the two-cell stage. Smc3 is a maternal gene with essential functions in the repair of spontaneous damage associated with DNA replication and subsequent chromosome segregation in zygotes, making cohesin a key protector of the zygotic genome.

Keywords:  Chromosome segregation; Cohesin; Developmental competence; Juvenile; Maternal-effect gene; Micronuclei; Mouse; SMC3; Spontaneous DNA damage; Zygote

DOI:  https://doi.org/10.1242/dev.199800
Cell Death Discov. 2021 Dec 20. 7(1): 395

Mitochondrial glutamine metabolism regulates sensitivity of cancer cells after chemotherapy via amphiregulin.

Sunsook Hwang, Seungyeon Yang, Minjoong Kim, Youlim Hong, Byungjoo Kim, Eun Kyung Lee, Seung Min Jeong.

The DNA damage response is essential for sustaining genomic stability and preventing tumorigenesis. However, the fundamental question about the cellular metabolic response to DNA damage remains largely unknown, impeding the development of metabolic interventions that might prevent or treat cancer. Recently, it has been reported that there is a link between cell metabolism and DNA damage response, by repression of glutamine (Gln) entry into mitochondria to support cell cycle arrest and DNA repair. Here, we show that mitochondrial Gln metabolism is a crucial regulator of DNA damage-induced cell death. Mechanistically, inhibition of glutaminase (GLS), the first enzyme for Gln anaplerosis, sensitizes cancer cells to DNA damage by inducing amphiregulin (AREG) that promotes apoptotic cell death. GLS inhibition increases reactive oxygen species production, leading to transcriptional activation of AREG through Max-like protein X (MLX) transcription factor. Moreover, suppression of mitochondrial Gln metabolism results in markedly increased cell death after chemotherapy in vitro and in vivo. The essentiality of this molecular pathway in DNA damage-induced cell death may provide novel metabolic interventions for cancer therapy.

DOI: https://doi.org/10.1038/s41420-021-00792-7
Talanta. 2021 Dec 15. pii: S0039-9140(21)01066-3. [Epub ahead of print]239 123144

Recent advances in biosensor for DNA glycosylase activity detection.

Yuzhen Ouyang, Yifan Liu, Yuan Deng, Hailun He, Jin Huang, Changbei Ma, Kemin Wang.

  Base excision repair (BER) is vital for maintaining the integrity of the genome under oxidative damage. DNA glycosylase initiates the BER pathway recognizes and excises the mismatched substrate base leading to the apurinic/apyrimidinic site generation, and simultaneously breaks the single-strand DNA. As the aberrant activity of DNA glycosylase is associated with numerous diseases, including cancer, immunodeficiency, and atherosclerosis, the detection of DNA glycosylase is significant from bench to bedside. In this review, we summarized novel DNA strategies in the past five years for DNA glycosylase activity detection, which are classified into fluorescence, colorimetric, electrochemical strategies, etc. We also highlight the current limitations and look into the future of DNA glycosylase activity monitoring.

Keywords:  Amplification; Biosensor; DNA glycosylase; Detection; Inhibitor

DOI:  https://doi.org/10.1016/j.talanta.2021.123144
Nucleic Acids Res. 2021 Dec 23. pii: gkab1223. [Epub ahead of print]

Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos.

Elena Lo Furno, Isabelle Busseau, Antoine Aze, Claudio Lorenzi, Cima Saghira, Matt C Danzi, Stephan Zuchner, Domenico Maiorano.

In early embryogenesis of fast cleaving embryos, DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient, implying the possible generation of mutations. We have analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster early embryos. We report the occurrence of a high mutation rate in Xenopus and show that it is dependent upon the translesion DNA synthesis (TLS) master regulator Rad18. Unexpectedly, we observed a homology-directed repair contribution of Rad18 in reducing the mutation load. Genetic invalidation of TLS in the pre-blastoderm Drosophila embryo resulted in reduction of both the hatching rate and single-nucleotide variations on pericentromeric heterochromatin in adult flies. Altogether, these findings indicate that during very early Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation.

DOI: https://doi.org/10.1093/nar/gkab1223
Life Sci. 2021 Dec 16. pii: S0024-3205(21)01229-7. [Epub ahead of print]289 120242

Chemotherapeutic drugs induce oxidative stress associated with DNA repair and metabolism modulation.

Yujie Zhang, Chunyang Ding, Wenkang Zhu, Xinyu Li, Techang Chen, Qingxi Liu, Sa Zhou, Tong-Cun Zhang, Wenjian Ma.

  Bulky DNA damage inducing chemotherapeutic cancer drugs such as cisplatin (CIS) and doxorubicin (DOX) are commonly used in the treatment of a variety of cancers. However, they often cause multi-organ toxicity, and the mechanisms underlying are not clear. Using cellular model, the present study showed that persistent endogenous reactive oxygen species (ROS) were stimulated after a single dose short treatment with CIS and DOX. ROS level correlated with the formation of DNA double-strand breaks (DSBs). Knockdown BRCA1, a key player involved in homologous recombination (HR), enhanced ROS accumulation. Whereas knockdown DNA-PKcs and overexpress BRCA1 to inhibit nonhomologous end-joining (NHEJ) repair pathway and restore HR can partially suppress ROS levels. These data indicated that ROS production is associated with DSB formation and repair which is likely a downstream event of DNA repair. Further studies showed that knockdown DNA repair regulators PP2A but not ATM, could partially reduce ROS too. The induction of ROS affected the level of proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Collectively, the present study reveals that DNA repair associated metabolism change and oxidative stress may be a direct cause of the severe side effects associated with genotoxic chemotherapy cancer drugs.

Keywords:  Chemotherapy; Cisplatin; DNA repair; Doxorubicin; Oxidative stress

DOI:  https://doi.org/10.1016/j.lfs.2021.120242
J Mol Biol. 2021 Dec 17. pii: S0022-2836(21)00647-1. [Epub ahead of print] 167410

The rate-limiting step of DNA synthesis by DNA polymerase occurs in the fingers-closed conformation.

Geraint W Evans, Timothy Craggs, Achillefs N Kapanidis.

  DNA polymerases maintain genomic integrity by copying DNA with high fidelity, part of which relies on the polymerase fingers opening-closing transition, a series ofconformational changes during the DNA synthesis reaction cycle. Fingers opening and closing has been challenging to study, mainly due to the need to synchronise molecular ensembles. We previously studied fingers opening-closing on single polymerase-DNA complexes using single-molecule FRET; however, our work was limited to pre-chemistry reaction steps. Here, we advance our analysis to extensible substrates, and observe DNA polymerase (Pol) conformational changes across theentireDNA polymerisation reaction in real-time, gaining direct access to an elusive post-chemistry step rate-limiting for DNA synthesis. Our results showed that Pol adopts the fingers-closed conformation during polymerisation, and that the post-chemistry rate-limiting step occurs in the fingers-closed conformation. We found thatfingers-opening in the Pol-DNA binary complex in the absence of polymerisation is slow (∼5.3 s-1), and comparable to the rate of fingers-opening after polymerisation (3.4 s-1); this indicates that the fingers-opening step itself could be largely responsible for the slow post-chemistry step, with the residual rate potentially accounted for by pyrophosphase release. We also observed that DNA chain-termination of the 3' end of the primer increases substantially the rate of fingers-opening in the Pol-DNA binary complex (5.3 → 29 s-1), demonstrating that the 3'-OH residue is important for the kinetics of fingers conformational changes. Our observations offer mechanistic insight and tools to offer mechanistic insight for all nucleic acid polymerases.

Keywords:  DNA polymerase; DNA synthesis; fingers opening-closing transition; real-time single molecule kinetics; single-molecule FRET

DOI:  https://doi.org/10.1016/j.jmb.2021.167410
Mol Cell. 2021 Dec 14. pii: S1097-2765(21)01027-3. [Epub ahead of print]

Autophosphorylation transforms DNA-PK from protecting to processing DNA ends.

Lan Liu, Xuemin Chen, Jun Li, Huaibin Wang, Christopher J Buehl, Noah J Goff, Katheryn Meek, Wei Yang, Martin Gellert.

  The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.

Keywords:  Artemis; DNA-PKcs; Ku70; Ku80; NHEJ; SCID; V(D)J recombination; hairpin

DOI:  https://doi.org/10.1016/j.molcel.2021.11.025
Mol Cancer Res. 2021 Dec 21. pii: molcanres.MCR-21-0581-E.2021. [Epub ahead of print]

Protein phosphatase 2A-dependent mitotic hnRNPA1 dephosphorylation and TERRA formation facilitate telomere capping.

Jiang-Dong Sui, Zheng Tang, Benjamin P C Chen, Ping Huang, Meng-Qi Yang, Nuo-Han Wang, Hao-Nan Yang, Hong-Lei Tu, Qing-Ming Jiang, Jing Zhang, Ying Wang, Yong-Zhong Wu.

The heterogeneous nuclear ribonucleoprotein A1(hnRNPA1), telomeric repeat-containing RNA (TERRA), and protection of telomeres 1 (POT1) have been reported to orchestrate to displace replication protein A (RPA) from telomeric overhangs, ensuring orderly telomere replication and capping. Our previous studies further demonstrated that DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-dependent hnRNPA1 phosphorylation plays a crucial role in the promotion of hnRNPA1 binding to telomeric overhangs and RPA displacement during G2/M phases. However, it is unclear that how the subsequent exchange between hnRNPA1 and POT1 is orchestrated. Here we report that the protein phosphatase 2A (PP2A) depends on its scaffold subunit, which is called PPP2R1A, to interact with and dephosphorylate hnRNPA1 in the late M phase. Furthermore, PP2A-mediated hnRNPA1 dephosphorylation and TERRA accumulation act in concert to promote the hnRNPA1-to-POT1 switch on telomeric single-stranded DNA. Consequently, defective PPP2R1A results in ATR-mediated DNA damage response at telomeres as well as induction of fragile telomeres. Combined inhibition of ATR and PP2A induces entry into a catastrophic mitosis and leads to synthetic lethality of tumor cells. In addition, PPP2R1A levels correlate with clinical stages and prognosis of multiple types of cancers. Taken together, our results indicate that PP2A is critical for telomere maintenance. Implications:This study demonstrates that the PP2A-dependent hnRNPA1 dephosphorylation and TERRA accumulation facilitates the formation of the protective capping structure of newly replicated telomeres, thus exerting essential oncogenic role in tumorigenesis.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0581
JCI Insight. 2021 Dec 21. pii: e154402. [Epub ahead of print]

RB expression confers sensitivity to CDK4/6 inhibitor-mediated radiosensitization across breast cancer subtypes.

Andrea M Pesch, Nicole H Hirsh, Anna R Michmerhuizen, Kassidy M Jungles, Kari Wilder-Romans, Benjamin C Chandler, Meilan Liu, Lynn M Lerner, Charles A Nino, Connor Ward, Erin F Cobain, Theodore S Lawrence, Lori J Pierce, James M Rae, Corey W Speers.

  Standard radiation (RT) therapy does not reliably provide locoregional control for women with multi-node positive and triple-negative (TNBC) breast cancers. We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor positive (ER+) cells, but also TNBC that express retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB wild-type TNBC (n=4, rER 1.49 - 2.22), but failed to radiosensitize RB-null TNBC (n=3, rER: 0.84 - 1.00). RB expression predicted response to CDK4/6i + RT (R2=0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88 - 1.13) after RB1 knockdown in isogenic and non-isogenic models. CDK4/6i suppressed homologous recombination (HR) in RB wild-type cells, but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB re-expression. Radiosensitization was independent of non-homologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts, but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult to control RB-intact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.

Keywords:  Breast cancer; DNA repair; Oncology; Radiation therapy

DOI:  https://doi.org/10.1172/jci.insight.154402
Neoplasia. 2021 Dec 21. pii: S1476-5586(21)00107-X. [Epub ahead of print]24(2): 76-85

MLH1 mediates cytoprotective nucleophagy to resist 5-Fluorouracil-induced cell death in colorectal carcinoma.

Shaista Manzoor, Maha Saber-Ayad, Azzam A Maghazachi, Qutayba Hamid, Jibran Sualeh Muhammad.

  Colorectal Cancer (CRC) with Microsatellite instability (MSI) and mutLhomolog-1 (MLH1) gene deficiency are less aggressive than MLH1 proficient cancers. MLH1 is involved in several cellular processes, but its connection with the autophagy-dependent cellular response towards anticancer drugs remains unclear. In this study, we aimed to investigate the interaction between MLH1 and the autophagy marker LC3, which facilitated nucleophagy induction, and its potential role in determining sensitivity to 5-Fluorouracil (5-FU) induced cell death. To examine the role of MLH1 in DNA-damage-induced nucleophagy in CRC cells, we utilized a panel of MLH1 deficient and MLH1 proficient CRC cell lines. We included a parental HCT116 cell line (MLH1-/-) and its isogenic cell line HCT116 MLH1+/- in which a single allele of the MLH1 gene was introduced using CRISPR-Cas9 gene editing. We observed that MLH1 proficient cells were less sensitive to the 5-FU-induced cytotoxic effect. The 5-FU induced DNA damage led to LC3 up-regulation, which was dependent on MLH1 overexpression. Moreover, immunofluorescence and immunoprecipitation data showed LC3 and MLH1 were colocalized in CRC cells. Consequently, MLH1 dependent 5-FU-induced DNA damage contributed to the formation of micronuclei. These micronuclei colocalize with autolysosome, indicating a cytoprotective role of MLH1 dependent nucleophagy. Interestingly, siRNA knockdown of MLH1 in HCT116 MLH1+/- prevented LC3 upregulation and micronuclei formation. These novel data are the first to show an essential role of MLH1 in mediating the chemoresistance and survival of cancer cells by increasing the LC3 expression and inducing nucleophagy in 5-FU treated CRC cells.

Keywords:  Autophagy; CRISPR-Cas9; Chemoresistance; Colorectal cancer; LC3; Lamin; Microsatellite instability; Mismatch Repair; Nucleophagy; SIRT1

DOI:  https://doi.org/10.1016/j.neo.2021.12.003
J Chromatogr A. 2021 Dec 07. pii: S0021-9673(21)00862-1. [Epub ahead of print]1663 462740

On-flow magnetic particle activity assay for the screening of human purine nucleoside phosphorylase inhibitors.

I A T Ximenes, M Albino, C Sangregorio, Q B Cass, M C de Moraes.

  Human purine nucleoside phosphorylase (HsPNP) catalyzes reversible phosphorolysis of nucleosides and deoxynucleosides in the purine cascade. HsPNP has been a target on behalf of the development of new leads for the treatment of a variety of T-cell mediated disorders. Several studies on the HsPNP are focused on the identification of effective, safe, and selective inhibitors. Therefore, this study describes the development of direct, simple, reliable, and inexpensive enzymatic assays to screen HsPNP inhibitors. Initially, HsPNP was covalently immobilized on the surface of magnetic particles (MPs). Due to the versatility of the MPs as solid support for enzyme immobilization, two different methods to monitor the enzyme activity are presented. Firstly, the activity of HsPNP-MPs was assessed offline by HPLC-DAD quantifying the formed hypoxanthine. Then, HsPNP-MPs were trapped in a peek tube, furnishing a microreactor which was inserted on-flow in an HPLC-DAD system to monitor the enzyme activity by the hypoxanthine quantification. Kinetic assays provided KMapp values for the inosine substrate of 488.2 ± 49.1 and 1084 ± 111 µM for the offline and on-flow assays, respectively. For the first time, kinetic studies for Pi as substrate using the HsPNP-MPs exhibits a Michaelis-Menten kinetic, yielding KMapp values for offline and on-flow of 521.2 ± 62.9 µM and 601 ± 66.5 µM, respectively. Inhibition studies conducted with a fourth generation immucillin derivative (DI4G) were employed as proof of concept to validate the use of the HsPNP-MPs assays for screening purposes. Additionally, a small library containing 11 compounds was used to assess the selectivity of the developed assays. The results showed that both presented assays can be applied to selectively recognizing and characterizing HsPNP inhibitors. Particularly, the on-flow method exhibited a high throughput and performance because of its automation and represents an easy and practical approach to reuse the HsPNP-MPs. Besides, this novel enzyme activity assay model can be further applied to other biological targets.

Keywords:  Bioaffinity chromatography; Immobilized enzyme; Magnetic particles; On-flow enzymatic assay; Purine nucleoside phosphorylase; Screening assay

DOI:  https://doi.org/10.1016/j.chroma.2021.462740
Bioorg Chem. 2021 Dec 11. pii: S0045-2068(21)00927-5. [Epub ahead of print]119 105549

Design, synthesis and biological evaluation studies of novel small molecule ENPP1 inhibitors for cancer immunotherapy.

Mukesh Gangar, Sandeep Goyal, Digambar Raykar, Princy Khurana, Ashwita M Martis, Avijit Goswami, Ishani Ghoshal, Ketul V Patel, Yadav Nagare, Santosh Raikar, Apurba Mukherjee, Rajath Cyriac, Jean-François Paquin, Aditya Kulkarni.

  Ecto-nucleotide pyrophosphatase/phosphodiesterases 1 (ENPP1 or NPP1), is an attractive therapeutic target for various diseases, primarily cancer and mineralization disorders. The ecto-enzyme is located on the cell surface and has been implicated in the control of extracellular levels of nucleotide, nucleoside and (di) phosphate. Recently, it has emerged as a critical phosphodiesterase that hydrolyzes cyclic 2'3'- cGAMP, the endogenous ligand for STING (STimulator of INterferon Genes). STING plays an important role in innate immunity by activating type I interferon in response to cytosolic 2'3'-cGAMP. ENPP1 negatively regulates the STING pathway and hence its inhibition makes it an attractive therapeutic target for cancer immunotherapy. Herein, we describe the design, optimization and biological evaluation studies of a series of novel non-nucleotidic thioguanine based small molecule inhibitors of ENPP1. The lead compound 43 has shown good in vitro potency, stability in SGF/SIF/PBS, selectivity, ADME properties and pharmacokinetic profile and finally potent anti-tumor response in vivo. These compounds are a good starting point for the development of potentially effective cancer immunotherapy agents.

Keywords:  ENPP1 inhibitors; Immune checkpoint inhibitors; Immune-oncology; Lung cancer; STING

DOI:  https://doi.org/10.1016/j.bioorg.2021.105549