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Summary 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., Graphical abstract, Highlights • SLFN11 expression is high in leukemia and confers response to DNA targeted agents • SLFN11 expression is driven by the innate immune IFN-JAK signaling pathway • Leukemia cell lines with JAK-GOF-mutations express high SLFN11 downstream from the AKT/ERK-ETS pathway • JAK and ETS inhibitors suppress SLFN11 expression, Cell biology; Immunology; Molecular biology

Poly(ADP-ribose) polymerase inhibitors (PARPis) have recently been approved for the treatment of ovarian and breast cancers with BRCA mutations, as well as for maintenance therapies regardless of BRCA mutation for ovarian and primary peritoneal cancers that previously responded to platinum-based chemotherapy. The rationale of these indications is derived from the facts that cancer cells with BRCA mutations are defective in homologous recombination (HR), which confers synthetic lethality with PARPis, and that some of the sensitivity-determining factors for PARPis are shared with platinums. Although BRCA1 and BRCA2 are central for HR, more players within and beyond HR are emerging as response determinants to PARPis. Furthermore, there are similarities as well as differences in the DNA lesions and repair pathways induced by PARPis, platinums, and camptothecin topoisomerase 1 (TOP1) inhibitors. Here we review the sensitivity-determining factors for PARPis and the rationale for using PARPis as single agents and in combination therapy for cancers.

Significance Schlafen-11 (SLFN11) inactivation leads to chemoresistance of a broad range of DNA-damaging agents. We uncover an expanded Ataxia Telangiectasia- and Rad3-related (ATR)-mediated signaling network that overcomes chemoresistance by an unbiased genome-wide RNAi screen in SLFN11-knockout cells and is validated with clinically developing ATR/CHK1 inhibitors. ATR inhibition induces CDT1 phosphorylation, leading to mitotic catastrophe cell death in SLFN11-deficient cells. We identify a key role of SLFN11 for CDT1 degradation by binding to DDB1–CUL4CDT2 ubiquitin ligase during DNA damage, thereby blocking replication reactivation by which SLFN11-deficient cells cause chemoresistance. Our findings provide a rationale for combination therapy with ATR inhibitor and DNA-targeted drugs, and reveal how SLFN11 irreversibly arrests replication during DNA damage acting as a cofactor of CUL4CDT2 ubiquitin ligase., 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.

Poly(ADP-ribose) polymerase inhibitors (PARPis) are DNA-damaging agents that trap PARP-DNA complexes and interfere with DNA replication. Three PARPis - olaparib, niraparib, and rucaparib - were recently approved by the FDA for the treatment of breast and ovarian cancers. These PARPis, along with 2 others (talazoparib and veliparib), are being evaluated for their potential to treat additional malignancies, including prostate cancers. While lack of PARP-1 confers high resistance to PARPis, it has not been established whether or not the levels of PARP-1 directly correlate with tumor response. In this issue of the JCI, Makvandi and coworkers describe an approach to address this question using [18F]FluorThanatrace, an [18F]-labeled PARP-1 inhibitor, for PET. The tracer was taken up by patient tumor tissue and appeared to differentiate levels of PARP-1 expression; however, future studies should be aimed at determining if this tracer can be used to stratify patient response to PARPi therapy.

Schlafen 11 (SLFN11) sensitizes cells to a broad range of anti-cancer drugs including platinum derivatives (cisplatin and carboplatin), inhibitors of topoisomerases (irinotecan, topotecan, doxorubicin, daunorubicin, mitoxantrone and etoposide), DNA synthesis inhibitors (gemcitabine, cytarabine, hydroxyurea and nucleoside analogues), and poly(ADPribose) polymerase (PARP) inhibitors (olaparib, rucaparib, niraparib and talazoparib). In spite of their different primary mechanisms of action, all these drugs damage DNA during S-phase, activate the intra-S-phase checkpoint and induce replication fork slowing and stalling with single-stranded DNA segments coated with replication protein A. Such situation with abnormal replication forks is known as replication stress. SLFN11 irreversibly blocks replication in cells under replication stress, explaining why SLFN11-positive cells are markedly more efficiently killed by DNA-targeting drugs than SLFN11-negative cells. SLFN11 is inactivated in ~50% of cancer cell lines and in a large fraction of tumors, and is linked with the native immune, interferon and T-cells responses, implying the translational relevance of measuring SLFN11 expression as a predictive biomarker of response and resistance in patients. SLFN11 is also a plausible epigenetic target for reactivation by inhibitors of histone deacetylases (HDAC), DNA methyltransferases (DNMT) and EZH2 histone methyltransferase and for combination of these epigenetic inhibitors with DNA-targeting drugs in cells lacking SLFN11 expression. In addition, resistance due to lack of SLFN11 expression in tumors is a potential indication for cell-cycle checkpoint inhibitors in combination with DNA-targeting therapies.

Purpose: Irinotecan and topotecan are used to treat a variety of different cancers. However, they have limitations, including chemical instability and severe side effects. To overcome these limitations, we developed the clinical indenoisoquinolines: LMP400 (indotecan), LMP776 (indimitecan), and LMP744. The purpose of the study is to build the molecular rationale for phase II clinical trials. Experimental Design: CellMinerCDB (http://discover.nci.nih.gov/cellminercdb) was used to mine the cancer cell lines genomic databases. The causality of Schlafen11 (SLFN11) was validated in isogenic cell lines. Because topoisomerase I (TOP1)-mediated replication DNA damage is repaired by homologous recombination (HR), we tested the “synthetic lethality” of HR-deficient (HRD) cells. Survival and cell-cycle alterations were performed after drug treatments in isogenic DT40, DLD1, and OVCAR cell lines with BRCA1, BRCA2, or PALB2 deficiencies and in organoids cultured from prostate cancer patient-derived xenografts with BRCA2 loss. We also used an ovarian orthotopic allograft model with BRCA1 loss to validate the efficacy of LMP400 and olaparib combination. Results: CellMinerCDB reveals that SLFN11, which kills cells undergoing replicative stress, is a dominant drug determinant to the clinical indenoisoquinolines. In addition, BRCA1-, BRCA2-, and PALB2-deficient cells were hypersensitive to the indenoisoquinolines. All 3 clinical indenoisoquinolines were also synergistic with olaparib, especially in the HRD cells. The synergy between LMP400 and olaparib was confirmed in the orthotopic allograft model harboring BRCA1 loss. Conclusions: Our results provide a rationale for molecularly designed clinical trials with the indenoisoquinolines as single agents and in combination with PARP inhibitors in HRD cancers expressing SLFN11.

Precision medicine is an unmet need for DNA-damaging agents with potentially severe side effects. Loss of Schlafen 11 (SLFN11) expression is frequently detected in ~50% cancer cell lines of the NCI, the Broad Institute cancer cell line panel (CCLE), and the Genomics of Drug Sensitivity in Cancer project (GDSC). Deficiency of SLFN11 expression causes chemoresistance to a broad range of DNA-damaging agents, including topoisomerase I (TOP1) inhibitors, topoisomerase II (TOP2) inhibitors, alkylating agents, DNA synthesis inhibitors and PARP inhibitors. Moreover, gene silencing of SLFN11 is observed in small cell lung cancer patient-derived xenograft models with acquired drug resistance. To identify synthetic lethal therapeutic targets to overcome chemoresistance in SLFN11 deficient cells, we performed a genome-wide RNAi screen with the human druggable genome siRNA library by using camptothecin (CPT), a TOP1 inhibitor, in SLFN11 wild-type (WT) and knock-out (KO) prostate cancer DU145 cells. Gene Ontology analysis identified the inhibition of ATR-mediated DNA repair pathway genes (ATR, CHK1, BRCA2 and RPA1) is synergistic with CPT in SLFN11 KO cells, whereas inhibition of the RNA metabolism-related genes (POLR2, PSMD3, POLR2F and PSMD11) had higher combination effects in SLFN11 WT cells. The synergistic effects by depletion of ATR-mediated DNA repair pathway in SLFN11 KO cells were validated by additional siRNAs-mediated cell viability assays. To determine whether inhibition of ATR-mediated pathway can be applied clinically, we tested the new clinical ATR (M4344) and CHK1 (SRA737) inhibitors. Treatment with non-toxic-doses of M4344 and SRA737 reversed drug resistance of the SLFN11 KO cells to TOP1 inhibitors [CPT, and clinically used topotecan and LMP400 (indotecan)]. Additionally, the same synergistic effects by combination of the ATR/CHK1inhibitors and CPT were detected in isogenic CCRF-CEM SLFN11 WT and KO leukemic lymphoblasts cells and non-isogenic DMS114 and H446 small cell lung cancer cells. We also confirmed synergy with ATR/CHK1 inhibitors in combination of other clinical DNA-damaging agents (TOP2 inhibitor: etoposide, alkylating agent: cisplatin, and PARP inhibitor: talazoparib). Molecular changes induced by the combination treatment were examined in the cell cycle by assessing DAPI and EdU incorporation, cell death with Annexin V, and DNA damage response by confocal microscopy with M4344 and CPT. Co-treatment with ATR inhibitor and CPT resulted in G2/M arrest and apoptotic cell death, and formation of micronuclei and fragmented nuclei in SLFN11 KO cells, compared with SLFN11 WT cells. Collectively, our results provide a new therapeutic rationale for the clinical development of combination treatments of chemotherapeutic DNA-targeted agents with ATR/CHK1 inhibitors based on SLFN11 status. Citation Format: Ukhyun Jo, Yasuhisa Murai, Junko Murai, Shar-yin N Huang, Sirisha Chakka, Lu Chen, Ken Cheng, Yves Pommier. The novel ATR inhibitor M4344 and CHK1 inhibitor SRA737 overcome chemoresistance in SLFN11-negative cells in combination treatment with DNA-damaging agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-121.

Poly(ADP-ribose) polymerase (PARP) inhibition serves as a potent therapeutic option eliciting synthetic lethality in cancers harboring homologous recombination (HR) repair defects, such as BRCA mutations. However, the development of resistance to PARP inhibitors (PARPis) poses a clinical challenge. Restoration of HR competency is one of the many molecular factors contributing to PARPi resistance. Combination therapy with cell cycle checkpoint (ATR, CHK1, and WEE1) inhibitors is being investigated clinically in many cancers, particularly in ovarian cancer, to enhance the efficacy and circumvent resistance to PARPis. Ideally, inhibition of ATR, CHK1 and WEE1 proteins will abrogate G2 arrest and subsequent DNA repair via restored HR in PARPi-treated cells. Replication fork stabilization has recently been identified as a potential compensatory PARPi resistance mechanism, found in the absence of restored HR. ATR, CHK1, and WEE1 each possess different roles in replication fork stabilization, providing different mechanisms to consider when developing combination therapies to avoid continued development of drug resistance. This review examines the impact of ATR, CHK1, and WEE1 on replication fork stabilization. We also address the therapeutic potential for combining PARPis with cell cycle inhibitors and the possible consequence of combination therapies which do not adequately address both restored HR and replication fork stabilization as PARPi resistance mechanisms.

Contrary to other anticancer targets, topoisomerase I (TOP1) is targeted by only one chemical class of FDA-approved drugs: topotecan and irinotecan, the derivatives of the plant alkaloid, camptothecin. The indenoisoquinolines LMP400, LMP744, and LMP776 are novel noncamptothecin TOP1 inhibitors in clinical trial, which overcome the limitations of camptothecins. To further improve metabolic stability, their methoxy groups have been replaced by fluorine, as in the fluoroindenoisoquinolines NSC 781517 (LMP517), NSC 779135 (LMP135), and NSC 779134 (LMP134). We tested the induction and stability of TOP1 cleavage complexes (TOP1cc), and the induction and persistence of DNA damage measured by histone H2AX phosphorylation (γH2AX) compared with their parent compounds LMP744 and LMP776 in leukemia CCRF-CEM and colon carcinoma HCT116 cells. The fluoroindenoisoquinolines induced TOP1cc and γH2AX at nanomolar concentrations, and at higher levels than the parent indenoisoquinolines. The fluoroindenoisoquinoline LMP135 showed greater antitumor activity than topotecan in small-cell lung cancer cell H82 xenografts. It was also more potent than topotecan in the NCI-60 cancer cell line panel. Bioinformatics tools (http://discover.nci.nih.gov/cellminercdb) were used to investigate the following: (i) the correlations of fluoroindenoisoquinolines activity with other drugs, and (ii) genomic determinants of response in the NCI-60. The activity of the fluoroindenoisoquinolines was mostly correlated with camptothecin derivatives and the parent indenoisoquinolines, consistent with TOP1 targeting. Genomic analyses and activity assays in CCRF-CEM SLFN11–deleted cells showed that SLFN11 expression is a dominant determinant of response to LMP135. This study shows the potential value of the fluoroindenoisoquinolines for further development as novel anticancer agents targeting TOP1. Mol Cancer Ther; 17(8); 1694–704. ©2018 AACR.

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.

The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases.

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1’s ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1’s role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme., This work was supported by the JSPS KAKENHI Grant Number (JP16K12598, JP16H02957 and JP16H01314 to KH, JP16H06306 to ST and JP16J02252 to RA), the JSPS Core-to-Core Program (A) Advanced Research Networks (to ST), the Takeda Science Foundation and Yamada Science Foundation (to KH), and the Center for Cancer Research, Intramural Program of the US National Cancer Institute (Z01 BC 006150 to KA and YP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Approximately half of high-grade serous epithelial ovarian cancers incur alterations in genes of homologous recombination (BRCA1, BRCA2, RAD51C, Fanconi anemia genes), and the rest incur alterations in other DNA repair pathways at high frequencies. Such cancer-specific gene alterations can confer selective sensitivity to DNA damaging agents such as cisplatin and carboplatin, topotecan, etoposide, doxorubicin, and gemcitabine. Originally presumed to inhibit DNA repair, PARP inhibitors that have recently been approved by the FDA for the treatment of advanced ovarian cancer also act as DNA damaging agents by inducing PARP-DNA complexes. These DNA damaging agents induce different types of DNA lesions that require various DNA repair genes for the repair, but commonly induce replication fork slowing or stalling, also referred to as replication stress. Replication stress activates DNA repair checkpoint proteins (ATR, CHK1), which prevent further DNA damage. Hence, targeting DNA repair genes or DNA repair checkpoint genes augments the anti-tumor activity of DNA damaging agents. This review describes the rational basis for using DNA repair and DNA repair checkpoint inhibitors as single agents. The review also presents the strategies combining these inhibitors with DNA damaging agents for ovarian cancer therapy based on specific gene alterations.

SLFN11 is a recently discovered protein with a putative DNA/RNA helicase function. First identified in association with the maturation of thymocytes, SLFN11 was later causally associated, by two independent groups, with the resistance to DNA damaging agents such as topoisomerase I and II inhibitors, platinum compounds, and other alkylators, making it an attractive molecule for biomarker development. Later, SLFN11 was linked to antiviral response in human cells and interferon production, establishing a potential bond between immunity and chemotherapy. Recently, we demonstrated the potential role of SLN11 as a biomarker to predict sensitivity to the carboplatin/taxol combination in ovarian cancer. The present manuscript reports on the first international monothematic workshop on SLFN11. Several researchers from around the world, directly and actively involved in the discovery, functional characterization, and study of SLFN11 for its biomarker and medicinal properties gathered to share their views on the current knowledge advances concerning SLFN11. The aim of the manuscript is to summarize the authors’ interventions and the main take-home messages resulting from the workshop.

RING finger protein 4 (RNF4) represents a subclass of ubiquitin ligases that target proteins modified by the small ubiquitin-like modifier (SUMO) for ubiquitin-mediated degradation. We disrupted the RNF4 gene in chicken DT40 cells and found that the resulting RNF4(-/-) cells gradually lost proliferation capability. Strikingly, this compromised proliferation was associated with an unprecedented cellular effect: the gradual decrease in the number of intact chromosomes. In the 6 weeks after gene targeting, there was a 25% reduction in the DNA content of the RNF4(-/-) cells. Regarding trisomic chromosome 2, 60% of the RNF4(-/-) cells lost one homologue, suggesting that DNA loss was mediated by whole chromosome loss. To determine the cause of this chromosome loss, we examined cell-cycle checkpoint pathways. RNF4(-/-) cells showed a partial defect in the spindle assembly checkpoint, premature dissociation of sister chromatids, and a marked increase in the number of lagging chromosomes at anaphase. Thus, combined defects in SAC and sister chromatid cohesion may result in increased lagging chromosomes, leading to chromosome loss without accompanying chromosome gain in RNF4(-/-) cells. We therefore propose that RNF4 plays a novel role in preventing the loss of intact chromosomes and ensures the maintenance of chromosome integrity.

Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I–, alkylation-, and chain terminator–induced DNA damage, whereas TDP2 repairs topoisomerase II–induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors. Mol Cancer Ther; 13(8); 2116–26. ©2014 AACR.

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.

To relax DNA supercoiling, topoisomerase I (TOP1) induces DNA cleavage complexes (TOP1cc). These TOP1cc can be trapped by camptothecin, leading to replication induced DNA double-strand breaks (DSB). Widely used, camptothecin derivatives have known limitations. It is now possible to overcome these limitations with the non-camptothecin indenoisoquinolines currently in clinical trials (LMP400, LMP776 and LMP744). Homologous recombination (HR) and its key components BRCA1, BRCA2 and PALB2 are needed to repair DSB induced by TOP1 inhibitors (Maede, Y et al. 2014). It has been shown that BRCA1 is a determinant of response to camptothecins in addition to Olaparib. Also, Schlafen11 (SLFN11) expression has been demonstrated to be a highly penetrant determinant of response to camptothecin derivatives (Zoppoli, G et al. 2012). To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether HR components deficiency or SLFN11 expression are determinants of LMPs response. Using isogenic cell lines, we assessed the survival after treatment with the LMPs. We found that SLFN11-positives cells are 10-times hypersensitive to LMPs compare to WT cells. Also, BRCA1-, BRCA2- and PALB2-deficient cells are 3- to 5- times hypersensitive to the LMPs. Adding Olaparib led to a greater cell death, especially in HR-deficient (HRD) cells. Our results show that the indenoisoquinolines are active at nanomolar concentrations and that HR deficiency and SLFN11 expression are strong drug response determinants. They also demonstrate that the LMPs synergize with Olaparib. These findings provide a rationale for personalized treatment and further clinical trials with the indenoisoquinolines in HR-deficient cancers. Citation Format: Yves Pommier, Laetitia Marzi, Zoe Waever Ohler, Ludmila Szabova, Shyam Sharan, Junko Murai, Mark Cushman. Indotecan (LMP400), Imidotecan (LMP776) and LMP744: A new class of non-camptothecin TOP1 inhibitors selective for cancer cells with homologous recombination deficiencies and high SLFN11 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4855.

SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.

All PARP inhibitors in clinical development (veliparib, olaparib, niraparib, rucaparib, talazoparib) are potent submicromolar competitive NAD+ inhibitors for PARP1 and PARP2, thereby blocking PARylation reactions [i.e. formation of poly(ADPribose) polymers]. In addition, PARP trapping, which determines the anticancer activity of PARP inhibitors as single agents, is drug-specific, and PARP inhibitors can be ranked according to their PARP trapping potency: Talazoparib >> niraparib ˜ olaparib ˜ rucaparib > veliparib. The highly synergistic effects of PARP inhibitors in combination with alkylating agent (temozolomide or methyl methanesulfonate, MMS) and topoisomerase I (Top1) inhibitors (camptothecins and indenoisoquinolines) are well documented. Both classes of drugs induce DNA single-strand breaks sensed by PARP. Yet, the molecular mechanisms of synergy are different. For alkylating agents (temozolomide and MMS), both PARP trapping and PARylation inhibition account for the synergy, whereas for Top1 inhibitors, there is no involvement of PARP trapping and it is PARylation inhibition that deters the coupling of PARP with the repair enzyme, tyrosyl-DNA phosphodiesterase TDP1. In this chapter, we will review the differences between PARP inhibitors and the rationale for choosing among different PARP inhibitors in combination with alkylating agents or Top1 inhibitors.

The putative DNA/RNA helicase, Schlafen 11 (SLFN11), is a recently discovered determinant of sensitivity to DNA damaging agents such as platinums, topoisomerase I and II inhibitors (camptothecins, etoposide, doxorubicin), cisplatin, gemcitabine (1-3) and PARP inhibitors (olaparib, talazoparib) (4). Because SLFN11 expression is suppressed in ~45% cancer cell lines, SLFN11 inactivation is potentially one of the most prevalent mechanisms of resistance to DNA damaging agents. However, the molecular mechanisms of SLFN11 action in the DNA damage response have not been solved. Using isogenic cell lines of SLFN11-knockout and SLFN11-overexpression, we will show that SLFN11 binds to chromatin in response to camptothecin within 2 hours. ChIP-seq analysis reveals that SLFN11 preferentially binds to replication origins under camptothecin treatment. We identify two helicases DHX9 and MCM3, which are involved in replication and origin firing, as binding partners of SLFN11 by IP-MS analysis. Cell cycle analyses revealed that SLFN11 inhibits replication in response to camptothecin, which irreversibly lasts until apoptosis is exerted. Notably, the S-phase arrest by SLFN11 is independent of ATR-mediated S-phase checkpoint. Unscheduled origin firing by ATR inhibition under camptothecin treatment is suppressed in the presence of SLFN11. Hence, SLFN11 enforces irreversible S-phase arrest in damaged cells possibly by inhibiting dormant origin firing and inactivating the MCM helicase in conjunction with DXH9. We conclude that the irreversible S-phase arrest by SLFN11 leads to cell death in response to DNA damaging agents, which is a novel mechanism of how cancer cells are killed by DNA damaging agents. 1. Sousa FG, Matuo R, Tang SW, Rajapakse VN, Luna A, Sander C, et al. Alterations of DNA repair genes in the NCI-60 cell lines and their predictive value for anticancer drug activity. DNA Repair (Amst) 2015;28:107-15. 2. Zoppoli G, Regairaz M, Leo E, Reinhold WC, Varma S, Ballestrero A, et al. Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents. Proc Natl Acad Sci U S A 2012;109(37):15030-5. 3. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012;483(7391):603-307. 4. Murai J, Feng Y, Yu GK, Ru Y, Tang SW, Shen Y, et al. Resistance to PARP inhibitors by SLFN11 inactivation can be overcome by ATR inhibition. Oncotarget 2016. Citation Format: Junko Murai, Yves Pommier. Schlafen 11 (SLFN11) induces lethal S-phase arrest in response to DNA damage: A novel mechanism of how cancer cells are killed by DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5875. doi:10.1158/1538-7445.AM2017-5875

To relax DNA supercoiling during transcription and replication, topoisomerase I (Top1) induces transient DNA cleavage complexes, which are trapped by anticancer drugs, leading to DNA double-strand breaks (DSB) that need to be repaired by homologous recombination (HR). BRCA1, BRCA2 and PALB2, which are key components for HR, lead to “synthetic lethality” with PARP inhibitors. Topotecan and irinotecan (camptothecin derivatives) are the only FDA-approved Top1 inhibitors. In spite of their wide usage they are plagued by their chemical instability, being drug efflux substrates, having short half-life, and dose-limiting bone marrow and gastro intestinal toxicity. It is now possible to overcome those limitations with the non-camptothecin indenoisoquinolines (LMP400, LMP776 and LMP744), which are in clinical trials. To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether the LMPs present a “synthetic lethality” toward BRCA1, BRCA2 or PALB2 deficiency, and whether this selectivity could be enhanced by combining them with the recently approved PARP inhibitor, olaparib.Using isogenic DT40 cell lines, with BRCA1, BRCA2 or PALB2 deficiencies, we assessed the role of HR in the cellular responses to the LMPs. Survival and cell cycle modifications were tested after treatment with the LMPs as single agents, as well as in combination with olaparib. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times hypersensitive to the LMPs (IC50’s for LMP400 and LMP744= 10 nM for HR-deficient cells vs. 45 nM for WT cells, and IC50 for LMP776 around 5 nM vs. 18 nM for WT cells). Cell cycle analyses confirmed the death of these HR-deficient cells. Moreover, combination treatments showed a significant synergy between each of the three LMPs and olaparib (Combination index Citation Format: Laetitia Marzi, Keli Agama, Zoe Weaver Ohler, Ludmila Szabova, Shyam Sharan, Junko Murai, Muthana Al Abo, Yves Pommier. Indotecan (LMP400), imidotecan (LMP776) and LMP744: A new class of non-camptothecin Top1 inhibitors selective for homologous recombination deficient (HRD) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2794. doi:10.1158/1538-7445.AM2017-2794

TDP1 and TDP2 were discovered and named based on the fact they process 3′- and 5′-DNA ends by excising irreversible protein tyrosyl-DNA complexes involving topoisomerases I and II, respectively. Yet, both enzymes have an extended spectrum of activities. TDP1 not only excises trapped topoisomerases I (Top1 in the nucleus and Top1mt in mitochondria), but also repairs oxidative damage-induced 3′-phosphoglycolates and alkylation damage-induced DNA breaks, and excises chain terminating anticancer and antiviral nucleosides in the nucleus and mitochondria. The repair function of TDP2 is devoted to the excision of topoisomerase II- and potentially topoisomerases III-DNA adducts. TDP2 is also essential for the life cycle of picornaviruses (important human and bovine pathogens) as it unlinks VPg proteins from the 5′-end of the viral RNA genome. Moreover, TDP2 has been involved in signal transduction (under the former names of TTRAP or EAPII). The DNA repair partners of TDP1 include PARP1, XRCC1, ligase III and PNKP from the base excision repair (BER) pathway. By contrast, TDP2 repair functions are coordinated with Ku and ligase IV in the non-homologous end joining pathway (NHEJ). This article summarizes and compares the biochemistry, functions, and post-translational regulation of TDP1 and TDP2, as well as the relevance of TDP1 and TDP2 as determinants of response to anticancer agents. We discuss the rationale for developing TDP inhibitors for combinations with topoisomerase inhibitors (topotecan, irinotecan, doxorubicin, etoposide, mitoxantrone) and DNA damaging agents (temozolomide, bleomycin, cytarabine, and ionizing radiation), and as novel antiviral agents.

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.

Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1-/-) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1-/- cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1-/- cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.

The putative DNA/RNA helicase, SLFN11, is a recently discovered determinant of response to DNA damaging agents including topoisomerase inhibitors (camptothecin, irinotecan, topotecan, doxorubicin and etoposide) and cisplatin. SLFN11 is inactivated in approximately 40% of cancer cell lines (NCI-60 and CCLE). Until now, the molecular mechanisms of SLFN11 action in response to DNA damage have been unknown. Using isogenic SLFN11-knockout cells as well as SLFN11-overexpressing cells, we will show that SLFN11 irreversibly arrests cells in S-phase without cell cycle recovery under camptothecin treatment, whereas SLFN11-negative cells continue cell cycle progression. Notably, the action of SLFN11 is independent of ATR-mediated S-phase checkpoint. Co-immunostaining of 5-Ethynyl Uridine (EU) and gamma-H2AX revealed that SLFN11 blocks RNA synthesis upon replicative damage. Comprehensive analysis of cell cycle genes revealed that genes that drive S-phase progression are selectively turned-off in SLFN11-positive cells, whereas p53-dependent genes remain induced by camptothecin. Accordingly, critical proteins for S-phase progression such as cyclin A, cyclin B, CDK1 and CDK2 were suppressed in SLFN11-positive but not in SLFN11-negative cells, which causality links SLFN11-mediated S-phase arrest in camptothecin-treated cells with inactivation of cell cycle-driving pathways. This mechanism is supported by the fact that the CDK inhibitor roscovitine recapitulates the S-phase arrest even in SLFN11-deficient cells. Finally, by contrast to normal SLFN11, overexpression of SLFN11 helicase-dead mutant failed to block RNA synthesis and to kill camptothecin-treated cells, indicating that the helicase activity of SLFN11 is necessary for its RNA synthesis regulatory function. Our study reveals a novel cell cycle regulation mechanism by SLFN11 that links DNA damage and transcriptional responses in cancer cells with replicative DNA damage. Citation Format: Junko Murai, Sai-wen Tang, Yves Pommier. Schlafen 11 (SLFN11) blocks RNA synthesis upon replicative damage, a novel mechanism for killing cancer cells in response to DNA damaging agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3736.

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.

The five clinical PARP inhibitors (veliparib, olaparib, niraparib, rucaparib and talazoparib) are potent submicromolar competitive NAD+ inhibitors for PARP1 and PARP2, thereby blocking PARylation reactions [i.e. formation of poly(ADPribose) polymers]. In addition, PARP trapping, which determines the anticancer activity of PARP inhibitor as single agents, is drug-specific, and PARP inhibitors can be ranked according to their PARP trapping potency: talazoparib >> niraparib ≈ olaparib ≈ rucaparib > veliparib. We will review evidence that PARP trapping is the primary cytotoxic mechanism of PARP inhibitors as single agents and that cancer-specific DNA repair alterations as well as SLFN11 expression determine the cytotoxicity of trapped PARP-DNA complexes in cancers beyond BRCA inactivation. Used in combination in cellular models, PARP inhibitors are highly synergistic with alkylating agent (temozolomide or methyl methanesulfonate) and topoisomerase I (Top1) inhibitors (camptothecin and its clinical derivatives (topotecan and irinotecan) and the non-camptothecin indenoisoquinoline Top1 inhibitors in clinical development (LMP400, LMP776 and LMP744). Both alkylating agents and Top1 inhibitors induce DNA single-strand breaks sensed by PARP. Yet, the molecular mechanisms of synergy are different. For alkylating agents (temozolomide and MMS), both PARP trapping and PARylation inhibition account for the synergy, whereas for Top1 inhibitors, there is no involvement of PARP trapping and it is PARylation inhibition that deters the coupling of PARP with the repair enzyme, tyrosyl-DNA phosphodiesterase, TDP1. We will review the molecular pharmacology differences between PARP inhibitors as single agents and the rationale for choosing among different PARP inhibitors in combination with alkylating agents or Top1 inhibitors based on trapping vs. catalytic inhibition. Citation Format: Yves G. Pommier, Junko Murai. PARP trapping versus PARP catalytic inhibition and coupling with TDP1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL05-04.

The USP1/UAF1 complex deubiquitinates the Fanconi anemia protein FANCD2, thereby promoting homologous recombination and DNA cross-link repair. How USP1/UAF1 is targeted to the FANCD2/FANCI heterodimer has remained unknown. Here we show that UAF1 contains a tandem repeat of SUMO-like domains in its C terminus (SLD1 and SLD2). SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 sequence of UAF1 or mutation of the SIM on FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination and DNA repair. The USP1/UAF1 complex also deubiquitinates PCNA-Ub, and deubiquitination requires the PCNA-binding protein hELG1. The SLD2 sequence of UAF1 binds to a SIM on hELG1, thus targeting the USP1/UAF1 complex to its PCNA-Ub substrate. We propose that the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD–SIM interactions coordinates homologous recombination and translesion DNA synthesis.

Bone undergoes remodeling consisting of osteoclastic bone resorption followed by osteoblastic bone formation throughout life. Although the effects of bone morphogenetic protein (BMP) signals on osteoblasts have been studied extensively, the function of BMP signals in osteoclasts has not been fully elucidated. To delineate the function of BMP signals in osteoclasts during bone remodeling, we deleted BMP receptor type IA (Bmpr1a) in an osteoclast-specific manner using a knock-in Cre mouse line to the cathepsin K locus (CtskCre/+;Bmpr1aflox/flox, designated as Bmpr1aΔOc/ΔOc). Cre was specifically expressed in multinucleated osteoclasts in vivo. Cre-dependent deletion of the Bmpr1a gene occurred at 4 days after cultivation of bone marrow macrophages obtained from Bmpr1aΔOc/ΔOc with RANKL. These results suggested that Bmpr1a was deleted after formation of osteoclasts in Bmpr1aΔOc/ΔOc mice. Expression of bone-resorption markers increased, thus suggesting that BMPRIA signaling negatively regulates osteoclast differentiation. Trabeculae in tibia and femurs were thickened in 3.5-, 8-, and 12-week-old Bmpr1aΔOc/ΔOc mice. Bone histomorphometry revealed increased bone volume associated with increased osteoblastic bone-formation rates (BFR) in the remodeling bone of the secondary spongiosa in Bmpr1aΔOc/ΔOc tibias at 8 weeks of age. For comparison, we also induced an osteoblast-specific deletion of Bmpr1a using Col1a1-Cre. The resulting mice showed increased bone volume with marked decreases in BFR in tibias at 8 weeks of age. These results indicate that deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation, thus suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone-formation activity during bone remodeling. © 2011 American Society for Bone and Mineral Research

Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1⁻/⁻, UAF1⁻/⁻/⁻, and USP1⁻/⁻ UAF1⁻/⁻/⁻ chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ.

Anti-poly(ADP-ribose)polymerase (PARP) drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. Yet, several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes; both olaparib and niraparib act as PARP poisons at pharmacological concentrations (Murai et al., Cancer Res, 2012). Here, we evaluate the novel PARP inhibitor, BMN 673, and compare its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically-modified chicken DT40 and human cancer cell lines. We show that BMN 673, olaparib, and rucaparib are similarly potent at inhibiting PARP catalytic activity. At the same time, BMN 673 is ∼100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as a single agent than olaparib, while olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient, and that BMN 673 is more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sulfonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor to date with the highest efficiency at trapping PARP-DNA complexes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A257. Citation Format: Junko Murai, Shar-yin N. Huang, Amelie Renaud, Yiping Zhang, Jiuping Ji, Shunichi Takeda, Joel Morris, Beverly Teicher, James H. Doroshow, Yves Pommier. Stereospecific trapping of PARP-DNA complexes by BMN 673 and comparison with olaparib and rucaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A257.

We recently showed that poly(ADP-ribose) polymerase (PARP) inhibitors act by a novel allosteric mechanism of action that involves the trapping of PARP-DNA complexes in addition to their previously established NAD-competitive catalytic inhibitory mechanism). We also demonstrated that PARP inhibitors differ in their ability to trap PARP-DNA complexes, with olaparib being more effective than veliparib in spite of their comparable potency as catalytic PARP inhibitors). Here, by using PARP knockout cells, olaparib and veliparib, we tested the role of PARP trapping in the potentiating effect of PARP inhibitors with the alkylating agent temozolomide and the topoisomerase I inhibitor, camptothecin. Using isogenic chicken DT40 cells, we show that PARP-dependent potentiation of temozolomide is markedly greater for olaparib than veliparib, and goes beyond knocking out PARP-1. On the other hand, in the case of camptothecin, knocking out PARP-1 has a greater effect than veliparib or olaparib. In human DU145 prostate cancer and SF295 glioblastoma cells, olaparib is markedly more potent than veliparib in potentiating the cytotoxicity of temozolomide whereas both olaparib and veliparib are almost comparable in the case of camptothecin. By measuring cellular PARP-DNA complexes), we find that in the case of temozolomide, olaparib is a least 10-fold more potent than veliparib at trapping PARP-DNA complexes. On the other hand, in the case of camptothecin, neither olaparib nor veliparib produced detectable trapping of PARP-DNA complexes. Finally, olaparib fails to sensitize DT40, DU145 or SF295 cells to cisplatin and does not induce detectable PARP-DNA complexes in cisplatin-treated cells. These results suggest that, in the case of temozolomide, olaparib trapping of PARP-DNA complexes is more toxic than inactivating PARP catalytic activity, which is not the case for topoisomerase I-induced DNA damage, where catalytic inactivation of PARP appears to be the sole enhancing mechanism. Citation Format: Yves G. Pommier, Junko Murai. Differential potentiation of temozolomide and camptothecin by the PARP inhibitors olaparib and veliparib in relationship with their PARP-DNA trapping abilities, and lack of impact of PARP inhibitors on cisplatin activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3421. doi:10.1158/1538-7445.AM2013-3421

Poly(ADPribose) (PAR) polymerase (PARP) inhibitors are in clinical trials with alkylating agents such as temozolomide and topoisomerase I (Top1) inhibitors such as camptothecin derivatives (topotecan and irinotecan). To elucidate the mechanism of action of PARP inhibitors and how they synergize with alkylating agents and Top1 inhibitors, we compared genetic PARP knockout versus chemical inhibition with the PARP inhibitor. DNA alkylation damage was assayed by treating wild type (WT) and PARP knockout chicken DT40 cells with methyl methane sulfonate (MMS) in the presence of oliparib or veliparib. Notably, PARP inhibitors produced a much greater cytotoxicity than knocking out the PARP gene in cells treated with low MMS concentrations that are not cytotoxic in WT cells. Moreover, knocking out PARP eliminated the cytotoxicity of the PARP inhibitors even in the absence of MMS treatment. These results suggested that PARP inhibitors not only act by abrogating the catalytic activity of PARP and blocking PAR polymer formation but also by poisoning PARP. To further investigate this possibility, we measured PARP binding to DNA. Accordingly, we found that both oliparib and veliparib trap PARP on cellular DNA. By contrast, in the case of Top1 inhibitors, the potentiation by PARP inhibitors was no greater than knocking out PARP. Together, these results demonstrate that oliparib and veliparib act primarily as catalytic PARP inhibitors for Top1-linked DNA damage, whereas, in the case of base damage, oliparib and veliparib also act by preventing the removal of PARP complexes from DNA, probably as a result of defective PARP auto-PARylation, thereby converting base damage and DNA nicks into tight PARP-DNA complexes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4689. doi:1538-7445.AM2012-4689

Objective: The USP1/UAF1 complex is a critical regulator of the FA pathway. The USP1/UAF1 complex deubiquitinates the Fanconi Anemia proteins, FANCD2/FANCI, and promotes homologous recombination and DNA crosslink repair. USP1/UAF1 also deubiquitinates PCNA-Ub, a key player in translesion DNA synthesis. This step requires the PCNA interacting protein, hELG1. The goal of our study is to identify the molecular mechanism of USP1/UAF1 complex targeting to the FANCD2-Ub/FANCI-Ub heterodimer and tothePCNA-Ub/hELG1 complex. Methods: Using bioinformatics, we identified a tandem repeat of SUMO-like domains (SLD) in the C-terminus of UAF1 (SLD1 and SLD2). A SLD sequence is a domain of approximately 100 amino acids with significant sequence identity and homology to the small ubiquitin-like modifier (SUMO). We tested the function of a mutant form of UAF1 lacking the SLD2 domain. Interestingly, this mutant UAF1 protein bound and stimulated the deubiquitinating activity of USP1 but failed to complement UAF1 knockout DT40 cells. We then discovered a SUMO-like-domain interacting motif (SIM) on both FANCI and hELG1. We therefore investigated the functional importance of the SLD-SIM interaction in regulating the Fanconi anemia pathway. Results: The SLD2 domain of UAF1 is dispensable for activating USP1 in vitro. However, complementation analyses using UAF1 knockout DT40 cells showed that the SLD2 is critical for the deubiquitination of USP1 substrates in vivo and for homologous recombination. We demonstrated that the SLD2 domain of UAF1 binds directly to the SIM sequence on FANCI and hELG1, and thereby regulates the deubiquitination of FANCD2-Ub and PCNA-Ub respectively. Disruption of these SLD-SIM interactions blocked the deubiquitination of FANCD2-Ub and/or PCNA-Ub, rendering cells DNA repair deficient. We also modeled the 3-D structure of SLD2 and identified critical residues involved in SLD-SIM interaction. Conclusions: Our study demonstrates that unique SLD-SIM interactions mediate the targeting of the USP1/UAF1 deubiquitinating complex to its monoubiquitinated substrates, FANCD2/FANCI and hELG1/PCNA, and thereby coordinate homologous recombination. This is a novel mechanism of DNA repair regulation, and it may have relevance to other DNA repair pathways. The SLD-SIM interaction sites are potential drug target sites for agonists or antagonists of the FA pathway. They may be useful in screening drugs for the treatment of FA patients and cancer patients from the general (non-FA) population. This abstract is also presented as Poster B38. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR4.