Allosteric Activation of Ubiquitin-Specific Proteases by ß-Propeller Proteins UAF1 and WDR20.

Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinating enzymes, whose catalytic competency is often modulated by their binding partners through unknown mechanisms. Here we report on a series of crystallographic and biochemical analyses of an evolutionarily conserved deubiquitinase, USP12, which is activated by two ß-propeller proteins, UAF1 and WDR20. Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far from its catalytic center. Without increasing the substrate affinity of USP12, the two ß-propeller proteins potentiate the enzyme through different allosteric mechanisms. UAF1 docks at the distal end of the USP12 Fingers domain and induces a cascade of structural changes that reach a critical ubiquitin-contacting loop adjacent to the catalytic cleft. By contrast, WDR20 anchors at the base of this loop and remotely modulates the catalytic center of the enzyme. Our results provide a mechanistic example for allosteric activation of USPs by their regulatory partners.

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors.

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1-CUL4CDT2 ubiquitin ligase.

Replication-dependent cytotoxicity and Spartan-mediated repair of trapped PARP1-DNA complexes.

The antitumor activity of poly(ADP-ribose) polymerase inhibitors (PARPis) has been ascribed to PARP trapping, which consists in tight DNA-protein complexes. Here we demonstrate that the cytotoxicity of talazoparib and olaparib results from DNA replication. To elucidate the repair of PARP1-DNA complexes associated with replication in human TK6 and chicken DT40 lymphoblastoid cells, we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs. We find that SPRTN-deficient cells are hypersensitive to talazoparib and olaparib, but not to veliparib, a weak PARP trapper. SPRTN-deficient cells exhibit delayed clearance of trapped PARP1 and increased replication fork stalling upon talazoparib and olaparib treatment. We also show that SPRTN interacts with PARP1 and forms nuclear foci that colocalize with the replicative cell division cycle 45 protein (CDC45) in response to talazoparib. Additionally, SPRTN is deubiquitinated and epistatic with translesion synthesis (TLS) in response to talazoparib. Our results demonstrate that SPRTN is recruited to trapped PARP1 in S-phase to assist in the excision and replication bypass of PARP1-DNA complexes.

Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway.

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

Conditional degradation of SDE2 by the Arg/N-End rule pathway regulates stress response at replication forks.

Multiple pathways counteract DNA replication stress to prevent genomic instability and tumorigenesis. The recently identified human SDE2 is a genome surveillance protein regulated by PCNA, a DNA clamp and processivity factor at replication forks. Here, we show that SDE2 cleavage after its ubiquitin-like domain generates Lys-SDE2Ct, the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2Ct constitutes a short-lived physiological substrate of the Arg/N-end rule proteolytic pathway, in which UBR1 and UBR2 ubiquitin ligases mediate the degradation. The Arg/N-end rule and VCP/p97UFD1-NPL4 segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2Ct degradation upon UVC damage. Conversely, cells expressing the degradation-refractory K78V mutant, Val-SDE2Ct, fail to induce RPA phosphorylation and single-stranded DNA formation, leading to defects in PCNA-dependent DNA damage bypass and stalled fork recovery. Together, our study elucidates a previously unappreciated axis connecting the Arg/N-end rule and the p97-mediated proteolysis with the replication stress response, working together to preserve replication fork integrity.

PCNA-Dependent Cleavage and Degradation of SDE2 Regulates Response to Replication Stress.

Maintaining genomic integrity during DNA replication is essential for cellular survival and for preventing tumorigenesis. Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA replication, and posttranslational modification of PCNA plays a key role in coordinating DNA repair against replication-blocking lesions by providing a platform to recruit factors required for DNA repair and cell cycle control. Here, we identify human SDE2 as a new genome surveillance factor regulated by PCNA interaction. SDE2 contains an N-terminal ubiquitin-like (UBL) fold, which is cleaved at a diglycine motif via a PCNA-interacting peptide (PIP) box and deubiquitinating enzyme activity. The cleaved SDE2 is required for negatively regulating ultraviolet damage-inducible PCNA monoubiquitination and counteracting replication stress. The cleaved SDE2 products need to be degraded by the CRL4CDT2 ubiquitin E3 ligase in a cell cycle- and DNA damage-dependent manner, and failure to degrade SDE2 impairs S phase progression and cellular survival. Collectively, this study uncovers a new role for CRL4CDT2 in protecting genomic integrity against replication stress via regulated proteolysis of PCNA-associated SDE2 and provides insights into how an integrated UBL domain within linear polypeptide sequence controls protein stability and function.

Vaccination with ErbB-2 peptides prevents cancer stem cell expansion and suppresses the development of spontaneous tumors in MMTV-PyMT transgenic mice.

ErbB-2 has been implicated as a target for cancer-initiating cells in breast and other cancers. ErbB-2-directed peptide vaccines have been shown to be effective in prevention of spontaneous tumorigenesis of breast in neu transgenic mouse model, and cellular immunity is proposed as a mechanism for the anti-tumor efficacy. However, there has been no explanation as to how immunity suppresses tumorigenesis from the early stage carcinogenesis, when ErbB-2 expression in breast is low. Here, we investigated a peptide-based vaccine, which consists of two MHC class II epitopes derived from murine ErbB-2, to prevent the occurrence of spontaneous tumors in breast and assess immune impact on breast cancer stem cells. Female MMTV-PyMT transgenic mice were immunized with either ErbB-2 peptide vaccine, or a peptide from tetanus toxoid, or PBS in immune adjuvant. ErbB-2 peptides vaccine completely suppressed spontaneous breast tumors, and the efficacy was correlated with antigen-specific T-cell and antibody responses. In addition, immune serum from the mice of ErbB-2 vaccine group had an inhibitory effect on mammosphere-forming capacity and signaling through ErbB-2 and downstream Akt pathway in ErbB-2 overexpressing mouse mammary cancer cells. We provide evidence that multi-epitope class II peptides vaccine suppresses tumorigenesis of breast potentially by inhibiting the growth of cancer stem cells. We also suggest that a strategy of inducing strong immune responses using multi-epitope ErbB-2-directed helper vaccine might be useful in preventing breast cancer recurrence.

The Novel ATR Inhibitor M1774 Induces Replication Protein Overexpression and Broad Synergy with DNA-targeted Anticancer Drugs.

Ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase inhibitors are in clinical trials. Here we explored the molecular pharmacology and therapeutic combination strategies of the oral ATR inhibitor M1774 (Tuvusertib) with DNA-damaging agents (DDA). As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small cell lung cancer H146, H82, and DMS114 cell lines. M1774 also efficiently blocked the activation of the ATR-CHK1 checkpoint pathway caused by replication stress induced by TOP1 inhibitors. Combination with non-toxic dose of M1774 enhanced TOP1 inhibitor-induced cancer cell death by enabling unscheduled replication upon replicative damage, thereby increasing genome instability. Tandem mass tag-based quantitative proteomics uncovered that M1774, in the presence of DDA, forces the expression of proteins activating replication (CDC45) and G2-M progression (PLK1 and CCNB1). In particular, the fork protection complex proteins (TIMELESS and TIPIN) were enriched. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

Activity of the Ubiquitin-activating Enzyme Inhibitor TAK-243 in Adrenocortical Carcinoma Cell Lines, Patient-derived Organoids, and Murine Xenografts.

Current treatment options for metastatic adrenocortical carcinoma (ACC) have limited efficacy, despite the common use of mitotane and cytotoxic agents. This study aimed to identify novel therapeutic options for ACC. An extensive drug screen was conducted to identify compounds with potential activity against ACC cell lines. We further investigated the mechanism of action of the identified compound, TAK-243, its synergistic effects with current ACC therapeutics, and its efficacy in ACC models including patient-derived organoids and mouse xenografts. TAK-243, a clinical ubiquitin-activating enzyme (UAE) inhibitor, showed potent activity in ACC cell lines. TAK-243 inhibited protein ubiquitination in ACC cells, leading to the accumulation of free ubiquitin, activation of the unfolded protein response, and induction of apoptosis. TAK-243 was found to be effluxed out of cells by MDR1, a drug efflux pump, and did not require Schlafen 11 (SLFN11) expression for its activity. Combination of TAK-243 with current ACC therapies (e.g., mitotane, etoposide, cisplatin) produced synergistic or additive effects. In addition, TAK-243 was highly synergistic with BCL2 inhibitors (Navitoclax and Venetoclax) in preclinical ACC models including patient-derived organoids. The tumor suppressive effects of TAK-243 and its synergistic effects with Venetoclax were further confirmed in a mouse xenograft model. These findings provide preclinical evidence to support the initiation of a clinical trial of TAK-243 in patients with advanced-stage ACC. TAK-243 is a promising potential treatment option for ACC, either as monotherapy or in combination with existing therapies or BCL2 inhibitors. SIGNIFICANCE: ACC is a rare endocrine cancer with poor prognosis and limited therapeutic options. We report that TAK-243 is active alone and in combination with currently used therapies and with BCL2 and mTOR inhibitors in ACC preclinical models. Our results suggest implementation of TAK-243 in clinical trials for patients with advanced and metastatic ACC.

Replication-associated formation and repair of human topoisomerase IIIα cleavage complexes.

Topoisomerase IIIα (TOP3A) belongs to the conserved Type IA family of DNA topoisomerases. Here we report that human TOP3A is associated with DNA replication forks and that a "self-trapping" TOP3A mutant (TOP3A-R364W) generates cellular TOP3A DNA cleavage complexes (TOP3Accs). We show that trapped TOP3Accs that interfere with replication, induce DNA damage and genome instability. To elucidate how TOP3Accs are repaired, we explored the role of Spartan (SPRTN), the metalloprotease associated with DNA replication, which digests proteins forming DNA-protein crosslinks (DPCs). We find that SPRTN-deficient cells show elevated TOP3Accs, whereas overexpression of SPRTN lowers cellular TOP3Accs. SPRTN is deubiquitinated and epistatic with TDP2 in response to TOP3Accs. In addition, we found that MRE11 can excise TOP3Accs, and that cell cycle determines the preference for the SPRTN-TDP2 vs. the ATM-MRE11 pathways, in S vs. G2, respectively. Our study highlights the prevalence of TOP3Accs repair mechanisms to ensure normal DNA replication.

Schlafen 11 (SLFN11) Kills Cancer Cells Undergoing Unscheduled Re-replication.

Schlafen 11 (SLFN11) is an increasingly prominent predictive biomarker and a molecular sensor for a wide range of clinical drugs: topoisomerases, PARP and replication inhibitors, and platinum derivatives. To expand the spectrum of drugs and pathways targeting SLFN11, we ran a high-throughput screen with 1,978 mechanistically annotated, oncology-focused compounds in two isogenic pairs of SLFN11-proficient and -deficient cells (CCRF-CEM and K562). We identified 29 hit compounds that selectively kill SLFN11-proficient cells, including not only previously known DNA-targeting agents, but also the neddylation inhibitor pevonedistat (MLN-4924) and the DNA polymerase α inhibitor AHPN/CD437, which both induced SLFN11 chromatin recruitment. By inactivating cullin-ring E3 ligases, pevonedistat acts as an anticancer agent partly by inducing unscheduled re-replication through supraphysiologic accumulation of CDT1, an essential factor for replication initiation. Unlike the known DNA-targeting agents and AHPN/CD437 that recruit SLFN11 onto chromatin in 4 hours, pevonedistat recruited SLFN11 at late time points (24 hours). While pevonedistat induced unscheduled re-replication in SLFN11-deficient cells after 24 hours, the re-replication was largely blocked in SLFN11-proficient cells. The positive correlation between sensitivity to pevonedistat and SLFN11 expression was also observed in non-isogenic cancer cells in three independent cancer cell databases (NCI-60, CTRP: Cancer Therapeutics Response Portal and GDSC: Genomic of Drug Sensitivity in Cancer). The present study reveals that SLFN11 not only detects stressed replication but also inhibits unscheduled re-replication induced by pevonedistat, thereby enhancing its anticancer efficacy. It also suggests SLFN11 as a potential predictive biomarker for pevonedistat in ongoing and future clinical trials.

Structural, molecular, and functional insights into Schlafen proteins.

Schlafen (SLFN) genes belong to a vertebrate gene family encoding proteins with high sequence homology. However, each SLFN is functionally divergent and differentially expressed in various tissues and species, showing a wide range of expression in cancer and normal cells. SLFNs are involved in various cellular and tissue-specific processes, including DNA replication, proliferation, immune and interferon responses, viral infections, and sensitivity to DNA-targeted anticancer agents. The fundamental molecular characteristics of SLFNs and their structures are beginning to be elucidated. Here, we review recent structural insights into the N-terminal, middle and C-terminal domains (N-, M-, and C-domains, respectively) of human SLFNs and discuss the current understanding of their biological roles. We review the distinct molecular activities of SLFN11, SLFN5, and SLFN12 and the relevance of SLFN11 as a predictive biomarker in oncology.

TOP1-DNA Trapping by Exatecan and Combination Therapy with ATR Inhibitor.

Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol nanoparticles, and antibody-drug conjugates. Here, we report the molecular pharmacology of exatecan compared with the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ataxia telangiectasia and Rad3-related kinase (ATR) inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533, and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage, and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR) deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide-exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.

Integrative epigenomic analyses of small cell lung cancer cells demonstrates the clinical translational relevance of gene body methylation.

DNA methylation is a key regulator of gene expression and a clinical therapeutic predictor. We examined global DNA methylation beyond the generally used promoter areas in human small cell lung cancer (SCLC) and find that gene body methylation is a robust positive predictor of gene expression. Combining promoter and gene body methylation better predicts gene expression than promoter methylation alone including genes involved in the neuroendocrine classification of SCLC and the expression of therapeutically relevant genes including MGMT, SLFN11, and DLL3. Importantly, for super-enhancer (SE) covered genes such as NEUROD1 or MYC, using H3K27ac and NEUROD1, ASCL1, and POU2F3 ChIP-seq data, we show that genic methylation is inversely proportional to expression, thus providing a new approach to identify potential SE regulated genes involved in SCLC pathogenesis. To advance SCLC transitional research, these data are integrated into our web portal (https://discover.nci.nih.gov/SclcCellMinerCDB/) for open and easy access to basic and clinical investigators.

Precision Oncology with Drugs Targeting the Replication Stress, ATR, and Schlafen 11.

Precision medicine aims to implement strategies based on the molecular features of tumors and optimized drug delivery to improve cancer diagnosis and treatment. DNA replication is a logical approach because it can be targeted by a broad range of anticancer drugs that are both clinically approved and in development. These drugs increase deleterious replication stress (RepStress); however, how to selectively target and identify the tumors with specific molecular characteristics are unmet clinical needs. Here, we provide background information on the molecular processes of DNA replication and its checkpoints, and discuss how to target replication, checkpoint, and repair pathways with ATR inhibitors and exploit Schlafen 11 (SLFN11) as a predictive biomarker.

Schlafen 11 expression in human acute leukemia cells with gain-of-function mutations in the interferon-JAK signaling pathway.

Schlafen11 (SLFN11) is referred to as interferon (IFN)-inducible. Based on cancer genomic databases, we identified human acute myeloid and lymphoblastic leukemia cells with gain-of-function mutations in the Janus kinase (JAK) family as exhibiting high SLFN11 expression. In these cells, the clinical JAK inhibitors cerdulatinib, ruxolitinib, and tofacitinib reduced SLFN11 expression, but IFN did not further induce SLFN11 despite phosphorylated STAT1. We provide evidence that suppression of SLFN11 by JAK inhibitors is caused by inactivation of the non-canonical IFN pathway controlled by AKT and ERK. Accordingly, the AKT and ERK inhibitors MK-2206 and SCH77284 suppressed SLFN11 expression. Both also suppressed the E26 transformation-specific (ETS)-family genes ETS-1 and FLI-1 that act as transcription factors for SLFN11. Moreover, SLFN11 expression was inhibited by the ETS inhibitor TK216. Our study reveals that SLFN11 expression is regulated via the JAK, AKT and ERK, and ETS axis. Pharmacological suppression of SLFN11 warrants future studies.

Novel and Highly Potent ATR Inhibitor M4344 Kills Cancer Cells With Replication Stress, and Enhances the Chemotherapeutic Activity of Widely Used DNA Damaging Agents.

Although several ATR inhibitors are in development, there are unresolved questions regarding their differential potency, molecular signatures of patients with cancer for predicting activity, and most effective therapeutic combinations. Here, we elucidate how to improve ATR-based chemotherapy with the newly developed ATR inhibitor, M4344 using in vitro and in vivo models. The potency of M4344 was compared with the clinically developed ATR inhibitors BAY1895344, berzosertib, and ceralasertib. The anticancer activity of M4344 was investigated as monotherapy and combination with clinical DNA damaging agents in multiple cancer cell lines, patient-derived tumor organoids, and mouse xenograft models. We also elucidated the anticancer mechanisms and potential biomarkers for M4344. We demonstrate that M4344 is highly potent among the clinically developed ATR inhibitors. Replication stress (RepStress) and neuroendocrine (NE) gene expression signatures are significantly associated with a response to M4344 treatment. M4344 kills cancer cells by inducing cellular catastrophe and DNA damage. M4344 is highly synergistic with a broad range of DNA-targeting anticancer agents. It significantly synergizes with topotecan and irinotecan in patient-derived tumor organoids and xenograft models. Taken together, M4344 is a promising and highly potent ATR inhibitor. It enhances the activity of clinical DNA damaging agents commonly used in cancer treatment including topoisomerase inhibitors, gemcitabine, cisplatin, and talazoparib. RepStress and NE gene expression signatures can be exploited as predictive markers for M4344.

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.

Debulking of topoisomerase DNA-protein crosslinks (TOP-DPC) by the proteasome, non-proteasomal and non-proteolytic pathways.

Topoisomerases play a pivotal role in ensuring DNA metabolisms during replication, transcription and chromosomal segregation. To manage DNA topology, topoisomerases generate break(s) in the DNA backbone by forming transient enzyme-DNA cleavage complexes (TOPcc) with phosphotyrosyl linkages between DNA ends and topoisomerase catalytic tyrosyl residues. Topoisomerases have been identified as the cellular targets of a variety of anti-cancer drugs (e.g. topotecan, irinotecan, etoposide and doxorubicin, and antibiotics (e.g. ciprofloxacin and levofloxacin). These drugs, as well as other exogenous and endogenous agents, convert the transient TOPcc into persistent TOPcc, which we refer to as topoisomerase DNA-protein crosslinks (TOP-DPC) that challenge genome integrity and lead to cell death if left unrepaired. Proteolysis of the bulky protein component of TOP-DPC (debulking) is a poorly understood repair process employed across eukaryotes. TOP-DPC proteolysis can be achieved either by the ubiquitin-proteasome pathway (UPP) or by non-proteasomal proteases, which are typified by the metalloprotease SPRTN/WSS1. Debulking of TOP-DPC exposes the phosphotyrosyl bonds, hence enables tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) to access and cleave the bonds. In this review, we focus on current knowledge of the protease pathways for debulking TOP-DPC and highlighting recent advances in understanding the mechanisms regulating the proteolytic repair pathways. We also discuss the avenues that are being exploited to target the proteolytic repair pathways for improving the clinical outcome of topoisomerase inhibitors.