bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2022–04–03
24 papers selected by
Sean Rudd, Karolinska Institutet



  1. Nature. 2022 Mar 30.
      Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.
    DOI:  https://doi.org/10.1038/s41586-022-04578-4
  2. Nat Commun. 2022 Apr 01. 13(1): 1740
      The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by "USP1-trapping". We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.
    DOI:  https://doi.org/10.1038/s41467-022-29369-3
  3. Sci Adv. 2022 Apr;8(13): eabm0314
      Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.
    DOI:  https://doi.org/10.1126/sciadv.abm0314
  4. J Biol Chem. 2022 Mar 28. pii: S0021-9258(22)00316-7. [Epub ahead of print] 101876
      Deoxyguanosine kinase (dGK) is reported responsible for the phosphorylation of deoxyadenosine (dA) and deoxyguanosine (dG) in the mitochondrial purine salvage pathway. Anti-viral nucleoside analogs known as nucleoside reverse transcriptase inhibitors (NRTI's) must first be phosphorylated by host enzymes for the analog to become active. We address the possibility that NRTI purine analogs may be competitive inhibitors of dGK. From a group of such analogs we provide evidence that entecavir (ETV) competitively inhibited the phosphorylation of dG and dA in rat mitochondria. Mitochondria from the brain, heart, kidney, and liver showed a marked preference for phosphorylation of dG over dA (10-30-fold), and ETV over dA (2.5 - 4-fold). We found that ETV inhibited the phosphorylation of dG with an IC50 of 15.3 ± 2.2 μM and that ETV and dG were both potent inhibitors of dA phosphorylation with IC50s of 0.034 ± 0.007 and 0.028 ± 0.006 μM respectively. In addition, the phosphorylation of dG and ETV followed Michaelis-Menten kinetics and each competitively inhibited the phosphorylation of the other. We observed that the kinetics of dA phosphorylation were strikingly different from those of dG phosphorylation, with an exponentially lower affinity for dGK and no effect of dA on dG or ETV phosphorylation. Finally, in an isolated heart perfusion model, we demonstrated that dG, dA, and ETV were phosphorylated, and dG phosphorylation was inhibited by ETV. Taken together, these data demonstrate that dGK is inhibited by ETV and that the primary role of dGK is in the phosphorylation of dG rather than dA.
    Keywords:  mitochondria; mitochondrial disease; nucleoside/nucleotide biosynthesis; nucleoside/nucleotide metabolism
    DOI:  https://doi.org/10.1016/j.jbc.2022.101876
  5. Mitochondrion. 2022 Mar 25. pii: S1567-7249(22)00025-3. [Epub ahead of print]64 73-81
      The correlation between mitochondrial function and oncogenesis is complex and is not fully understood. Here we determine the importance of mitochondrial-linked pyrimidine synthesis for the aggressiveness of cancer cells. The enzyme dihydroorotate dehydrogenase (DHODH) links oxidative phosphorylation to de novo synthesis of pyrimidines. We demonstrate that an inhibition of DHODH results in a respiration-independent significant increase of anchorage-independent growth but does not affect DNA repair ability. Instead, we show an autophagy-independent increase of lysosomes. The results of this study suggest that inhibition of mitochondrial-linked pyrimidine synthesis in cancer cells results in a more aggressive tumor phenotype.
    Keywords:  DNA repair; Lysosome increase; Mitochondria; Mitochondrial-linked pyrimidine synthesis; Tumorigenesis
    DOI:  https://doi.org/10.1016/j.mito.2022.03.005
  6. Nucleic Acids Res. 2022 Mar 31. pii: gkac119. [Epub ahead of print]
      Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double-stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.
    DOI:  https://doi.org/10.1093/nar/gkac119
  7. Sci Rep. 2022 Mar 31. 12(1): 5422
      Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.
    DOI:  https://doi.org/10.1038/s41598-022-09308-4
  8. JCI Insight. 2022 Mar 29. pii: e152955. [Epub ahead of print]
      The bromodomain and extraterminal (BET) family of chromatin reader proteins bind to acetylated histones and regulate gene expression. The development of BET inhibitors (BETi) has expanded our knowledge of BET protein function beyond transcriptional regulation and has ushered several prostate cancer (PCa) clinical trials. However, BETi as a single-agent is not associated with anti-tumor activity in castration-resistant prostate cancer (CRPC) patients. We hypothesized that novel combinatorial strategies are likely to enhance the efficacy of BETi. Prior studies by our group and others have shown that BET proteins are essential for the repair of DNA double-strand breaks (DSBs) by the non-homologous end joining (NHEJ) as well as the homologous recombination (HR) DNA repair pathways. By using PCa patient-derived explants (PDEs) and xenograft models, we show that BETi treatment enhances the efficacy of radiation therapy (RT) and also overcomes radioresistance. Mechanistically, BETi potentiates the activity of RT by blocking the repair of DNA DSBs. We also report a synthetic lethal relationship between BETi and Topoisomerase I (TOP1) inhibitors (TOP1i). We show that the BETi, OTX015, synergizes with the new class of synthetic non-camptothecin TOP1i, LMP400 (indotecan), to block tumor growth in aggressive CRPC xenograft models. Mechanistically, BETi potentiates the anti-tumor activity of TOP1i by disrupting replication fork stability. Longitudinal analysis of patient tumors indicated that TOP1 transcript abundance increased as patients progressed from hormone-sensitive prostate cancer (HSPC) to CRPC. Consistent with this observation, TOP1 was highly expressed in metastatic CRPC (mCRPC) and its expression correlated with the expression of BET family genes-BRD4, BRD3 and BRD2. These studies open new avenues for the rational combinatorial treatment of aggressive PCa-particularly, cancers refractory to androgen signaling inhibitors.
    Keywords:  Cancer; Oncology; Radiation therapy; Urology
    DOI:  https://doi.org/10.1172/jci.insight.152955
  9. Front Mol Biosci. 2022 ;9 808036
      Human mitochondrial DNA contains more UV-induced lesions than the nuclear DNA due to lack of mechanism to remove bulky photoproducts. Human DNA polymerase gamma (Pol γ) is the sole DNA replicase in mitochondria, which contains a polymerase (pol) and an exonuclease (exo) active site. Previous studies showed that Pol γ only displays UV lesion bypassing when its exonuclease activity is obliterated. To investigate the reaction environment on Pol γ translesion activity, we tested Pol γ DNA activity in the presence of different metal ions. While Pol γ is unable to replicate through UV lesions on DNA templates in the presence of Mg2+, it exhibits robust translesion DNA synthesis (TLS) on cyclobutane pyrimidine dimer (CPD)-containing template when Mg2+ was mixed with or completely replaced by Mn2+. Under these conditions, the efficiency of Pol γ's TLS opposite CPD is near to that on a non-damaged template and is 800-fold higher than that of exonuclease-deficient Pol γ. Interestingly, Pol γ exhibits higher exonuclease activity in the presence of Mn2+ than with Mg2+, suggesting Mn2+-stimulated Pol γ TLS is not via suppressing its exonuclease activity. We suggest that Mn2+ ion expands Pol γ's pol active site relative to Mg2+ so that a UV lesion can be accommodated and blocks the communication between pol and exo active sites to execute translesion DNA synthesis.
    Keywords:  DNA polymerase gamma; TLS; UV lesion; metal-dependence; mitochondrial DNA
    DOI:  https://doi.org/10.3389/fmolb.2022.808036
  10. J Biol Chem. 2022 Mar 23. pii: S0021-9258(22)00301-5. [Epub ahead of print] 101861
      DNA polymerase eta (Pol η) is a eukaryotic member of the Y-family of DNA polymerase involved in translesion DNA synthesis (TLS) and genome mutagenesis. Recently, several TLS polymerases have been found to function in repair of DNA double-strand breaks (DSBs). However, the role of Pol η in promoting DSB repair remains to be well defined. Here, we demonstrated that Pol η could be targeted to etoposide (ETO)-induced DSBs and that depletion of Pol η in cells causes increased sensitivity to ETO. Intriguingly, depletion of Pol η also led to a NHEJ repair defect in a catalytic activity-independent manner. We further identified the scaffold protein Kap1 as a novel interacting partner of Pol η, the depletion of which resulted in impaired formation of Pol η and Rad18 foci after ETO treatment. Additionally, overexpression of Kap1 failed to restore Pol η focus formation in Rad18-deficient cells after ETO treatment. Interestingly, we also found that Kap1 bound to Rad18 in a Pol η-dependent manner, and moreover, depletion of Kap1 led to a significant reduction in Rad18/Pol η association, indicating that Kap1 forms a ternary complex with Rad18 and Pol η to stabilize Rad18/Pol η association. Our findings demonstrate that Kap1 could regulate the role of Pol η in ETO-induced DSB repair via facilitating Rad18 recruitment and stabilizing Rad18/Pol η association.
    DOI:  https://doi.org/10.1016/j.jbc.2022.101861
  11. Trends Biochem Sci. 2022 Mar 26. pii: S0968-0004(22)00060-3. [Epub ahead of print]
      Mutations in BRCA1 and BARD1 predispose carriers to breast and ovarian cancers. The BRCA1 and BARD1 proteins form a heterodimeric complex (BRCA1/BARD1) that regulates many biological processes, including transcription and DNA double-stranded break repair. These functions are mediated by the only known enzymatic activity of BRCA1/BARD1 in its capacity as an E3 ubiquitin ligase and its role as a central hub for many large protein complexes. But the mechanisms by which BRCA1/BARD1 interfaces with chromatin, where it exerts its major functions, have remained unknown. Here, we review recent advancements in structural and cellular biology that have provided critical insights into how BRCA1/BARD1 serves as both a nucleosome reader and writer to facilitate transcriptional regulation and DNA repair by homologous recombination.
    Keywords:  DNA damage repair; chromatin regulation; homologous recombination; transcriptional regulation; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.tibs.2022.03.001
  12. Nucleic Acids Res. 2022 Mar 29. pii: gkac188. [Epub ahead of print]
      Dual-inhibitors of PARP1 and PARP2 are promising anti-cancer drugs. In addition to blocking PARP1&2 enzymatic activity, PARP inhibitors also extend the lifetime of DNA damage-induced PARP1&2 foci, termed trapping. Trapping is important for the therapeutic effects of PARP inhibitors. Using live-cell imaging, we found that PARP inhibitors cause persistent PARP2 foci by switching the mode of PARP2 recruitment from a predominantly PARP1- and PAR-dependent rapid exchange to a WGR domain-mediated stalling of PARP2 on DNA. Specifically, PARP1-deletion markedly reduces but does not abolish PARP2 foci. The residual PARP2 foci in PARP1-deficient cells are DNA-dependent and abrogated by the R140A mutation in the WGR domain. Yet, PARP2-R140A forms normal foci in PARP1-proficient cells. In PARP1-deficient cells, PARP inhibitors - niraparib, talazoparib, and, to a lesser extent, olaparib - enhance PARP2 foci by preventing PARP2 exchange. This trapping of PARP2 is independent of auto-PARylation and is abolished by the R140A mutation in the WGR domain and the H415A mutation in the catalytic domain. Taken together, we found that PARP inhibitors trap PARP2 by physically stalling PARP2 on DNA via the WGR-DNA interaction while suppressing the PARP1- and PAR-dependent rapid exchange of PARP2.
    DOI:  https://doi.org/10.1093/nar/gkac188
  13. J Cell Sci. 2022 Mar 28. pii: jcs.259114. [Epub ahead of print]
      Senescence is an irreversible proliferation withdrawal that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 and RB family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the CDK inhibitor p21 and Chk1 controls D-type cyclin-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting Cyclin D1-CDK2/CDK4. The resulting G2 exit, which precedes appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and DNA damage foci reduction. In p53/RB-proficient cancer cells, compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely Cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to Cyclin D1 and Cyclin E1-CDK complexes and down-regulating CDK6, whereas Chk2 knockdown enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting DNA damage-induced senescence onset in G2.
    Keywords:  CDK6; Chk1; DNA damage checkpoints; G2 arrest after DNA damage; Senescence; p21
    DOI:  https://doi.org/10.1242/jcs.259114
  14. Nucleic Acids Res. 2022 Mar 29. pii: gkac185. [Epub ahead of print]
      Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3'-phospho-α,β-unsaturated aldehyde (3'-PUA), to form unstable but long-lived Schiff base DNA-protein cross-links at 3'-DNA termini (3'-PUA-protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1's intrinsic redox capacity, yielding a stable 3'-PUA-PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3'-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3'-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3'-PUA-protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3'-PUA-histone DPCs, but not 3'-PUA-PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3'-PUA-protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.
    DOI:  https://doi.org/10.1093/nar/gkac185
  15. Biochim Biophys Acta Bioenerg. 2022 Mar 24. pii: S0005-2728(22)00023-8. [Epub ahead of print]1863(5): 148554
      Mitochondria is a unique cellular organelle involved in multiple cellular processes and is critical for maintaining cellular homeostasis. This semi-autonomous organelle contains its circular genome - mtDNA (mitochondrial DNA), that undergoes continuous cycles of replication and repair to maintain the mitochondrial genome integrity. The majority of the mitochondrial genes, including mitochondrial replisome and repair genes, are nuclear-encoded. Although the repair machinery of mitochondria is quite efficient, the mitochondrial genome is highly susceptible to oxidative damage and other types of exogenous and endogenous agent-induced DNA damage, due to the absence of protective histones and their proximity to the main ROS production sites. Mutations in replication and repair genes of mitochondria can result in mtDNA depletion and deletions subsequently leading to mitochondrial genome instability. The combined action of mutations and deletions can result in compromised mitochondrial genome maintenance and lead to various mitochondrial disorders. Here, we review the mechanism of mitochondrial DNA replication and repair process, key proteins involved, and their altered function in mitochondrial disorders. The focus of this review will be on the key genes of mitochondrial DNA replication and repair machinery and the clinical phenotypes associated with mutations in these genes.
    Keywords:  Base-excision repair; CPEO; LIG3; POLG; mtDNA replication
    DOI:  https://doi.org/10.1016/j.bbabio.2022.148554
  16. iScience. 2022 Apr 15. 25(4): 104053
      Ubiquitination of proliferating cell nuclear antigen (PCNA) triggers pathways of DNA damage tolerance, including mutagenic translesion DNA synthesis, and comprises a cascade of reactions involving the E1 ubiquitin-activating enzyme Uba1, the E2 ubiquitin-conjugating enzyme Rad6, and the E3 ubiquitin ligase Rad18. We report here the discovery of a series of xanthenes that inhibit PCNA ubiquitination, Rad6∼ubiquitin thioester formation, and the Rad6-Rad18 interaction. Structure-activity relationship experiments across multiple assays reveal chemical and structural features important for different activities along the pathway to PCNA ubiquitination. The compounds that inhibit these processes are all a subset of the xanthen-3-ones we tested. These small molecules thus represent first-in-class probes of Rad6 function and the association of Rad6 and Rad18, the latter being a new inhibitory activity discovered for a small molecule, in the PCNA ubiquitination cascade and potential therapeutic agents to contain cancer progression.
    Keywords:  Cancer; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2022.104053
  17. Blood. 2022 Mar 25. pii: blood.2021014103. [Epub ahead of print]
      The Plant Homeodomain 6 gene (PHF6) encodes a nucleolar and chromatin-associated leukemia tumor suppressor with proposed roles in transcription regulation. However, specific molecular mechanisms controlled by PHF6 remain rudimentarily understood. Here we show that PHF6 engages multiple nucleosome remodeling protein complexes including NuRD, SWI/SNF and ISWI factors, the replication machinery and DNA repair proteins. Moreover, following DNA damage, PHF6 localizes to sites of DNA injury and its loss impairs the resolution of DNA breaks with consequent accumulation of single- and double-stranded DNA lesions. Native chromatin immunoprecipitation sequencing analyses reveal that PHF6 specifically associates with difficult to replicate heterochromatin at satellite DNA regions enriched in Histone H3 lysine 9 trimethyl marks (H3K9me3) and single molecule locus-specific analyses identify PHF6 as an important regulator of genomic stability at fragile sites. These results extend our understanding of the molecular mechanisms controlling HSC homeostasis and leukemia transformation by placing PHF6 at the crossroads of chromatin remodeling, replicative fork dynamics and DNA repair.
    DOI:  https://doi.org/10.1182/blood.2021014103
  18. Cell Death Dis. 2022 Apr 01. 13(4): 293
      Overexpression of histone deacetylases (HDACs) in cancer commonly causes resistance to genotoxic-based therapies. Here, we report on the novel mechanism whereby overexpressed class I HDACs increase the resistance of glioblastoma cells to the SN1 methylating agent temozolomide (TMZ). The chemotherapeutic TMZ triggers the activation of the DNA damage response (DDR) in resistant glioma cells, leading to DNA lesion bypass and cellular survival. Mass spectrometry analysis revealed that the catalytic activity of class I HDACs stimulates the expression of the E3 ubiquitin ligase RAD18. Furthermore, the data showed that RAD18 is part of the O6-methylguanine-induced DDR as TMZ induces the formation of RAD18 foci at sites of DNA damage. Downregulation of RAD18 by HDAC inhibition prevented glioma cells from activating the DDR upon TMZ exposure. Lastly, RAD18 or O6-methylguanine-DNA methyltransferase (MGMT) overexpression abolished the sensitization effect of HDAC inhibition on TMZ-exposed glioma cells. Our study describes a mechanism whereby class I HDAC overexpression in glioma cells causes resistance to TMZ treatment. HDACs accomplish this by promoting the bypass of O6-methylguanine DNA lesions via enhancing RAD18 expression. It also provides a treatment option with HDAC inhibition to undermine this mechanism.
    DOI:  https://doi.org/10.1038/s41419-022-04751-7
  19. Mol Cancer Res. 2022 Mar 29. pii: molcanres.MCR-21-0725-E.2021. [Epub ahead of print]
      Targeting the DNA damage response in combination with radiation enhances type I interferon (T1IFN)-driven innate immune signaling. It is not understood, however, whether DNA-dependent protein kinase (DNA-PK), the kinase critical for repairing the majority of radiation-induced DNA double-strand breaks in cancer cells, is immunomodulatory. We show that combining radiation with DNA-PK inhibition increases cytosolic double-stranded DNA and tumoral T1IFN signaling in a cGAS- and STING-independent, but an RNA POL III, RIG-I, and MAVS-dependent manner. Although DNA-PK inhibition and radiation also promote programmed death-ligand 1 (PD-L1) expression, the use of anti-PD-L1 in combination with radiation and DNA-PK inhibitor potentiates antitumor immunity in pancreatic cancer models. Our findings demonstrate a novel mechanism for the antitumoral immune effects of DNA-PK inhibitor and radiation that leads to increased sensitivity to anti-PD-L1 in poorly immunogenic pancreatic cancers. Implications: Our work nominates a novel therapeutic strategy as well as biomarkers of treatment resistance pertinent for future clinical trials combining M3814, radiation and αPD-L1 antibody in patients with pancreatic cancer.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-0725
  20. RSC Chem Biol. 2021 Feb 01. 2(1): 47-76
      Damaging DNA is a current and efficient strategy to fight against cancer cell proliferation. Numerous mechanisms exist to counteract DNA damage, collectively referred to as the DNA damage response (DDR) and which are commonly dysregulated in cancer cells. Precise knowledge of these mechanisms is necessary to optimise chemotherapeutic DNA targeting. New research on DDR has uncovered a series of promising therapeutic targets, proteins and nucleic acids, with application notably via an approach referred to as combination therapy or combinatorial synthetic lethality. In this review, we summarise the cornerstone discoveries which gave way to the DNA being considered as an anticancer target, and the manipulation of DDR pathways as a valuable anticancer strategy. We describe in detail the DDR signalling and repair pathways activated in response to DNA damage. We then summarise the current understanding of non-B DNA folds, such as G-quadruplexes and DNA junctions, when they are formed and why they can offer a more specific therapeutic target compared to that of canonical B-DNA. Finally, we merge these subjects to depict the new and highly promising chemotherapeutic strategy which combines enhanced-specificity DNA damaging and DDR targeting agents. This review thus highlights how chemical biology has given rise to significant scientific advances thanks to resolutely multidisciplinary research efforts combining molecular and cell biology, chemistry and biophysics. We aim to provide the non-specialist reader a gateway into this exciting field and the specialist reader with a new perspective on the latest results achieved and strategies devised.
    DOI:  https://doi.org/10.1039/d0cb00151a
  21. Cancer Sci. 2022 Mar 29.
      DNA high methylation is one of driving force for colorectal carcinoma (CRC) pathogenesis. Transcription factors (TFs) can determine cell fate and play fundamental roles in multistep process of tumorigenesis. Dysregulation of DNA methylation of TFs should be vital for the progression of CRC. Here, we demonstrated that TBX20, a T-box TF family protein, was downregulated with hypermethylation of promoter in early stage CRC tissues and correlated with a poor prognosis for CRC patients. Moreover, we identified PDZRN3 as the E3 ubiquitin ligase of TBX20 protein, which mediated the ubiquitination and degradation of TBX20. Furthermore, we revealed that TBX20 suppressed cell proliferation and tumor growth through impairing non-homologous DNA end joining (NHEJ)-mediated double-strand breaks (DSBs) repair by binding the middle domain of both Ku70 and Ku80 and thus inhibiting the recruitment of them on chromatin in CRC cells. Altogether, our results reveal the tumor-suppressive role of TBX20 by inhibiting NHEJ-mediated DNA repair in CRC cells, and provide a potential biomarker for predicting the prognosis of patients with early stage CRC and a therapeutic target for combination therapy.
    Keywords:  CRC; DNA methylation; Ku70; NHEJ; TBX20
    DOI:  https://doi.org/10.1111/cas.15348
  22. Nat Commun. 2022 Apr 01. 13(1): 1751
      The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.
    DOI:  https://doi.org/10.1038/s41467-022-29409-y
  23. Sci Transl Med. 2022 Mar 30. 14(638): eabc7480
      Residual cancer cells that survive drug treatments with targeted therapies act as a reservoir from which eventual resistant disease emerges. Although there is great interest in therapeutically targeting residual cells, efforts are hampered by our limited knowledge of the vulnerabilities existing in this cell state. Here, we report that diverse oncogene-targeted therapies, including inhibitors of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), KRAS, and BRAF, induce DNA double-strand breaks and, consequently, ataxia-telangiectasia mutated (ATM)-dependent DNA repair in oncogene-matched residual tumor cells. This DNA damage response, observed in cell lines, mouse xenograft models, and human patients, is driven by a pathway involving the activation of caspases 3 and 7 and the downstream caspase-activated deoxyribonuclease (CAD). CAD is, in turn, activated through caspase-mediated degradation of its endogenous inhibitor, ICAD. In models of EGFR mutant non-small cell lung cancer (NSCLC), tumor cells that survive treatment with small-molecule EGFR-targeted therapies are thus synthetically dependent on ATM, and combined treatment with an ATM kinase inhibitor eradicates these cells in vivo. This led to more penetrant and durable responses in EGFR mutant NSCLC mouse xenograft models, including those derived from both established cell lines and patient tumors. Last, we found that rare patients with EGFR mutant NSCLC harboring co-occurring, loss-of-function mutations in ATM exhibit extended progression-free survival on first generation EGFR inhibitor therapy relative to patients with EGFR mutant NSCLC lacking deleterious ATM mutations. Together, these findings establish a rationale for the mechanism-based integration of ATM inhibitors alongside existing targeted therapies.
    DOI:  https://doi.org/10.1126/scitranslmed.abc7480
  24. Genomics. 2022 Mar 26. pii: S0888-7543(22)00096-9. [Epub ahead of print] 110351
       BACKGROUND: Increasing evidence has reported the critical roles of PIEZO1 in organism. However, the knowledge of PIEZO1 in human cancers is still inadequate.
    METHODS: In silico analyses and experimental validation were performed to analyze PIEZO1's expression, prognostic values and potential upstream/downstream mechanism in breast cancer.
    RESULTS: PIEZO1 was significantly overexpressed in human breast cancer cell lines and tissue samples. PIEZO1 expression was statistically positively associated with malignant progression and short survival of breast cancer. miR-10b-5p downregulation was partially responsible for PIEZO1 upregulation in breast cancer. Further exploration revealed that PIEZO1 might exert its function by regulating purine metabolism (especially GUK1, POLD1 and APRT) in breast cancer.
    CONCLUSIONS: Collectively, the current study elucidated an important role of PIEZO1 in breast cancer and providing key clues for identifying PIEZO1 as a therapeutic target and prognostic biomarker in breast cancer.
    Keywords:  Breast cancer; PIEZO1; Prognosis; Purine metabolism; miR-10b-5p
    DOI:  https://doi.org/10.1016/j.ygeno.2022.110351