Roles of eukaryotic topoisomerases in transcription, replication and genomic stability.

Topoisomerases introduce transient DNA breaks to relax supercoiled DNA, remove catenanes and enable chromosome segregation. Human cells encode six topoisomerases (TOP1, TOP1mt, TOP2α, TOP2ß, TOP3α and TOP3ß), which act on a broad range of DNA and RNA substrates at the nuclear and mitochondrial genomes. Their catalytic intermediates, the topoisomerase cleavage complexes (TOPcc), are therapeutic targets of various anticancer drugs. TOPcc can also form on damaged DNA during replication and transcription, and engage specific repair pathways, such as those mediated by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 and by endonucleases (MRE11, XPF-ERCC1 and MUS81). Here, we review the roles of topoisomerases in mediating chromatin dynamics, transcription, replication, DNA damage repair and genomic stability, and discuss how deregulation of topoisomerases can cause neurodegenerative diseases, immune disorders and cancer.

Topoisomerase II-Induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity.

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.

Identification and characterization of topoisomerase III beta poisons.

We designed and carried out a high-throughput screen for compounds that trap topoisomerase III beta (TOP3B poisons) by developing a Comparative Cellular Cytotoxicity Screen. We found a bisacridine compound NSC690634 and a thiacyanine compound NSC96932 that preferentially sensitize cell lines expressing TOP3B, indicating that they target TOP3B. These compounds trap TOP3B cleavage complex (TOP3Bcc) in cells and in vitro and predominately act on RNA, leading to high levels of RNA-TOP3Bccs. NSC690634 also leads to enhanced R-loops in a TOP3B-dependent manner. Preliminary structural activity studies show that the lengths of linkers between the two aromatic moieties in each compound are critical; altering the linker length completely abolishes the trapping of TOP3Bccs. Both of our lead compounds share a similar structural motif, which can serve as a base for further modification. They may also serve in anticancer, antiviral, and/or basic research applications.

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT.

The recognition that cytosolic mitochondrial DNA (mtDNA) activates cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

Topoisomerase I-mediated cleavage at unrepaired ribonucleotides generates DNA double-strand breaks.

Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2',3'-cyclic phosphates at ribonucleotide sites. Here, we provide genetic and biochemical evidence that DNA double-strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. We show that RNase H2-deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1-induced damage at ribonuclelotide sites. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1-induced DNA nicks at ribonucleotide sites. Analysis of Top1-linked DNA from pull-down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2-deficient yeast cells, supporting this model. Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

TDP1 suppresses mis-joining of radiomimetic DNA double-strand breaks and cooperates with Artemis to promote optimal nonhomologous end joining.

The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3'-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, both a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3'-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS, but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining.

Parallel analysis of ribonucleotide-dependent deletions produced by yeast Top1 in vitro and in vivo.

Ribonucleotides are the most abundant non-canonical component of yeast genomic DNA and their persistence is associated with a distinctive mutation signature characterized by deletion of a single repeat unit from a short tandem repeat. These deletion events are dependent on DNA topoisomerase I (Top1) and are initiated by Top1 incision at the relevant ribonucleotide 3'-phosphodiester. A requirement for the re-ligation activity of Top1 led us to propose a sequential cleavage model for Top1-dependent mutagenesis at ribonucleotides. Here, we test key features of this model via parallel in vitro and in vivo analyses. We find that the distance between two Top1 cleavage sites determines the deletion size and that this distance is inversely related to the deletion frequency. Following the creation of a gap by two Top1 cleavage events, the tandem repeat provides complementarity that promotes realignment to a nick and subsequent Top1-mediated ligation. Complementarity downstream of the gap promotes deletion formation more effectively than does complementarity upstream of the gap, consistent with constraints to realignment of the strand to which Top1 is covalently bound. Our data fortify sequential Top1 cleavage as the mechanism for ribonucleotide-dependent deletions and provide new insight into the component steps of this process.

Lack of mitochondrial topoisomerase I (TOP1mt) impairs liver regeneration.

The liver has an exceptional replicative capacity following partial hepatectomy or chemical injuries. Cellular proliferation requires increased production of energy and essential metabolites, which critically depend on the mitochondria. To determine whether Top1mt, the vertebrate mitochondrial topoisomerase, is involved in this process, we studied liver regeneration after carbon tetrachloride (CCl4) administration. TOP1mt knockout (KO) mice showed a marked reduction in regeneration and hepatocyte proliferation. The hepatic mitochondrial DNA (mtDNA) failed to increase during recovery from CCl4 exposure. Reduced glutathione was also depleted, indicating increased reactive oxygen species (ROS). Steady-state levels of ATP, O2 consumption, mtDNA, and mitochondrial mass were also reduced in primary hepatocytes from CCl4-treated KO mice. To further test whether Top1mt acted by enabling mtDNA regeneration, we tested TOP1mt KO fibroblasts and human colon carcinoma HCT116 cells and measured mtDNA after 3-d treatment with ethidium bromide. Both types of TOP1mt knockout cells showed defective mtDNA regeneration following mtDNA depletion. Our study demonstrates that Top1mt is required for normal mtDNA homeostasis and for linking mtDNA expansion with hepatocyte proliferation.

PARP1-TDP1 coupling for the repair of topoisomerase I-induced DNA damage.

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3'-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1-PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.

Mapping topoisomerase sites in mitochondrial DNA with a poisonous mitochondrial topoisomerase I (Top1mt).

Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin OH, in the ribosomal genes (12S and 16S) and at the light strand replication origin OL. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.

Proteolytic degradation of topoisomerase II (Top2) enables the processing of Top2·DNA and Top2·RNA covalent complexes by tyrosyl-DNA-phosphodiesterase 2 (TDP2).

Eukaryotic type II topoisomerases (Top2α and Top2ß) are homodimeric enzymes; they are essential for altering DNA topology by the formation of normally transient double strand DNA cleavage. Anticancer drugs (etoposide, doxorubicin, and mitoxantrone) and also Top2 oxidation and DNA helical alterations cause potentially irreversible Top2·DNA cleavage complexes (Top2cc), leading to Top2-linked DNA breaks. Top2cc are the therapeutic mechanism for killing cancer cells. Yet Top2cc can also generate recombination, translocations, and apoptosis in normal cells. The Top2 protein-DNA covalent complexes are excised (in part) by tyrosyl-DNA-phosphodiesterase 2 (TDP2/TTRAP/EAP2/VPg unlinkase). In this study, we show that irreversible Top2cc induced in suicidal substrates are not processed by TDP2 unless they first undergo proteolytic processing or denaturation. We also demonstrate that TDP2 is most efficient when the DNA attached to the tyrosyl is in a single-stranded configuration and that TDP2 can efficiently remove a tyrosine linked to a single misincorporated ribonucleotide or to polyribonucleotides, which expands the TDP2 catalytic profile with RNA substrates. The 1.6-Å resolution crystal structure of TDP2 bound to a substrate bearing a 5'-ribonucleotide defines a mechanism through which RNA can be accommodated in the TDP2 active site, albeit in a strained conformation.

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs.

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl-DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs-acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)-we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3'-ends. We also show that Tdp1-/- cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1-/- cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.

Biochemical characterization of human tyrosyl-DNA phosphodiesterase 2 (TDP2/TTRAP): a Mg(2+)/Mn(2+)-dependent phosphodiesterase specific for the repair of topoisomerase cleavage complexes.

TDP2 is a multifunctional enzyme previously known for its role in signal transduction as TRAF and TNF receptor-associated protein (TTRAP) and ETS1-associated protein 2 (EAPII). The gene has recently been renamed TDP2 because it plays a critical role for the repair of topoisomerase II cleavage complexes (Top2cc) and encodes an enzyme that hydrolyzes 5'-tyrosine-DNA adducts that mimic abortive Top2cc. Here we further elucidate the DNA-processing activities of human recombinant TDP2 and its biochemical characteristics. The preferred substrate for TDP2 is single-stranded DNA or duplex DNA with a four-base pair overhang, which is consistent with the known structure of Top2cc or Top3cc. The k(cat)/K(m) of TDP1 and TDP2 was determined. It was found to be 4 × 10(5) s(-1)M(-1) for TDP2 using single-stranded 5'-tyrosyl-DNA. The processing of substrates as short as five nucleotides long suggests that TDP2 can directly bind DNA ends. 5'-Phosphodiesterase activity requires a phosphotyrosyl linkage and tolerates an extended group attached to the tyrosine. TDP2 requires Mg(2+) or Mn(2+) for efficient catalysis but is weakly active with Ca(2+) or Zn(2+). Titration with Ca(2+) demonstrates a two-metal binding site in TDP2. Sequence alignment suggests that TDP2 contains four conserved catalytic motifs shared by Mg(2+)-dependent endonucleases, such as APE1. Substitutions at each of the four catalytic motifs identified key residues Asn-120, Glu-152, Asp-262, and His-351, whose mutation to alanine significantly reduced or completely abolished enzymatic activity. Our study characterizes the substrate specificity and kinetic parameters of TDP2. In addition, a two-metal catalytic mechanism is proposed.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs DNA damage induced by topoisomerases I and II and base alkylation in vertebrate cells.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.

Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex.

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.

Cancer/Testis Antigen 55 is required for cancer cell proliferation and mitochondrial DNA maintenance.

Cancer/Testis Antigens (CTAs) represent a group of proteins whose expression under physiological conditions is restricted to testis but activated in many human cancers. Also, it was observed that co-expression of multiple CTAs worsens the patient prognosis. Five CTAs were reported acting in mitochondria and we recently reported 147 transcripts encoded by 67 CTAs encoding for proteins potentially targeted to mitochondria. Among them, we identified the two isoforms encoded by CT55 for whom the function is poorly understood. First, we found that patients with tumors expressing wild-type CT55 are associated with poor survival. Moreover, CT55 silencing decreases dramatically cell proliferation. Second, to investigate the role of CT55 on mitochondria, we first show that CT55 is localized to both mitochondria and endoplasmic reticulum (ER) due to the presence of an ambiguous N-terminal targeting signal. Then, we show that CT55 silencing decreases mtDNA copy number and delays mtDNA recovery after an acute depletion. Moreover, demethylation of CT55 promotor increases its expression, which in turn increases mtDNA copy number. Finally, we measured the mtDNA copy number in NCI-60 cell lines and screened for genes whose expression is strongly correlated to mtDNA amount. We identified CT55 as the second highest correlated hit. Also, we show that compared to siRNA scrambled control (siCtrl) treatment, CT55 specific siRNA (siCT55) treatment down-regulates aerobic respiration, indicating that CT55 sustains mitochondrial respiration. Altogether, these data show for first time that CT55 acts on mtDNA copy number, modulates mitochondrial activity to sustain cancer cell proliferation.

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.

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

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

SLFN11 Inactivation Induces Proteotoxic Stress and Sensitizes Cancer Cells to Ubiquitin Activating Enzyme Inhibitor TAK-243.

Schlafen11 (SLFN11) inactivation occurs in approximately 50% of cancer cell lines and in a large fraction of patient tumor samples, which leads to chemoresistance. Therefore, new therapeutic approaches are needed to target SLFN11-deficient cancers. To that effect, we conducted a drug screen with the NCATS mechanistic drug library of 1,978 compounds in isogenic SLFN11-knockout (KO) and wild-type (WT) leukemia cell lines. Here we report that TAK-243, a first-in-class ubiquitin activating enzyme UBA1 inhibitor in clinical development, causes preferential cytotoxicity in SLFN11-KO cells; this effect is associated with claspin-mediated DNA replication inhibition by CHK1 independently of ATR. Additional analyses showed that SLFN11-KO cells exhibit consistently enhanced global protein ubiquitylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and protein aggregation. TAK-243 suppressed global protein ubiquitylation and activated the UPR transducers PERK, phosphorylated eIF2α, phosphorylated IRE1, and ATF6 more effectively in SLFN11-KO cells than in WT cells. Proteomic analysis using biotinylated mass spectrometry and RNAi screening also showed physical and functional interactions of SLFN11 with translation initiation complexes and protein folding machinery. These findings uncover a previously unknown function of SLFN11 as a regulator of protein quality control and attenuator of ER stress and UPR. Moreover, they suggest the potential value of TAK-243 in SLFN11-deficient tumors. SIGNIFICANCE: This study uncovers that SLFN11 deficiency induces proteotoxic stress and sensitizes cancer cells to TAK-243, suggesting that profiling SLFN11 status can serve as a therapeutic biomarker for cancer therapy.