bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒01‒03
twenty-four papers selected by
Sean Rudd
Karolinska Institutet


  1. Nature. 2020 Dec 23.
    Shoshani O, Brunner SF, Yaeger R, Ly P, Nechemia-Arbely Y, Kim DH, Fang R, Castillon GA, Yu M, Li JSZ, Sun Y, Ellisman MH, Ren B, Campbell PJ, Cleveland DW.
      Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.
    DOI:  https://doi.org/10.1038/s41586-020-03064-z
  2. Cancer Discov. 2020 Dec 28. pii: CD-20-0387. [Epub ahead of print]
    Li J, Duran MA, Dhanota N, Chatila WK, Bettigole SE, Kwon J, Sriram RK, Humphries MP, Salto-Tellez M, James JA, Hanna MG, Melms JC, Vallabhaneni S, Litchfield K, Usaite I, Biswas D, Bareja R, Li HW, Martin ML, Dorsaint P, Cavallo JA, Li P, Pauli C, Gottesdiener L, DiPardo BJ, Hollmann TJ, Merghoub T, Wen HY, Reis-Filho JS, Riaz N, Su SM, Kalbasi A, Vasan N, Powell SN, Wolchok JD, Elemento O, Swanton C, Shoushtari AN, Parkes EE, Izar B, Bakhoum SF.
      Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS-STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune stimulatory metabolite whose breakdown products include the immune suppressor, adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wildtype ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part, through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti-PD1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune suppressive pathway.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-0387
  3. Cancer Discov. 2020 Dec 18. pii: CD-20-0790. [Epub ahead of print]
    Chung J, Maruvka YE, Sudhaman S, Kelly J, Haradhvala NJ, Bianchi V, Edwards M, Forster VJ, Nunes NM, Galati MA, Komosa M, Deshmukh S, Cabric V, Davidson S, Zatzman M, Light N, Hayes R, Brunga L, Anderson ND, Ho B, Hodel KP, Siddaway R, Morrissy AS, Bowers DC, Larouche V, Bronsema A, Osborn M, Cole KA, Opocher E, Mason G, Thomas GA, George B, Ziegler DS, Lindhorst S, Vanan M, Yalon-Oren M, Reddy AT, Massimino M, Tomboc P, Van Damme A, Lossos A, Durno C, Aronson M, Morgenstern DA, Bouffet E, Huang A, Taylor MD, Villani A, Malkin D, Hawkins CE, Pursell ZF, Shlien A, Kunkel TA, Getz G, Tabori U.
      Although replication repair deficiency, either by mismatch repair deficiency (MMRD) and/or loss of DNA polymerase proofreading, can cause hypermutation in cancer, microsatellite instability (MSI) is considered a hallmark of MMRD alone. By genome-wide analysis of tumors with germline and somatic deficiencies in replication repair, we reveal a novel association between loss of polymerase proofreading and MSI, especially when both components are lost. Analysis of indels in microsatellites (MS-indels) identified five distinct signatures (MS-sigs). MMRD MS-sigs are dominated by multi-base losses, while mutant-polymerase MS-sigs contain primarily single-base gains. MS-deletions in MMRD tumors depend on the original size of the microsatellite and converge to a preferred length, providing mechanistic insight. Finally, we demonstrate that MS-sigs can be a powerful clinical tool for managing individuals with germline MMRD and replication repair deficient cancers, as they can detect the replication repair deficiency in normal cells and predict their response to immunotherapy.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-0790
  4. JCI Insight. 2020 Dec 22. pii: 142149. [Epub ahead of print]
    Tothova Z, Valton AL, Gorelov R, Vallurupalli M, Krill-Burger JM, Holmes A, Landers CC, Haydu JE, Malolepsza E, Hartigan CR, Donahue M, Popova KD, Koochaki SHJ, Venev SV, Rivera JF, Chen E, Lage K, Schenone M, D'Andrea AD, Carr SA, Morgan EA, Dekker J, Ebert BL.
      The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer including myelodysplatic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of STAG2-mutant AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to PARP inhibition. We developed a mouse model of MDS in which Stag2 mutations arise as clonal secondary lesions in the background of clonal hematopoiesis driven by Tet2 mutations, and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which is associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and RPA proteins. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
    Keywords:  Epigenetics; Hematology; Leukemias; Mouse models; Oncology
    DOI:  https://doi.org/10.1172/jci.insight.142149
  5. Cell Rep. 2020 Dec 29. pii: S2211-1247(20)31522-9. [Epub ahead of print]33(13): 108533
    van den Berk P, Lancini C, Company C, Serresi M, Sanchez-Bailon MP, Hulsman D, Pritchard C, Song JY, Schmitt MJ, Tanger E, Popp O, Mertins P, Huijbers IJ, Jacobs H, van Lohuizen M, Gargiulo G, Citterio E.
      Altering ubiquitination by disruption of deubiquitinating enzymes (DUBs) affects hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. Here, we systematically inactivate DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNA (shRNA) screens. We find that multiple DUBs may be individually required for hematopoiesis and identify ubiquitin-specific protease 15 (USP15) as essential for HSC maintenance in vitro and in transplantations and Usp15 knockout (KO) mice in vivo. USP15 is highly expressed in human hematopoietic tissues and leukemias. USP15 depletion in murine progenitors and leukemia cells impairs in vitro expansion and increases genotoxic stress. In leukemia cells, USP15 interacts with and stabilizes FUS (fused in sarcoma), a known DNA repair factor, directly linking USP15 to the DNA damage response (DDR). Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSCs and leukemia cells.
    Keywords:  DNA damage response; FUS; HSC; RNAi; USP15; deubiquitinase; deubiquitinating enzymes; fused in sarcoma; genome integrity; hematopoietic stem cell; in vivo shRNA screen; leukemia
    DOI:  https://doi.org/10.1016/j.celrep.2020.108533
  6. Sci Adv. 2020 Dec;pii: eabb8626. [Epub ahead of print]6(51):
    Juhász S, Smith R, Schauer T, Spekhardt D, Mamar H, Zentout S, Chapuis C, Huet S, Timinszky G.
      Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the treatment of BRCA-deficient cancers, with treatments currently extending toward other homologous recombination defective tumors. In a genome-wide CRISPR knockout screen with olaparib, we identify ALC1 (Amplified in Liver Cancer 1)-a cancer-relevant poly(ADP-ribose)-regulated chromatin remodeling enzyme-as a key modulator of sensitivity to PARP inhibitor. We found that ALC1 can remove inactive PARP1 indirectly through binding to PARylated chromatin. Consequently, ALC1 deficiency enhances trapping of inhibited PARP1, which then impairs the binding of both nonhomologous end-joining and homologous recombination repair factors to DNA lesions. We also establish that ALC1 overexpression, a common feature in multiple tumor types, reduces the sensitivity of BRCA-deficient cells to PARP inhibitors. Together, we conclude that ALC1-dependent PARP1 mobilization is a key step underlying PARP inhibitor resistance.
    DOI:  https://doi.org/10.1126/sciadv.abb8626
  7. NAR Cancer. 2020 Dec;2(4): zcaa038
    Kumar RJ, Chao HX, Simpson DA, Feng W, Cho MG, Roberts VR, Sullivan AR, Shah SJ, Wozny AS, Fagan-Solis K, Kumar S, Luthman A, Ramsden DA, Purvis JE, Gupta GP.
      TP53 deficiency in cancer is associated with poor patient outcomes and resistance to DNA damaging therapies. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Using live cell imaging of DNA double-strand breaks (DSBs) and cell cycle state transitions, we show that p53-deficient cells exhibit accelerated repair of radiomimetic-induced DSBs arising in S phase. Low-dose DNA-dependent protein kinase (DNA-PK) inhibition increases the S-phase DSB burden in p53-deficient cells, resulting in elevated rates of mitotic catastrophe. However, a subset of p53-deficient cells exhibits intrinsic resistance to radiomimetic-induced DSBs despite DNA-PK inhibition. We show that p53-deficient cells under DNA-PK inhibition utilize DNA polymerase theta (Pol θ)-mediated end joining repair to promote their viability in response to therapy-induced DSBs. Pol θ inhibition selectively increases S-phase DSB burden after radiomimetic therapy and promotes prolonged G2 arrest. Dual inhibition of DNA-PK and Pol θ restores radiation sensitivity in p53-deficient cells as well as in p53-mutant breast cancer cell lines. Thus, combination targeting of DNA-PK- and Pol θ-dependent end joining repair represents a promising strategy for overcoming resistance to DNA damaging therapies in p53-deficient cancers.
    DOI:  https://doi.org/10.1093/narcan/zcaa038
  8. Cell Rep. 2020 Dec 22. pii: S2211-1247(20)31535-7. [Epub ahead of print]33(12): 108546
    Kotsantis P, Segura-Bayona S, Margalef P, Marzec P, Ruis P, Hewitt G, Bellelli R, Patel H, Goldstone R, Poetsch AR, Boulton SJ.
      Regulator of telomere length 1 (RTEL1) is an essential helicase that maintains telomere integrity and facilitates DNA replication. The source of replication stress in Rtel1-deficient cells remains unclear. Here, we report that loss of RTEL1 confers extensive transcriptional changes independent of its roles at telomeres. The majority of affected genes in Rtel1-/- cells possess G-quadruplex (G4)-DNA-forming sequences in their promoters and are similarly altered at a transcriptional level in wild-type cells treated with the G4-DNA stabilizer TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine). Failure to resolve G4-DNAs formed in the displaced strand of RNA-DNA hybrids in Rtel1-/- cells is suggested by increased R-loops and elevated transcription-replication collisions (TRCs). Moreover, removal of R-loops by RNaseH1 overexpression suppresses TRCs and alleviates the global replication defects observed in Rtel1-/- and Rtel1PIP_box knockin cells and in wild-type cells treated with TMPyP4. We propose that RTEL1 unwinds G4-DNA/R-loops to avert TRCs, which is important to prevent global deregulation in both transcription and DNA replication.
    Keywords:  G-quadruplexes; G4-DNA structures; R-loops; RTEL1; genome instability; replication stress; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2020.108546
  9. Cell Rep. 2020 Dec 22. pii: S2211-1247(20)31532-1. [Epub ahead of print]33(12): 108543
    Ko JH, Son MY, Zhou Q, Molnarova L, Song L, Mlcouskova J, Jekabsons A, Montagna C, Krejci L, Hasty P.
      DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.
    Keywords:  DNA damage tolerance; double-strand break repair; genomic instability; homologous recombination; replication fork maintenance
    DOI:  https://doi.org/10.1016/j.celrep.2020.108543
  10. Int J Mol Sci. 2020 Dec 13. pii: E9484. [Epub ahead of print]21(24):
    Denkiewicz-Kruk M, Jedrychowska M, Endo S, Araki H, Jonczyk P, Dmowski M, Fijalkowska IJ.
      The CMG complex (Cdc45, Mcm2-7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.
    Keywords:  CMGE helicase-polymerase complex; DNA polymerase epsilon; DNA replication fidelity; DRIM; GINS complex; PSF1/GINS1; Pol ε; Pol ζ; genetic instability; polymerase zeta; recombination; repeat tracts instability
    DOI:  https://doi.org/10.3390/ijms21249484
  11. Proc Natl Acad Sci U S A. 2020 Dec 29. 117(52): 33436-33445
    Wang R, Lenoir WF, Wang C, Su D, McLaughlin M, Hu Q, Shen X, Tian Y, Klages-Mundt N, Lynn E, Wood RD, Chen J, Hart T, Li L.
      Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4 As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.
    Keywords:  DNA polymerase; Fanconi anemia pathway; lesion bypass; whole genome fitness screens
    DOI:  https://doi.org/10.1073/pnas.2008821117
  12. PLoS Genet. 2020 Dec 28. 16(12): e1009256
    Whelan DR, Lee WTC, Marks F, Kong YT, Yin Y, Rothenberg E.
      Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to sample preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize individual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways.
    DOI:  https://doi.org/10.1371/journal.pgen.1009256
  13. Cancers (Basel). 2020 Dec 23. pii: E28. [Epub ahead of print]13(1):
    Wendel SO, Snow JA, Bastian T, Brown L, Hernandez C, Burghardt E, Kahn A, Murthy V, Neill D, Smith ZC, Ault K, Tawfik O, Wu C, Wallace NA.
      High risk genus α human papillomaviruses (α-HPVs) express two versatile oncogenes (α-HPV E6 and E7) that cause cervical cancer (CaCx) by degrading tumor suppressor proteins (p53 and RB). α-HPV E7 also promotes replication stress and alters DNA damage responses (DDR). The translesion synthesis pathway (TLS) mitigates DNA damage by preventing replication stress from causing replication fork collapse. Computational analysis of gene expression in CaCx transcriptomic datasets identified a frequent increased expression of TLS genes. However, the essential TLS polymerases did not follow this pattern. These data were confirmed with in vitro and ex vivo systems. Further interrogation of TLS, using POLη as a representative TLS polymerase, demonstrated that α-HPV16 E6 blocks TLS polymerase induction by degrading p53. This doomed the pathway, leading to increased replication fork collapse and sensitivity to treatments that cause replication stress (e.g., UV and Cisplatin). This sensitivity could be overcome by the addition of exogenous POLη.
    Keywords:  Cisplatin; Cisplatin resistance; cervical cancer; polymerase eta; translesion synthesis
    DOI:  https://doi.org/10.3390/cancers13010028
  14. Cell Rep. 2020 Dec 29. pii: S2211-1247(20)31548-5. [Epub ahead of print]33(13): 108559
    Chansel-Da Cruz M, Hohl M, Ceppi I, Kermasson L, Maggiorella L, Modesti M, de Villartay JP, Ileri T, Cejka P, Petrini JHJ, Revy P.
      The MRE11-RAD50-NBS1 complex plays a central role in response to DNA double-strand breaks. Here, we identify a patient with bone marrow failure and developmental defects caused by biallelic RAD50 mutations. One of the mutations creates a null allele, whereas the other (RAD50E1035Δ) leads to the loss of a single residue in the heptad repeats within the RAD50 coiled-coil domain. This mutation represents a human RAD50 separation-of-function mutation that impairs DNA repair, DNA replication, and DNA end resection without affecting ATM-dependent DNA damage response. Purified recombinant proteins indicate that RAD50E1035Δ impairs MRE11 nuclease activity. The corresponding mutation in Saccharomyces cerevisiae causes severe thermosensitive defects in both DNA repair and Tel1ATM-dependent signaling. These findings demonstrate that a minor heptad break in the RAD50 coiled coil suffices to impede MRE11 complex functions in human and yeast. Furthermore, these results emphasize the importance of the RAD50 coiled coil to regulate MRE11-dependent DNA end resection in humans.
    Keywords:  DNA repair; MRE11; MRN; RAD50; coiled-coil; immunodeficiency; nuclease activity; replication
    DOI:  https://doi.org/10.1016/j.celrep.2020.108559
  15. Cell. 2020 Dec 18. pii: S0092-8674(20)31614-7. [Epub ahead of print]
    Shen JZ, Qiu Z, Wu Q, Finlay D, Garcia G, Sun D, Rantala J, Barshop W, Hope JL, Gimple RC, Sangfelt O, Bradley LM, Wohlschlegel J, Rich JN, Spruck C.
      Repetitive elements (REs) compose ∼50% of the human genome and are normally transcriptionally silenced, although the mechanism has remained elusive. Through an RNAi screen, we identified FBXO44 as an essential repressor of REs in cancer cells. FBXO44 bound H3K9me3-modified nucleosomes at the replication fork and recruited SUV39H1, CRL4, and Mi-2/NuRD to transcriptionally silence REs post-DNA replication. FBXO44/SUV39H1 inhibition reactivated REs, leading to DNA replication stress and stimulation of MAVS/STING antiviral pathways and interferon (IFN) signaling in cancer cells to promote decreased tumorigenicity, increased immunogenicity, and enhanced immunotherapy response. FBXO44 expression inversely correlated with replication stress, antiviral pathways, IFN signaling, and cytotoxic T cell infiltration in human cancers, while a FBXO44-immune gene signature correlated with improved immunotherapy response in cancer patients. FBXO44/SUV39H1 were dispensable in normal cells. Collectively, FBXO44/SUV39H1 are crucial repressors of RE transcription, and their inhibition selectively induces DNA replication stress and viral mimicry in cancer cells.
    Keywords:  FBXO44; H3K9me3; SUV39H1; immunotherapy; repetitive elements
    DOI:  https://doi.org/10.1016/j.cell.2020.11.042
  16. Structure. 2020 Dec 15. pii: S0969-2126(20)30467-6. [Epub ahead of print]
    Bythell-Douglas R, Deans AJ.
      The Bloom syndrome complex is a DNA damage repair machine. It consists of several protein components which are functional in isolation, but interdependent in cells for the maintenance of accurate homologous recombination. Mutations to any of the genes encoding these proteins cause numerous physical and developmental markers as well as phenotypes of genome instability, infertility, and cancer predisposition. Here we review the published structural and biochemical data on each of the components of the complex: the helicase BLM, the type IA topoisomerase TOP3A, and the OB-fold-containing RMI and RPA subunits. We describe how each component contributes to function, interacts with each other, and the DNA that it manipulates/repairs.
    Keywords:  BLM helicase; Bloom syndrome; DNA repair; RMI; double Holliday junction; homologous recombination; structural; topoisomerase III alpha
    DOI:  https://doi.org/10.1016/j.str.2020.11.020
  17. Nat Rev Mol Cell Biol. 2020 Dec 22.
    Covarrubias AJ, Perrone R, Grozio A, Verdin E.
      Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for redox reactions, making it central to energy metabolism. NAD+ is also an essential cofactor for non-redox NAD+-dependent enzymes, including sirtuins, CD38 and poly(ADP-ribose) polymerases. NAD+ can directly and indirectly influence many key cellular functions, including metabolic pathways, DNA repair, chromatin remodelling, cellular senescence and immune cell function. These cellular processes and functions are critical for maintaining tissue and metabolic homeostasis and for healthy ageing. Remarkably, ageing is accompanied by a gradual decline in tissue and cellular NAD+ levels in multiple model organisms, including rodents and humans. This decline in NAD+ levels is linked causally to numerous ageing-associated diseases, including cognitive decline, cancer, metabolic disease, sarcopenia and frailty. Many of these ageing-associated diseases can be slowed down and even reversed by restoring NAD+ levels. Therefore, targeting NAD+ metabolism has emerged as a potential therapeutic approach to ameliorate ageing-related disease, and extend the human healthspan and lifespan. However, much remains to be learnt about how NAD+ influences human health and ageing biology. This includes a deeper understanding of the molecular mechanisms that regulate NAD+ levels, how to effectively restore NAD+ levels during ageing, whether doing so is safe and whether NAD+ repletion will have beneficial effects in ageing humans.
    DOI:  https://doi.org/10.1038/s41580-020-00313-x
  18. Mol Cell. 2020 Dec 22. pii: S1097-2765(20)30904-7. [Epub ahead of print]
    Luengo A, Li Z, Gui DY, Sullivan LB, Zagorulya M, Do BT, Ferreira R, Naamati A, Ali A, Lewis CA, Thomas CJ, Spranger S, Matheson NJ, Vander Heiden MG.
      Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD+/NADH ratio. This change in NAD+/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD+ to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD+ is in excess of the demand for ATP.
    Keywords:  Aerobic Glycolysis; Cell Metabolism; Fermentation; NAD+; PDK; Warburg Effect
    DOI:  https://doi.org/10.1016/j.molcel.2020.12.012
  19. Cell Metab. 2020 Dec 17. pii: S1550-4131(20)30660-4. [Epub ahead of print]
    Jeong S, Savino AM, Chirayil R, Barin E, Cheng Y, Park SM, Schurer A, Mullarky E, Cantley LC, Kharas MG, Keshari KR.
      A significant increase in dietary fructose consumption has been implicated as a potential driver of cancer. Metabolic adaptation of cancer cells to utilize fructose confers advantages for their malignant growth, but compelling therapeutic targets have not been identified. Here, we show that fructose metabolism of leukemic cells can be inhibited by targeting the de novo serine synthesis pathway (SSP). Leukemic cells, unlike their normal counterparts, become significantly dependent on the SSP in fructose-rich conditions as compared to glucose-rich conditions. This metabolic program is mediated by the ratio of redox cofactors, NAD+/NADH, and the increased SSP flux is beneficial for generating alpha-ketoglutarate from glutamine, which allows leukemic cells to proliferate even in the absence of glucose. Inhibition of PHGDH, a rate-limiting enzyme in the SSP, dramatically reduces leukemia engraftment in mice in the presence of high fructose, confirming the essential role of the SSP in the metabolic plasticity of leukemic cells.
    Keywords:  in vivo isotope tracing; metabolic flux; redox; serine synthesis pathway
    DOI:  https://doi.org/10.1016/j.cmet.2020.12.005
  20. Cancer Discov. 2020 Dec 18. pii: CD-20-1065. [Epub ahead of print]
    Fresquet V, Garcia-Barchino MJ, Larrayoz MJ, Celay J, Vicente C, Fernandez-Galilea M, Larrayoz MJ, Calasanz MJ, Panizo C, Junza A, Han J, Prior C, Fortes P, Pio R, Oyarzabal J, Martínez-Baztán Á, Paiva B, Moreno-Aliaga MJ, Odero MD, Agirre X, Yanes O, Prósper F, Martinez-Climent JA.
      During millions of years, endogenous retroelements have remained transcriptionally silent within mammalian genomes by epigenetic mechanisms. Modern anti-cancer therapies targeting the epigenetic machinery awaken retroelement expression, inducing anti-viral responses that eliminate tumors through mechanisms not completely understood. Here we find that massive binding of epigenetically-activated retroelements by RIG-I and MDA5 viral sensors promotes ATP hydrolysis and depletes intracellular energy, driving tumor killing independently of immune signaling. Energy depletion boosts compensatory ATP production by switching glycolysis to mitochondrial oxidative phosphorylation, thereby reversing the Warburg effect. However, hyperfunctional succinate dehydrogenase in mitochondrial electron transport chain generates excessive oxidative stress that unleashes RIP1-mediated necroptosis. To maintain ATP generation, hyperactive mitochondrial membrane blocks intrinsic apoptosis by increasing BCL2 dependency. Accordingly, drugs targeting BCL2-family proteins and epigenetic inhibitors yield synergistic responses in multiple cancer types. Thus, epigenetic therapy kills cancer cells by rewiring mitochondrial metabolism upon retroelement activation, which primes mitochondria to apoptosis by BH3-mimetics.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-1065
  21. Cancer Res. 2020 Dec 28. pii: canres.2121.2020. [Epub ahead of print]
    Dai M, Boudreault J, Wang N, Poulet S, Daliah G, Yan G, Moamer A, Burgos SA, Sabri S, Ali S, Lebrun JJ.
      Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacological inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated pro-metastatic inflammatory cytokine signaling whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in pre-established cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-2121
  22. Cancers (Basel). 2020 Dec 10. pii: E3710. [Epub ahead of print]12(12):
    Zhou Y, Takacs GP, Lamba JK, Vulpe C, Cogle CR.
      Refractory disease is a major challenge in treating patients with acute myeloid leukemia (AML). Whereas the armamentarium has expanded in the past few years for treating AML, long-term survival outcomes have yet to be proven. To further expand the arsenal for treating AML, we searched for druggable gene targets in AML by analyzing screening data from a lentiviral-based genome-wide pooled CRISPR-Cas9 library and gene knockout (KO) dependency scores in 15 AML cell lines (HEL, MV411, OCIAML2, THP1, NOMO1, EOL1, KASUMI1, NB4, OCIAML3, MOLM13, TF1, U937, F36P, AML193, P31FUJ). Ninety-four gene KOs met the criteria of (A) specifically essential to AML cell survival, (B) non-essential in non-AML cells, and (C) druggable according to three-dimensional (3D) modeling or ligand-based druggability scoring. Forty-four of 94 gene-KOs (47%) had an already-approved drug match and comprised a drug development list termed "deKO." Fifty of 94 gene-KOs (53%) had no drug in development and comprised a drug discovery list termed "disKO." STRING analysis and gene ontology categorization of the disKO targets preferentially cluster in the metabolic processes of UMP biosynthesis, IMP biosynthesis, dihydrofolate metabolism, pyrimidine nucleobase biosynthesis, vitellogenesis, and regulation of T cell differentiation and hematopoiesis. Results from this study serve as a testable compendium of AML drug targets that, after validation, may be translated into new therapeutics.
    Keywords:  CRISPR; acute myeloid leukemia; screening; target identification
    DOI:  https://doi.org/10.3390/cancers12123710
  23. J Biol Chem. 2020 Dec 23. pii: jbc.RA120.014455. [Epub ahead of print]
    Popov AV, Endutkin AV, Yatsenko DD, Yudkina AV, Barmatov AE, Makasheva KA, Raspopova DY, Diatlova EA, Zharkov DO.
      DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemo- and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structure. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine-DNA glycosylase (OGG1). In parallel, we have experimentally characterized the activity, thermostability and DNA binding in a subset of these mutant proteins. Among the analyzed variants of OGG1, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance.
    Keywords:  DNA damage; DNA glycosylases; DNA repair; OGG1; cancer; genetic polymorphism; molecular dynamics; protein function prediction; structure-function
    DOI:  https://doi.org/10.1074/jbc.RA120.014455
  24. Acta Pharm Sin B. 2020 Dec;10(12): 2259-2271
    Yin Y, Chen F.
      Since accelerated metabolism produces much higher levels of reactive oxygen species (ROS) in cancer cells compared to ROS levels found in normal cells, human MutT homolog 1 (MTH1), which sanitizes oxidized nucleotide pools, was recently demonstrated to be crucial for the survival of cancer cells, but not required for the proliferation of normal cells. Therefore, dozens of MTH1 inhibitors have been developed with the aim of suppressing cancer growth by accumulating oxidative damage in cancer cells. While several inhibitors were indeed confirmed to be effective, some inhibitors failed to kill cancer cells, complicating MTH1 as a viable target for cancer eradication. In this review, we summarize the current status of developing MTH1 inhibitors as drug candidates, classify the MTH1 inhibitors based on their structures, and offer our perspectives toward the therapeutic potential against cancer through the targeting of MTH1.
    Keywords:  AI, 7-azaindole; AID, 7-azaindazole; AP, aminopyrimidine; AQ, amidoquinolines; AZ, 2-aminoquinazoline; Anticancer; CETSA, cellular thermal shift assay; CR, cyclometalated ruthenium; DDR, DNA damage response; DNA repair; F, fragment; FP, farnesyl phenolic; IC50, half-maximal inhibitory concentrations; Inhibitor; MMR, DNA mismatch repair; MTH1; MTH1, human MutT homolog 1; NSCLC, non-small cell lung cancer; Oxidized nucleotide; P, purinone; PDT, photodynamic therapy; PM, purinone macrocycle; Pu, purine; ROS, reactive oxygen species; TLR7, Toll-like receptor 7; TPP, thermal proteome profiling; TS-FITGE, thermal stability shift-based fluorescence difference in two-dimensional gel electrophoresis
    DOI:  https://doi.org/10.1016/j.apsb.2020.02.012