Journal articles on the topic 'Ligase IV Inhibitor SCR7'
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Anuchina, Arina A., Milyausha I. Zaynitdinova, Anna G. Demchenko, Nadezhda A. Evtushenko, Alexander V. Lavrov, and Svetlana A. Smirnikhina. "Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7." International Journal of Molecular Sciences 24, no. 7 (April 4, 2023): 6704. http://dx.doi.org/10.3390/ijms24076704.
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This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.
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Greco, George E., Yoshihiro Matsumoto, Rhys C. Brooks, Zhengfei Lu, Michael R. Lieber, and Alan E. Tomkinson. "SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV." DNA Repair 43 (July 2016): 18–23. http://dx.doi.org/10.1016/j.dnarep.2016.04.004.
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Calimeri, Teresa, Daniele Caracciolo, Nicola Amodio, Mehmet Kemal Samur, Marzia Leotta, Mariateresa Fulciniti, Marco Rossi, et al. "Targeting Aberrant Non-Homologous End Joining in Multiple Myeloma: Role of the Classical and Alternative Pathways in Genomic Instability." Blood 124, no. 21 (December 6, 2014): 3417. http://dx.doi.org/10.1182/blood.v124.21.3417.3417.
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Abstract Multiple Myeloma (MM) is characterized by the growth of malignant plasma cells harboring numerous genomic aberrations. The molecular basis driving MM genomic instability is still largely unknown. The ability to repair DNA damages is essential for the maintenance of its integrity, especially the double-strand breaks (DSBs) which are mainly repaired by Non Homologous End Joining (NHEJ). We have investigated NHEJ pathway in myeloma and observed a significant association between up-regulated NHEJ pathway-related gene expression and poor overall survival in two large datasets (IFM and Arkansas) in myeloma. We have also observed a higher end joining (EJ) activity in MM cell lines compared to normal cells using a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene to measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency. Moreover, we confirmed an increased NHEJ activity in several primary patient myeloma cells at different disease stage. Based on this rationale, since an altered NHEJ has been linked to genomic instability and its inhibition leading to eventual cell death, we hypothesized that the aberrant NHEJ can be used as a potential therapeutic target in MM. To address the relevance of NHEJ inhibition in MM cell proliferation and survival, we used SCR7, an inhibitor of Ligase IV (Lig-IV) which is essential for ligation of the double strand breaks following their recognition by the KU70/KU80 heterodimer and the recruitment of DNA-PKcs. We tested 4 different MM cell lines (U266, R8226, MM1s and Dox40), however, except for some level of inhibition in Dox40 (IC50, between 50 and 100 uM at 72 hours), the other cell line growth was not significantly affected (R8226 - IC30 at the concentration; and U266 and MM1s did not reach IC30). The same data were confirmed by Annexin V/7AAD staining and Caspase assay. Interestingly, expression of Lig-IV estimated by western blot analysis, inversely correlated with MM cells sensitivity suggesting that higher protein concentration may require higher drug levels for inhibition. Consistent with this result, we observed a strong inhibition of the NHEJ pathway by ku86-directed shRNAs, which was able to induce cell death in the more resistant MM cell line u266. Subsequently we used the dual gene plasmid-based assay to evaluate the effect of sub-lethal dose (20 uM) of SCR7 on NHEJ in 3 MM cell lines (u266, R8226 and MM1s) and observed an increased recombination activity in 2 of them. We also confirmed these data with another NHEJ inhibitor, NU7441, which target DNA-PK; and by using ku86-shRNA in U266 cell line. Moreover we observed an accumulation of unrepaired DSBs at the genome level as demonstrated by an increased γ-H2AX by western blotting. These results suggested the possibility that the inhibition of the NHEJ by blocking Lig-IV could activate the alternative NHEJ pathway (a-NHEJ), which is more error-prone compared to the classical NHEJ (c-NHEJ). To confirm this hypothesis further, we treated MM cell lines with sub-lethal dose of NU7441 (2.5 uM), Benzamide (2.5 uM), an inhibitor of PARP, which is one of the main protein involved in the a-NHEJ, or both. The different modulation observed with single and combination treatments, along with the ability of NU7441 to revert sensitivity to Benzamide in R8226 cells, suggested that inhibition of the classical pathway could switch on the a-NHEJ and indicated its basal activity at least in this cell line. Ongoing study is assessing the influence of such compounds on NHEJ in primary MM cells and their impact on acquisition of new genomic changes. In conclusion, our data confirm the aberrant activation of NHEJ in MM, and suggest the potential role for both classical and more error-prone a-NHEJ pathways in inducing genomic instability, which may require a dual inhibition to trigger myeloma cell death. Disclosures No relevant conflicts of interest to declare.
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Greco, George E., Zane A. Conrad, Alycia M. Johnston, Qingyao Li, and Alan E. Tomkinson. "Synthesis and structure determination of SCR7, a DNA ligase inhibitor." Tetrahedron Letters 57, no. 29 (July 2016): 3204–7. http://dx.doi.org/10.1016/j.tetlet.2016.06.037.
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Kotnis, Ashwin, and Rita Mulherkar. "Novel inhibitor of DNA ligase IV with a promising cancer therapeutic potential." Journal of Biosciences 39, no. 3 (April 29, 2014): 339–40. http://dx.doi.org/10.1007/s12038-014-9433-0.
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Sallmyr, Annahita, and Feyruz V. Rassool. "Up-Regulated WRN and DNA Ligase IIIα Are Involved in Alternative NHEJ Repair Pathway of DNA Double Strand Breaks (DSB) in Chronic Myeloid Leukemia (CML)." Blood 110, no. 11 (November 16, 2007): 1016. http://dx.doi.org/10.1182/blood.v110.11.1016.1016.
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Abstract The oncogenic BCR-ABL in CML produces increased reactive oxygen species (ROS) leading to DSB and aberrant repair. We have previously shown that CML cells demonstrate an increased frequency of errors of non homologous end-joining (NHEJ). DSB are repaired by two major pathways, homologous recombination (HR) and NHEJ, the dominant pathway in eukaryotic cells, also known as DNA-PK dependent NHEJ (D-NHEJ). Recent reports have identified alternative or “back-up” NHEJ pathways (B-NHEJ) that are highly error-prone, and may explain the altered DSB repair reported in CML. To determine the mechanism for the aberrant NHEJ repair in CML, we examined steady state levels of D-NHEJ proteins, including Ku70/86, DNA-PKcs, Artemis and DNA Ligase IV/XRCC4 in four different BCR-ABL positive CML cell lines compared with three lymphoblastoid cell lines established from normal individuals and one BCR-ABL negative CML cell line. We find that two key components of D-NHEJ, Artemis (4–7 fold) and DNA Ligase IV (2–3 fold) are down-regulated, compared with controls. These data suggest that D-NHEJ repair is compromised in CML. To determine whether alternative NHEJ repair plays a role in the aberrant repair of DSB in CML cells, we next examined expression levels of DNA Ligase IIIα/XRCC1, PARP and other proteins known to be associated with NHEJ repair, such as the protein found to be deleted in Werner’s syndrome, WRN. We find that WRN and DNA Ligase IIIα are increased (3–6 fold) in BCR-ABL-positive CML compared with control cell lines. Importantly, DNA Ligase IIIα/XRCC1 forms a complex with WRN, suggesting that it may be a new member of the alternative repair pathway. To confirm that up-regulation of DNA Ligase IIIα and WRN are elicited by BCR-ABL, we examined the levels of these proteins in primary samples (N=4) from patients with different levels of BCR-ABL, following treatment with the tyrosine kinase inhibitor Gleevec. WRN and DNA Ligase IIIα are down regulated in patient samples where BCR-ABL levels are significantly decreased. Furthermore, we confirmed that these up-regulated proteins are involved in DSB repair in CML cells because they co-localize to induced DSB in BCR-ABL-positive cell lines stably transfected with DSB-containing DRneo plasmid, using fluorescence in situ hybridization (FISH) co-immunostaining. Importantly we show that siRNA down-regulation of WRN and DNA Ligase IIIα leads to elevated levels of unrepaired DSB and a decreased frequency of DSB repair efficiency in CML cells. In addition siRNA down-regulation of WRN leads to large deletions at the site of repair, while siRNA down-regulation of DNA Ligase IIIα results in an increased frequency of misrepair. Finally, we determined whether “correction” of main NHEJ pathway proteins in CML can lead to a decrease in the frequency of errors of end-joining repair. Over-expression of Artemis using pcDNA constructs in CML cells leads to more correct end-joining, compared with vector transfected controls. We conclude that down-regulation of Artemis and DNA Ligase IV leads to compensatory up-regulation of alternative repair pathways in BCR-ABL-positive CML cells, and suggest a role for a new protein complex in CML, in protecting and joining DNA ends, thus ensuring the survival of CML cells. Inhibition of alternative NHEJ repair may be explored in combination with other agents as a therapeutic strategy in CML.
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Chou, Mei-Chia, Yuan-Jia Lee, Yao-Ting Wang, Shi-Yie Cheng, and Hsueh-Ling Cheng. "Cytotoxic and Anti-Inflammatory Triterpenoids in the Vines and Leaves of Momordica charantia." International Journal of Molecular Sciences 23, no. 3 (January 19, 2022): 1071. http://dx.doi.org/10.3390/ijms23031071.
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The vines and leaves of Momordica charantia L. are used as herbal medicines to treat inflammation-related disorders. However, their safety profile remains uncharacterized, and the constituents in their extracts that exert anti-inflammatory and adverse effects remain unclear. This study isolated the characteristic cucurbitane-type triterpenoid species in the vines and leaves of M. charantia L. and analyzed their cytotoxicity, anti-inflammatory effects, and underlying mechanisms. Four structurally related triterpenoids—momordicines I, II, IV, and (23E) 3β,7β,25-trihydroxycucurbita-5,23-dien-19-al (TCD)—were isolated from the triterpenoid-rich fractions of extracts from the vines and leaves of M. charantia. Momordicine I was cytotoxic on normal cells, momordicine II exerted milder cytotoxicity, and momordicine IV and TCD had no obvious adverse effects on cell growth. TCD had anti-inflammatory activity both in vivo and in vitro. In lipopolysaccharide-stimulated RAW 264.7 cells, TCD inhibited the inhibitor kappa B kinase/nuclear factor-κB pathway and enhanced the expression of nuclear factor erythroid 2-related factor 2, heme oxygenase-1, and glutamate-cysteine ligase modifier subunit through the extracellular signal-regulated kinase1/2 and p38. Thus, the vines and leaves of M. charantia should be used with caution. An extraction protocol that can enrich TCD but remove momordicine I would likely enhance the safety of the extract.
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Devassy, Greeshma, Ranjith Ramachandran, Kottarapat Jeena, Vijayabhaskar R. Junnuthula, Vindya K. Gopinatha, Cheripelil Manju, Maneesh Manohar, Shantikumar V. Nair, Sathees C. Raghavan, and Manzoor Koyakutty. "Simultaneous release of two drugs from polymer nano-implant inhibits recurrence in glioblastoma spheroids." Precision Nanomedicine 2, no. 1 (February 1, 2019): 218–29. http://dx.doi.org/10.33218/prnano2(1).181122.1.
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Local implant-based delivery of rationally selected combination of chemotherapeutics has some major advantages for the treatment of glioblastoma such as: (a) 100 % bio-availability locally in brain can be achieved at the tumor site (b) avoid systemic leakage and associated toxicity, and (c) simultaneous inhibition of multiple, mutually exclusive cancer mechanisms is possible. Here, we report a polymeric brain implant capable of delivering two different drugs in recur-rent glioma cells. We have selected a combination of clinically used DNA alkylating agent, Te-mozolomide, and a DNA mismatch repair protein (Ligase IV) inhibitor, SCR-7, and delivered simultaneously into tumor spheroids formed by rat glioma cells, C6. The dual-drug loaded polymeric wafer, prepared by lyophilization method, could deliver both the drugs in a controlled fashion. To test the efficacy of this system, we have optimized an in vitro recurrent model of glioma spheroids wherein, the implant released both the drugs in a sustained fashion, thereby continuously exposing the cells to DNA methylation while inhibiting the DNA repair pathways. This leads to synergistic toxicity and inhibition of tumor recurrence for extended duration compared to free drug combination.
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Cha, Sang-Wook. "Generating Nonmosaic Mutants in Xenopus Using CRISPR–Cas in Oocytes." Cold Spring Harbor Protocols 2022, no. 6 (July 8, 2021): pdb.prot106989. http://dx.doi.org/10.1101/pdb.prot106989.
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In CRISPR–Cas9 genome editing, double-strand DNA breaks (DSBs) primarily undergo repair through nonhomologous end joining (NHEJ), which produces insertion or deletion of random nucleotides within the targeted region (indels). As a result, frameshift mutation-mediated loss-of-function mutants are frequently produced. An alternative repair mechanism, homology-directed repair (HDR), can be used to fix DSBs at relatively low frequency. By injecting a DNA-homology repair construct with the CRISPR–Cas components, specific nucleotide sequences can be introduced within the target region by HDR. We have taken advantage of the fact that Xenopus oocytes have much higher levels of HDR than eggs to increase the effectiveness of creating precise mutations. We introduced the oocyte host transfer technique, well established for knockdown of maternal mRNA for loss-of-function experiments, to CRISPR–Cas9-mediated genome editing. The host-transfer technique is based on the ability of Xenopus oocytes to be isolated, injected with CRISPR–Cas components, and cultured in vitro for up to 5 d before fertilization. During these 5 d, CRISPR–Cas components degrade, preventing further alterations to the paternal or maternal genomes after fertilization and resulting in heterozygous, nonmosaic embryos. Treatment of oocytes with a DNA ligase IV inhibitor, which blocks the NHEJ repair pathway, before fertilization further improves the efficiency of HDR. This method allows straightforward generation of either nonmosaic F0 heterozygous indel mutant Xenopus or Xenopus with efficient, targeted insertion of small DNA fragments (73–104 nt). The germline transmission of mutations in these animals allows homozygous mutants to be obtained one generation (F1) sooner than previously reported.
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Shima, Yutaka, Takito Shima, Tomoki Chiba, Tatsuro Irimura, and Issay Kitabayashi. "PML Protects HIPK2 and p300 from SCF-Mediated Ubiquitin-Dependent Degradation To Activate Transcription." Blood 110, no. 11 (November 16, 2007): 2653. http://dx.doi.org/10.1182/blood.v110.11.2653.2653.
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Abstract The Pml gene is the target of t(15;17) chromosome translocation in acute promyelocytic leukemia. PML protein is known to localize in discrete nuclear speckles, named PML nuclear bodies (NBs). In NBs, PML interacts with several transcription factors, such as p53 and AML1, and their co-activators, such as HIPK2 and p300. PML activates transcription of their target genes. PML is thought to stabilize transcription factor complex and function as a mediator in transcription activation, but little is known about the molecular mechanism by which PML activates transcription. To clarify the role of PML in transcription regulation, we purified the PML complex and identified a novel F-box protein (FBP), Skp1, and Cullin1 (Cul1) in the PML complex by LC/MS/MS analysis. FBPs form SCF ubiquitin ligase complexes with Skp1, Cul1 and ROC1 and mediate recognition of specific substrates for ubiquitination. We found that the FBP that we identified here also forms a SCF complex with Skp1, Cul1 and ROC1. To identify substrates for the SCF complex, we tested several proteins that could bind to PML, and found that the FBP promotes degradation of HIPK2 and p300. These degradations were inhibited in the presence of a proteasome inhibitor, MG132. The FBP stimulated ubiquitination of HIPK2. These results suggest that the SCF promotes degradation of these proteins by the ubiquitin-proteasome pathway. The fact that the SCF is a part of the PML complex suggests that PML plays a role in the SCF-mediated degradation of HIPK2 and p300 by the ubiquitin-proteasome pathway. In order to clarify the role of PML in degradation of HIPK2 and p300, we tested effects of PML on the degradation and found that PML inhibited the SCF-mediated degradation of HIPK2 and p300 without inhibition of ubiquitination. To clarify roles of HIPK2, PML IV and the FBP in p53-dependent transcription, we performed reporter analysis using the MDM2 promoter in H1299 cells. Since the FBP promotes degradation of HIPK2, we initially thought that the FBP might inhibit activation of p53-dependent transcription by HIPK2 and PML IV. However, the FBP, HIPK2 and PML synergistically stimulated the p53-dependent transcriptional activation. Taken together our data suggest that the SCF-induced ubiquitination of transcription co-activators HIPK2 and p300 plays a critical role in transcriptional regulation, and that PML stimulates transcription by protecting HIPK2 and p300 from ubiquitin-dependent degradation.
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Abraham, Shaji, Leonard C. Edelstein, Chad A. Shaw, Pierrette Andre, Xianguo Kong, Carol T. Dangelmaier, Alexander Tsygankov, Paul F. Bray, Satya P. Kunapuli, and Steven E. McKenzie. "Platelet FcγRIIA Signaling Results in Ubiquitination and Cellular Translocation of Activated Syk." Blood 124, no. 21 (December 6, 2014): 2761. http://dx.doi.org/10.1182/blood.v124.21.2761.2761.
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Abstract Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thrombosis syndromes, such as heparin-induced thrombocytopenia (HIT). FcγRIIA is also the major transmembrane signaling adapter for αIIbβ3 outside-in signaling. In HIT, antibody to heparin/PF4 is necessary but not sufficient for disease to occur. Inter-individual variation in platelet activation via FcγRIIA contributes to HIT risk, but the molecular basis for the variation is incompletely understood. In our PRAX1 study of platelet reactivity and RNA expression (Edelstein, Nature Med 2013; Simon, Blood 2014), we identified differentially expressed mRNAs from healthy donors with different platelet reactivity to FcγRIIA stimulation. We observed significant differential expression of molecules involved in ubiquitination processes in relation to platelet reactivity to FcγRIIA stimulation. Syk is a protein tyrosine kinase and the major signaling node downstream of platelet receptors that use immunotyrosine activation motif (ITAM) signaling, such as FcγRIIA, GPVI and CLEC-2. We previously reported Syk ubiquitination following GPVI stimulation, and the role of c-cbl as the E3 ubiquitin ligase (Dangelmaier, Blood 2005). Ubiquitination is an important post-translational modification that modulates signal transduction by regulating the activity, subcellular localization or stability of proteins. We tested the hypothesis that ubiquitination participates in signaling, and examined ubiquitination of Syk downstream of platelet FcγRIIA activation. Using both washed human platelets and HEL cells, we observed ubiquitination of Syk upon FcγRIIA engagement by cross-linking IV.3 mAb (10 ug/ml) with goat anti-mouse Fab’2 (30 ug/ml). Both tyrosine phosphorylation and ubiquitination of Syk occurred within 15 sec, peaked by 1-3 min and decreased thereafter. The pattern of ubiquitination was consistent with 1 to 3 Ub molecules per Syk molecule. Ubiquitinated-Syk (Ub-Syk) was increased in the presence of PR-619, a deubiquitinating enzyme inhibitor, confirming ubiquitination of Syk. Ub-Syk associates with the cytoskeletal-rich platelet fraction, membrane skeleton fraction and with cytosolic fraction in detergent lysed platelets that were fractionated by lower g-forces (15,500 x g) and higher g-forces (100,000 x g). This suggests that Ub-Syk is translocated into all cellular compartments upon platelet activation. Ub-Syk was absent upon pre-treatment with Src-family kinase inhibitor PP2 (10 uM), but minimally affected in the presence of Syk inhibitor PRT318 (1 uM), in both platelets and HEL cells, as compared to DMSO treated control cells. Further, phosphorylation of c-cbl was inhibited strongly by PP2, but only slightly inhibited by PRT318, suggesting that ubiquitination of Syk depends on Src kinase activity. Of note, Ub-Syk was not degraded by the proteasome, since no accumulation of Ub-Syk was observed by pretreatment with proteasome inhibitors MG132 or Epoxomicin in either platelets or HEL cells, compared to control cells. In conclusion, Syk is ubiquitinated upon cross-linking platelet FcγRIIA and is translocated to all major subcellular compartments. Since ubiquitinated Syk is activation-dependent and not subject to proteasomal degradation, it likely serves as a novel adapter molecule for protein-protein interactions in mediating platelet activation via FcγRIIA. Disclosures No relevant conflicts of interest to declare.
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Chauhan, Dharminder, Ze Tian, Benjamin Nicholson, Bin Zhou, Teru Hideshima, Nikhil Munshi, Paul Richardson, and Kenneth C. Anderson. "Deubiquitylating Enzyme USP-7, a Novel Therapeutic Target in Multiple Myeloma." Blood 114, no. 22 (November 20, 2009): 610. http://dx.doi.org/10.1182/blood.v114.22.610.610.
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Abstract Abstract 610 Background and Rationale: Ubiquitin-Proteasome Signaling (UPS) pathway is mediated via a large number of components, suggesting many potential sites of pharmacological intervention. Therapeutic targeting of UPS is exemplified by the recent FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM). As with other agents, however, dose-limiting toxicities and the development of resistance limit its long-term utility. Recent efforts have been devoted to discovery and development of small molecule inhibitors of the major components of UPS, such as E1-conjugating enzyme inhibitors, E3 ubiquitin ligase inhibitors (e.g inhibitors of TRAF6, SCF-p27, POSH, MDM2-P53) or Deubiquitylating enzyme (DUB) inhibitor. Therapeutic strategies using these small molecule inhibitors will allow for specific targeting of the UPS and therefore may be less likely than proteasome inhibitors to trigger off-target activities and associated toxicities. In this context, Ubiquitin Specific Protease-7 (USP-7), a deubiquitylating enzyme, is of particular interest since it regulates activity of key biological signaling pathways such as p53, the transcription factor FOXO-4, Cdk inhibitor p27, and PI3K/Akt. In the present study, we examined the anti-tumor activity of a novel USP-7 inhibitor P5091 (Progenra, Inc., Malvern, PA) in MM cells using both in vitro and in vivo model systems. Results: We first confirmed the functional specificity of P5091 using different experimental strategies: 1) Cell-free based experiments using a novel reporter (Ub-PLA2)-based assay showed a potent, specific and selective deubiquitylating activity of P5091 against USP-7 (EC50 = 5.6 mM) versus other DUBs and proteases (EC50 25 to > 50 mM); 2) Treatment of MM.1S MM cells with P5091 blocked USP-7 deubiquitylating activity, without altering proteasome activity; and 3) treatment of HCT-116 (wild type) cells with P5091 markedly decreased viability; conversely, knockout of USP-7 in HCT-116 conferred resistance to P5091 (P < 0.001; n=2). Taken together, these data demonstrate that P5091 targets USP-7. We next examined the effects of P5091 in MM cells. Protein expression analysis showed that USP-7 is highly expressed in MM cells. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, U266, KMS-12BM, and INA-6) and primary patient cells for 72h significantly decreased their viability (IC50 range 5-10 mM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for P5091. P5091-induced apoptosis was confirmed in MM.1S and RPMI-8226 cells, as evidenced by marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, transfection of USP-7 siRNA, but not scrambled (genome control) siRNA, inhibits growth of MM.1S cells, similar to USP-7 inhibitor P5091 (P < 0.001; n=2). In addition, p5091 triggered apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies further showed that P5091-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of Noxa, PUMA, and Bid; 3) downregulation of MM cell growth and survival proteins: Akt, Bclxl, phospho-S6 ribosomal protein, and c-Myc; 4) inhibition of migration of MM cells and angiogenesis; and 5) suppression of USP-7 specific substrate HDM2 with concurrent increased expression of CDK inhibitor p21. Importantly, blockade of HDM2 and p21 using siRNA strategy significantly abrogates P5091-induced MM.1S cell death (P value <0.001; n=2). We next examined the in vivo efficacy of p5091 using human plasmacytoma xenograft mouse model. Treatment of tumor-bearing mice with P5091 (10 mg/kg IV, twice a week for three weeks), but not vehicle alone, significantly (P < 0.001) inhibits MM tumor growth and prolongs survival of these mice (P < 0.001; 13.6 days prologation of survival in mice receiving P5091 vs. vehicle-treated mice). The concentrations of P5091 were well tolerated by mice, without significant weight loss. Conclusions: These preclinical studies will allow for the development of next generation UPS-based therapies and provide the rationale for novel therapeutics targeting USP7 to improve patient outcome in MM. Disclosures: Chauhan: Progenra, Inc: Consultancy. Nicholson:Progenra, Inc: Employment.
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Nakamura, Shingen, Masahiro Abe, Qu Cui, Ayako Nakano, Asuka Oda, Hiroe Amou, Akishige Ikegame, et al. "Facilitation of Osteoblast Differentiation by ATF-4 Accumulation through Proteasome Inhibition." Blood 114, no. 22 (November 20, 2009): 1823. http://dx.doi.org/10.1182/blood.v114.22.1823.1823.
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Abstract Abstract 1823 Poster Board I-849 The proteasome inhibitor bortezomib, a novel anti-myeloma (MM) agent, has recently drawn considerable attention to its anabolic actions on bone formation in patients with MM. Bortezomib was reported to enhance the activity of Runx2/cbfa1, an essential transcription factor for osteoblast (OB) induction, in mesenchymal stem cells to induce OB differentiation. However, because over-expression of Runx2 unexpectedly suppresses terminal OB differentiation or mineralization, there may be critical factors involved in OB differentiation in concert with Runx2 to achieve terminal OB differentiation in the treatment with bortezomib. Proteasome inhibition accumulates a variety of proteins and induces ER stress or unfolded protein response. Among proteins induced by ER stress, activating transcription factor-4 (ATF-4) plays a critical role in OB differentiation. ATF-4 is expressed in osteoprogenitors and preOBs following Runx2, and acts in concert with Runx2 to facilitate terminal maturation of OBs. However, it is unknown whether a change in ATF-4 protein levels plays any role in OB differentiation induced by proteasome inhibition. In the present study, we therefore explored the role of ATF-4 in OB differentiation by proteasome inhibition in Runx2-expressing immature OB lineage cells. Bortezomib dose-dependently increased ATF-4 protein levels in primary bone marrow stromal cells and ST-2 stromal and MC3T3-E1 preosteoblastic cell lines at concentrations higher than 10 nM as early as 3 hours. Because serum bortezomib levels reach around 100 nM (Cmax) with T1/2 of 3 hours after iv injection at therapeutic doses, bortezomib treatment in MM patients is expected to enhance ATF-4 protein levels in OB lineage cells. Interestingly, bortezomib treatment did not change mRNA levels of ATF-4 as well as βTrCP1, E3 ligase for ATF-4. Because translation of ATF-4 mRNA is triggered by ER stress response, it is plausible that the ATF-4 accumulation by bortezomib is mediated by the suppression of proteasomal degradation with subsequent induction of ER stress response. MM cell-derived factors and TGF-β released from bone by enhanced bone resorption suppress OB differentiation in MM bone lesions. Treatment with bortezomib was able to accumulate ATF-4 in the presence of MM conditioned media (CM) or TGF-β to the levels similar to those without MM CM nor TGF-β. Furthermore, bortezomib enhanced promotor activity of osteocalcin, a marker of mature OBs, as well as BMP-2-induced mineralized nodule formation in MC3T3-E1 cells, and these effects of bortezomib were suppressed by ATF-4 siRNA. These results demonstrate that bortezomib treatment accumulate ATF-4, and suggest that the effect of bortezomib on OB differentiation is mediated via an accumulation of ATF-4 protein in OB lineage cells. We have previously demonstrated that OB differentiation is suppressed in MM bone lesions, and that differentiated OBs suppress MM cell growth and survival. Thus, resumption of bone formation by bortezomib may further suppress MM cell growth in concert with its direct anti-MM actions. Disclosures No relevant conflicts of interest to declare.
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Abraham, Shaji, Carol T. Dangelmaier, Yuhang Zhou, Leonard C. Edelstein, Paul F. Bray, Satya P. Kunapuli, and Steven E. McKenzie. "Syk Is Regulated Downstream Of FcγRIIA In Platelets By Transient Tyrosine Phosphorylation and Ubiquitylation." Blood 122, no. 21 (November 15, 2013): 4737. http://dx.doi.org/10.1182/blood.v122.21.4737.4737.
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Platelet FcγRIIA contributes to the pathophysiology of heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis syndromes. Activation of FcγRIIA results in tyrosine phosphorylation of Syk in human platelets, but little is known about ubiquitylation of Syk. Protein ubiquitylation has been shown to regulate physiological and pathological cellular processes by regulating signaling networks. In the Platelet RNA and Expression-1 (PRAX1) study, we studied platelets from 154 healthy subjects and identified increased expression of mRNAs encoding proteins involved in ubiquitylation in platelets that were highly reactive to FcγRIIA stimulation. Previously, we (CD, SPK) reported that platelet stimulation via GPVI/FcRγ results in transient Syk phosphorylation and ubiquitylation, involving c-cbl and TULA-2. Briefly, c-Cbl acts as an E3 ligase transferring ubiquitin to the lysine residue of the target protein in the ubiquitylation reaction and TULA-2 is a dephosphorylating enzyme that removes phosphate group from ubiquitinated syk. In this study, we investigated Syk tyrosine phosphorylation and ubiquitylation downstream of platelet FcγRIIA engagement. FcγRIIA on washed human platelets was activated by cross-linking with IV.3 antibody (10ug/ml) and goat anti-mouse antibody (GAM) (30ug/ml). At specific time intervals following activation, platelets were lysed and the lysates were immunoblotted for total Syk and for pY525/526 phospho-Syk. We observed phosphorylation and ubiquitylation of Syk within 15 sec, and peaking within 1 to 3 min. The MW of the Syk species was consistent with 1 to 3 ubiquitin (Ub) moieties per Syk molecule. Alternatively, platelet FcγRIIA was activated via anti-CD9; Ub-Syk was observed at 15 sec, while ubiqutinated and phosphorylated Syk was observed at 2 min. Additional ubiquitinated bands appeared by 2 min; all diminished after 5 min. To understand if the ubiquitinated Syk was degraded by the proteasomal system, human platelets were pre-treated with proteasomal inhibitors (MG132 or Epoxomicin) followed by activation of FcγRIIA. There was no accumulation of Ub-Syk observed for up to 5 minutes after FcγRIIA stimulation in the presence of proteasomal inhibitors compared to controls, indicating that Ub-Syk is not degraded by the proteasomes and inactivated. Activation of FcγRIIA by cross linking with IV.3/GAM in HEL cells, a cell line model, showed a transient increase in ubiquitylation and phosphorylation of Syk within 15 sec which decreased by 3 min. To understand if activation of Syk by upstream Src-family kinases is necessary for ubiquitylation in this model system, SFK inhibitor (PP2) (10uM) was pre-incubated with HEL cells followed by FcγRIIA activation. Western blot analysis showed elimination of phosphorylated and ubiquitinated Syk upon activation of FcγRIIA in HEL cells in presence of PP2. In contrast, ubiquitylation and phosphorylation of Syk was observed in control cells treated with PP3 or vehicle (DMSO) and as well as in untreated cells. HEL cells were also pretreated with proteasomal inhibitors (MG132 or Epoxomicin) followed by activation of FcγRIIA. There was no accumulation of phosphorylated and ubiquitinated Syk observed for up to 10 min of stimulation of FcγRIIA in HEL cells in presence of proteasomal inhibitors compared to controls indicating no degradation of Ub-syk by proteasomal complex. Finally, we directly compared the ubiquitylation and phosphorylation of Syk in GPVI/FcRγ-stimulated vs.FcγRIIA-stimulated platelets from the same donors. Of note, FcγRIIA-stimulated platelets demonstrated both different kinetics and extent of Syk ubiquitylation than did GPVI/FcRγ-stimulated platelets. In this study, we demonstrated that FcγRIIA signaling results in transient Syk tyrosine phosphorylation and ubiquitylation in platelets and HEL cells. The ubiquitinated Syk is not destined for proteasomal degradation. Limited ubiquitylation of proteins has been noted to modulate downstream signaling events and protein-protein interactions. Further studies are needed to decipher the molecular partners of ubiquitinated Syk following FcγRIIA activation, and to elucidate the differences between GPVI/FcRγ and FcγRIIA signaling via Syk. Disclosures: No relevant conflicts of interest to declare.
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Raghavan, Sathees C., Ujjayinee Ray, Anjana Elizabeth Jose, Rohini Suresh, Uthara Kaloor, Hassan A. Swarup, Mridula Nambiar, and Sathees C. Raghavan. "Water-soluble SCR7 Can Abrogate DNA End Joining and Induce Cancer Cell Death." Clinical Oncology and Research, July 16, 2020, 1–7. http://dx.doi.org/10.31487/j.cor.2020.07.09.
Full textAbstract:
Small molecule inhibitors targeting DNA repair pathways in cancer cells is a novel and promising approach in cancer therapy, which can improve current therapeutic regimen. Although various attempts have been made for designing inhibitors against DNA damage response and repair proteins, reports on Nonhomologous End Joining (NHEJ) inhibitors are limited. Of the several chemical moieties identified, SCR7 and its oxidized form are novel and potent DNA Ligase IV inhibitors involved in the abrogation of DNA end joining thereby leading to cell death. In the present study, we have synthesized sodium salt of SCR7 to generate a water-soluble version of the molecule, referred to as water-soluble SCR7 (WS-SCR7). WS-SCR7 inhibits NHEJ in Ligase IV dependent manner, with a subtle effect on Ligase III at higher concentration. No effect on Ligase I mediated joining was observed. WS-SCR7 shows cytotoxicity in cancer cell lines, leading to induction of apoptosis in a dose-dependent manner.
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Hu, Zheng, Zhaoying Shi, Xiaogang Guo, Baishan Jiang, Guo Wang, Dixian Luo, Yonglong Chen, and Yuan-Shan Zhu. "Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR–Cas9 and ssODN in human cancer cells." Cell & Bioscience 8, no. 1 (February 19, 2018). http://dx.doi.org/10.1186/s13578-018-0200-z.
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Gupta, Navin, Takuya Matsumoto, Ken Hiratsuka, Edgar Garcia Saiz, Pierre Galichon, Tomoya Miyoshi, Koichiro Susa, Narihito Tatsumoto, Michifumi Yamashita, and Ryuji Morizane. "Modeling injury and repair in kidney organoids reveals that homologous recombination governs tubular intrinsic repair." Science Translational Medicine 14, no. 634 (March 2, 2022). http://dx.doi.org/10.1126/scitranslmed.abj4772.
Full textAbstract:
Kidneys have the capacity for intrinsic repair, preserving kidney architecture with return to a basal state after tubular injury. When injury is overwhelming or repetitive, however, that capacity is exceeded and incomplete repair results in fibrotic tissue replacing normal kidney parenchyma. Loss of nephrons correlates with reduced kidney function, which defines chronic kidney disease (CKD) and confers substantial morbidity and mortality to the worldwide population. Despite the identification of pathways involved in intrinsic repair, limited treatments for CKD exist, partly because of the limited throughput and predictivity of animal studies. Here, we showed that kidney organoids can model the transition from intrinsic to incomplete repair. Single-nuclear RNA sequencing of kidney organoids after cisplatin exposure identified 159 differentially expressed genes and 29 signal pathways in tubular cells undergoing intrinsic repair. Homology-directed repair (HDR) genes including Fanconi anemia complementation group D2 ( FANCD2 ) and RAD51 recombinase ( RAD51 ) were transiently up-regulated during intrinsic repair but were down-regulated in incomplete repair. Single cellular transcriptomics in mouse models of obstructive and hemodynamic kidney injury and human kidney samples of immune-mediated injury validated HDR gene up-regulation during tubular repair. Kidney biopsy samples with tubular injury and varying degrees of fibrosis confirmed loss of FANCD2 during incomplete repair. Last, we performed targeted drug screening that identified the DNA ligase IV inhibitor, SCR7, as a therapeutic candidate that rescued FANCD2/RAD51-mediated repair to prevent the progression of CKD in the cisplatin-induced organoid injury model. Our findings demonstrate the translational utility of kidney organoids to identify pathologic pathways and potential therapies.
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Fronik, Philipp, Michael Gutmann, Petra Vician, Mirjana Stojanovic, Alexander Kastner, Petra Heffeter, Christine Pirker, Bernhard K. Keppler, Walter Berger, and Christian R. Kowol. "A platinum(IV) prodrug strategy to overcome glutathione-based oxaliplatin resistance." Communications Chemistry 5, no. 1 (April 6, 2022). http://dx.doi.org/10.1038/s42004-022-00661-z.
Full textAbstract:
AbstractClinical efficacy of oxaliplatin is frequently limited by severe adverse effects and therapy resistance. Acquired insensitivity to oxaliplatin is, at least in part, associated with elevated levels of glutathione (GSH). In this study we report on an oxaliplatin-based platinum(IV) prodrug, which releases L-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase, the rate-limiting enzyme in GSH biosynthesis. Two complexes bearing either acetate (BSO-OxOAc) or an albumin-binding maleimide (BSO-OxMal) as second axial ligand were synthesized and characterized. The in vitro anticancer activity of BSO-OxOAc was massively reduced in comparison to oxaliplatin, proving its prodrug nature. Nevertheless, the markedly lower intracellular oxaliplatin uptake in resistant HCT116/OxR cells was widely overcome by BSO-OxOAc resulting in distinctly reduced resistance levels. Platinum accumulation in organs of a colorectal cancer mouse model revealed higher tumor selectivity of BSO-OxMal as compared to oxaliplatin. This corresponded with increased antitumor activity, resulting in significantly enhanced overall survival. BSO-OxMal-treated tumors exhibited reduced GSH levels, proliferative activity and enhanced DNA damage (pH2AX) compared to oxaliplatin. Conversely, pH2AX staining especially in kidney cells was distinctly increased by oxaliplatin but not by BSO-OxMal. Taken together, our data provide compelling evidence for enhanced tumor specificity of the oxaliplatin(IV)/BSO prodrug.
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Anbalagan, Selvakumar, Cecilia Ström, Jessica A. Downs, Penny A. Jeggo, David McBay, Anna Wilkins, Kai Rothkamm, Kevin J. Harrington, John R. Yarnold, and Navita Somaiah. "TP53 modulates radiotherapy fraction size sensitivity in normal and malignant cells." Scientific Reports 11, no. 1 (March 29, 2021). http://dx.doi.org/10.1038/s41598-021-86681-6.
Full textAbstract:
AbstractRecent clinical trials in breast and prostate cancer have established that fewer, larger daily doses (fractions) of radiotherapy are safe and effective, but these do not represent personalised dosing on a patient-by-patient basis. Understanding cell and molecular mechanisms determining fraction size sensitivity is essential to fully exploit this therapeutic variable for patient benefit. The hypothesis under test in this study is that fraction size sensitivity is dependent on the presence of wild-type (WT) p53 and intact non-homologous end-joining (NHEJ). Using single or split-doses of radiation in a range of normal and malignant cells, split-dose recovery was determined using colony-survival assays. Both normal and tumour cells with WT p53 demonstrated significant split-dose recovery, whereas Li-Fraumeni fibroblasts and tumour cells with defective G1/S checkpoint had a large S/G2 component and lost the sparing effect of smaller fractions. There was lack of split-dose recovery in NHEJ-deficient cells and DNA-PKcs inhibitor increased sensitivity to split-doses in glioma cells. Furthermore, siRNA knockdown of p53 in fibroblasts reduced split-dose recovery. In summary, cells defective in p53 are less sensitive to radiotherapy fraction size and lack of split-dose recovery in DNA ligase IV and DNA-PKcs mutant cells suggests the dependence of fraction size sensitivity on intact NHEJ.