The mTOR Inhibitor Everolimus Overcomes CXCR4-Mediated Resistance to HDAC Inhibitor Panobinostat through Inhibition of p21 and Mitosis Regulators, Sensitizing MM Cells to DNA-Damaged Induced Apoptosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 891-891
Author(s):  
Katia Beider ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Evgenia Rosenberg ◽  
Hila Magen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Cell Lines ◽  
Hdac Inhibitor ◽  
Mtor Inhibitor ◽  
Mtor Inhibition ◽  
Induced Apoptosis

Abstract Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it. Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p<0.001) increased their resistance to panobinostat, pointing to the role of the CXCR4 axis in HDACi response. Notably, similar expression levels of class I HDACs (HDAC1-3) were detected in MM cells with either low or high CXCR4. Interaction with BM stromal cells that represent the source of CXCL12 also protected MM cells from panobinostat-induced apoptosis, further strengthening a role for CXCR4 downstream pathway. Decreased sensitivity to cytotoxic effect was concomitant with reduced histone (H3K9 and H4K8) acetylation in response to panobinostat treatment. In addition, resistance to HDACi was associated with the reversible G0/G1 cell growth arrest, whereas sensitivity was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators involved in CXCR4-mediated HDACi resistance revealed the pro-survival AKT/mTOR pathway to be regulated by both CXCR4 over-expression and interaction with BMSCs. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance and induced synergistic cell death of MM cell lines and primary MM cells, but not of normal mononuclear cells (CI<0.4). This effect was concurrent with the increase in DNA double strand breaks, histone H2AX phosphorylation, loss of Dψm, cytochrome c release, caspase 3 activation and PARP cleavage. The increase in DNA damage upon combinational treatment was not secondary to the apoptotic DNA fragmentation, as it occurred similarly when apoptosis onset was blocked by caspase inhibitor z-VAD-fmk. Kinetics studies also confirmed that panobinostat-induced DNA damage preceded apoptosis induction. Strikingly, combined panobinostat/everolimus treatment resulted in sustained DNA damage and irreversible suppression of MM cell proliferation accompanied by robust apoptosis, in contrast to the modest effects induced by single agent. Gene expression analysis revealed distinct genetic profiles of single versus combined exposures. Whereas panobinostat increased the expression of cell cycle inhibitors GADD45G and p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated DNA repair genes, including RAD21, Ku70, Ku80 and DNA-PKcs. Furthermore, combined treatment markedly decreased both mRNA and protein expression of anti-apoptotic factors survivin and BCL-XL, checkpoint regulator CHK1, and G2/M-specific factors PLK1, CDK1 and cyclin B1, therefore suppressing the DNA damage repair and inhibiting mitotic progression. Given the anti-apoptotic role of p21, the synergistic lethal effect of everolimus could be attributed to its ability to suppress p21 induction by panobinostat ensuing the shift in the DNA damage response toward apoptosis. Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis. Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals DR5 Knockout Mice Are Compromised in Radiation-Induced Apoptosis

2005 ◽  
Vol 25 (5) ◽  
pp. 2000-2013 ◽  
Author(s):  
Niklas Finnberg ◽  
Joshua J. Gruber ◽  
Peiwen Fei ◽  
Dorothea Rudolph ◽  
Anka Bric ◽  
...  
Keyword(s):  
Cell Death ◽  
Null Mouse ◽  
Organ Specific ◽  
Radiation Induced ◽  
Induced Apoptosis ◽  
The Brain ◽  
Necrosis Factor

ABSTRACT DR5 (also called TRAIL receptor 2 and KILLER) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called TRAIL and Apo2 ligand). DR5 is a transcriptional target of p53, and its overexpression induces cell death in vitro. However, the in vivo biology of DR5 has remained largely unexplored. To better understand the role of DR5 in development and in adult tissues, we have created a knockout mouse lacking DR5. This mouse is viable and develops normally with the exception of having an enlarged thymus. We show that DR5 is not expressed in developing embryos but is present in the decidua and chorion early in development. DR5-null mouse embryo fibroblasts expressing E1A are resistant to treatment with TRAIL, suggesting that DR5 may be the primary proapoptotic receptor for TRAIL in the mouse. When exposed to ionizing radiation, DR5-null tissues exhibit reduced amounts of apoptosis compared to wild-type thymus, spleen, Peyer's patches, and the white matter of the brain. In the ileum, colon, and stomach, DR5 deficiency was associated with a subtle phenotype of radiation-induced cell death. These results indicate that DR5 has a limited role during embryogenesis and early stages of development but plays an organ-specific role in the response to DNA-damaging stimuli.

Abstract 1950: Role of Superoxide, Nitric Oxide and Peroxynitrite in Doxorubicin-induced Cell Death in vitro and in vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Partha Mukhopadhyay ◽  
Mohanraj Rajesh ◽  
Sandor Bátkai ◽  
György Haskó ◽  
Csaba Szabo ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Superoxide Generation ◽  
Cytochrome C Release ◽  
Mitochondrial Superoxide ◽  
Induced Apoptosis ◽  
Peroxynitrite Scavengers

Although doxorubicin (DOX) is one of the most potent antitumor agents available, its clinical use is limited because of the risk of severe cardiotoxicity often leading to irreversible congestive heart failure. Apoptotic cell death is a key component in DOX-induced cardiotoxicity, but its trigger(s) and mechanisms are poorly understood. Here, we explore the role of peroxynitrite (a reactive oxidant produced from the diffusion-controlled reaction between nitric oxide and superoxide anion) in DOX-induced cell death. Using a well-established in vivo mouse model of DOX-induced acute heart failure, we demonstrate marked increases in myocardial apoptosis (caspase-3 and 9 gene expression, caspase 3 activity, cytochrome-c release, and TUNEL), iNOS but not eNOS and nNOS expression, 3-nitrotyrosine formation and a decrease in myocardial contractility following DOX treatment. Pre-treatment of mice with peroxynitrite scavengers markedly attenuated DOX-induced myocardial cell death and dysfunction without affecting iNOS expression. DOX induced increased superoxide generation and nitrotyrosine formation in the mitochondria, dissipation of mitochondrial membrane potential, apoptosis (cytochrome-C release, annexin V staining, caspase activation, nuclear fragmentation), and disruption of actin cytoskeleton structure in cardiac-derived H9c2 cells. Selective iNOS inhibitors attenuated DOX-induced apoptosis, without affecting increased mitochondrial superoxide generation, whereas NO donors increased DOX-induced cell death in vitro . The peroxynitrite scavengers FeTMPyP and MnTMPyP markedly reduced both DOX- or peroxynitrite-induced nitrotyrosine formation and cell death in vitro , without affecting DOX-induced increased mitochondrial superoxide formation. Thus, peroxynitrite is a major trigger of DOX-induced apoptosis, and its effective neutralization can be of significant therapeutic benefit.

Induction of Apoptosis and Paradoxical Activation of NF-κB by Trichostatin A in Plasma Cell Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4851-4851
Author(s):  
Keiko Asakura ◽  
Hideo Uchida ◽  
Akihiro Muto ◽  
Yoshitaka Miyakawa ◽  
Yasuo Ikeda ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Lines ◽  
Plasma Cell ◽  
Hdac Inhibitor ◽  
Antitumor Effect ◽  
Trichostatin A ◽  
Hdac Inhibitors ◽  
Plasma Cell Myeloma ◽  
Induced Apoptosis

Abstract Plasma cell myeloma (PCM) is a mature B cell malignancy characterized by monoclonal gammopathy and destructive bone lesions. Although recent development of therapeutic drugs including thalidomide or proteasome inhibitor may prolong survival time, PCM is still an incurable disease. To improve the treatment outcome of PCM, the development of novel, pathophysiology-based therapies are needed. Histone acetylation plays a role in transcriptional regulation by altering the chromatin structure, which allows the transcription factors to access the DNA. Histone acetyltransferases (HATs) are involved in tumor suppression and inhibition of histone deacetylases (HDACs) results in antitumor effect. HDAC inhibitors have also reported to induce apoptosis or differentiation of tumor cells. Trichostatin A (TSA) is one of the HDAC inhibitors, and it has shown to be effective in vitro against leukemic cells with characteristic chromosomal translocations. Although none of the disease-specific genetic alterations involving aberrant transcription factors in PCM have been reported, previous studies have demonstrated the antitumor effect of HDAC inhibitors on PCM. Accordingly, to address the detailed molecular mechanisms of HDAC inhibitor-induced apoptosis, we examined the in vitro effects of TSA on PCM cell lines. By using flow cytometric analyses, we found that cell cycle arrest at G1 phase and apoptosis were induced in various PCM cell lines, RPMI8226, IM9, and U266 treated with 100nM of TSA for 24 hours. Especially, the viability of TSA-treated RPMI8226 cells was strongly suppressed to approximately 30% compared to the cells without TSA. Nuclear factor (NF) -κB is thought to be a key molecule of cellular growth in PCM; therefore, we studied the functional alterations of NF-κB in PCM cell lines stimulated with TSA. Immunoblot and electrophoretic mobility shift assays demonstrated that TSA augmented the nuclear localization of NF-κB, which resulted in the increased DNA binding activity and upregulation of downstream target gene expression such as ICAM-1. Increased phosphorylation of Iκ-Bα by TSA was observed simultaneously. Transient transfection assays showed that the transcriptional activity of NF-κB was upregulated more than 4-fold by TSA, and the synergistic effects of TSA with TNF-α or PMA were observed. Interestingly, an NF-κB-specific inhibitor, SN50, induced apoptosis of PCM cell lines, and TSA showed a synergism with SN50. These data may provide a notion that increased activity of NF-κB by TSA could be a compensatory response against proapoptotic signals induced by TSA. Additionally, we investigated other molecules responsible for TSA-stimulated apoptosis, and found the downregulation of Mcl-1, Bcl-XL, XIAP proteins, as well as dephosphorylation of Akt and cleavage of Bid proteins in a time-dependent manner. These results suggest that either PI 3-K/Akt pathway or JNK-dependent pathway could be involved in the TSA-induced apoptosis. In conclusion, HDAC inhibitors will be promising agents to improve survival of patients with PCM, and the combination of HDAC inhibitor and NF-κB inhibitor may provide a novel therapeutic strategy against PCM.

Bim Is the Critical, and Bad and Bmf Are the Ancillary BH3-Only Proteins for Imatinib Mesylate-Induced Apoptosis of Bcr/Abl-Positive Leukemias.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2876-2876
Author(s):  
Junya Kuroda ◽  
Hamsa Puthalakath ◽  
Philippe Bouillet ◽  
Mark S. Cragg ◽  
Priscilla N. Kelly ◽  
...  
Keyword(s):  
Cell Death ◽  
Imatinib Mesylate ◽  
Cell Lines ◽  
Cell Killing ◽  
Leukemic Cells ◽  
Imatinib Treatment ◽  
Killing Activity ◽  
Induced Apoptosis ◽  
Transformed Cell Lines

Abstract Imatinib mesylate (imatinib) exerts the anti-Philadelphia-positive (Ph1+) leukemia activity both by the inhibition of cell proliferation and by the induction of apoptosis. Recent studies demonstrate that the induction of cell death is essential for eradication of Ph1+ leukemic clones in imatinib treatment; however, the molecular mechanisms have not yet been clearly described. By examining the effect of imatinib on parental K562 and subclones overexpressing either Bcl-2, Bcl-XL or a dominant interfering mutant of FADD/MORT1, which blocks death receptor apoptosis signalling, we found that imatinib triggers apoptosis exclusively via the Bcl-2 family-regulated intrinsic apoptotic pathway. We investigated the involvement of BH3-only proteins as apoptotic initiators in imatinib-induced cell death, because the cell life-or-death decision is arbitrated by the balance between pro-apoptotic BH3 only-proteins and anti-apoptotic Bcl-2 proteins. We found that imatinib treatment upregulated Bim in Ph1+ leukemic cell lines and bcr-c-abl transformed murine fetal liver cells (FLCs)-derived cell lines both by transcriptional and post-translational mechanisms. Imatinib also activated Bad through dephosphorylation and upregulated Bmf transcriptionally. To examine the role of Bim in imatinib-induced apoptosis, we examined the cell killing activity of imatinib in subclones of K562 and BV173 Ph1+ cells expressing abnormally reduced levels of Bim using stable RNA interference system. This revealed that the cell killing activity of imatinib largely dependent on Bim expression levels in these cell lines, although significant apoptosis was still evident. To further define the role of Bim, Bad and Bmf in imatinib-induced cell death, we examined the effect of imatinib on retrovirally bcr-c-abl transformed cell lines derived from FLCs from wild type C57BL/6, Bim-/-, Bad-/-, Bim-/-Bad-/- double KO and Bcl-2 transgenic fetuses. The bim-/-bcr-c-abl+ FLCs were shown to be more resistant to imatinib-induced cell death than wt.bcr-c-abl+ FLCs, however, bim-/-bcr-c-abl+ FLCs were eventually induced into cell death, indicating that Bim is not the only initiator of apoptosis. The bad-/-bcr-c-abl+ FLCs were also partially resistant to imatinib-induced cell death. Intriguingly, like in vav.bcl-2.bcr-c-abl+ FLCs, the cell death induction by imatinib (~5.0μM) was largely abrogated in bim-/-bad-/-bcr-c-abl+ FLCs, indicating that Bim collaborates with Bad for the apoptotic induction by imatinib. Importantly, we found that Bim was inducible by ex vivo imatinib treatment in primary Ph1+ leukemic cells only from clinically good responders but not from patients refractory to imatinib treatment. Collectively, these results demonstrate that Bim is the critical but not the only initiator required for imatinib-induced apoptosis of Bcr/Abl-positive hematopoietic cells; Bad and Bmf may be the ancillary BH3-only proteins in this process. Our results provide evidence for the therapeutic significance of regulation of BH3-only proteins, particularly Bim, for the eradication of Ph1+ leukemic cells.

In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4459-4459
Author(s):  
Morris Kletzel ◽  
Sarah C. Tallman ◽  
Marie Olszewski ◽  
Wei Huang
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

The Telomerase Inhibitor RHPS4 Induces Telomere Shortening, DNA-Damage and Cell Death in AML Cells and Chemosensitises Cells to Daunorubicin.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2760-2760
Author(s):  
Monica Pallis ◽  
Dotun Ojo ◽  
Jaineeta Richardson ◽  
John Ronan ◽  
Malcolm Stevens ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Telomere Length ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Cell Types ◽  
Telomere Shortening ◽  
Damage Response

Abstract Abstract 2760 Poster Board II-736 The quadruplex ligand RHPS4 is the lead compound in a drug discovery program at the University of Nottingham. It has been shown to bind to telomeres and inhibit telomerase, and subsequently induces growth arrest in progenitor cells from cancer cell lines whilst sparing normal haematopoietic progenitor cells. We explored its in vitro effects in AML cells, which are reported generally to have considerably shorter telomeres than normal CD34+ cells. AML cell lines were grown for 21 days in suspension culture. Primary samples were cultured for 14 days in semi-solid medium. Telomere length was measured by Southern blotting. γH2A.X was used to identify a DNA damage response, and cell viability was measured flow cytometrically with 7-amino actinomycin D. As reported in other tumour cell types, sensitivity to RHPS4 was found to be greatest in those AML cells with the shortest telomeres. In the OCI-AML3 cell line 0.3 μM RHPS4 inhibited cell growth by 50% in a 21 day clonogenic assay, accompanied by shortening of telomeres from 2.6 Kb to <1 Kb. Molm 13 cells (initial telomere length 3.2kB) also underwent telomere shortening in the presence of 0.3 μM RHPS4 (2.8Kb), whereas TF1a and U937 (both with initial telomere lengths approximately 6.5 kB) were insensitive at that concentration. After 6 days at 0.3 μM, RHPS4 was cytostatic, but at higher concentrations (1 μM) the drug was found to induce a substantial DNA damage response and loss of viability to OCI-AML3 cells. Moreover 0.3 μM RHPS4 enhanced the γH2A.X expression and cell death induced by the chemotherapy drug daunorubicin in these cells. Using 14 day clonogenic assays in primary AML samples (n=6), we found that the IC50 for RHPS4 alone was 0.7 μM. However, in the presence of 0.3 μM RHPS4, the median IC50 to daunorubicin was reduced from 19 nM to 5.5 nM. In conclusion we have determined that RHPS4 has telomere-shortening, cytostatic, cytotoxic and chemosensitising properties in AML cells. Disclosures: Stevens: Pharminox Ltd: director and shareholder of Pharminox Ltd which has a financial interest in RHPS4.

Role Of Sirtuin-6 In Maintenance Of Genomic Instability In Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 276-276
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
Chirag Acharya ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Genomic Instability ◽  
Cell Lines ◽  
Dsb Repair ◽  
Dna Damaging Agents ◽  
Genotoxic Agents ◽  
Equity Ownership

Abstract Background Deregulation of the DNA damage response (DDR) signaling machinery underlies genomic instability, leading to cancer development and clonal evolution. Multiple Myeloma (MM) remains an incurable disease characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DDR machinery. Mammalian NAD+-dependent deacetylase sirtuin-6 (SIRT6) is emerging as new protein involved in multiple pathways, including maintenance of genome integrity. Methods A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in this study. Blood and BM samples from healthy volunteers and MM patients were obtained after informed consent and mononuclear cells (MNCs) separated by Ficoll-Paque density sedimentation. Patient MM cells were isolated from BM MNCs by CD138-positive selection. Lentiviral delivery was used for expression and knock-down of SIRT6 in MM cell lines. The biologic impact of SIRT6 phenotype was evaluated using cell growth, viability and apoptosis assays. DNA Double-Strand Breaks (DSB) repair occurring via homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways was assessed using a transient direct repeat (DR)-GFP/I-SceI system. Results A comparative gene expression analysis of 414 newly-diagnosed uniformly-treated MM patients showed high levels of SIRT6 mRNA in MM patients versus MGUS or normal donors; moreover, in active MM elevated SIRT6 expression correlated with adverse clinical outcome. Due to its prognostic significance, we further evaluated its role in MM biology. We found higher SIRT6 nuclear expression in MM cell lines and primary cells compared to PBMCs from healthy donors. Targeting SIRT6 by specific shRNA increased MM cell survival by reducing DNA repair efficiency (HR and NHEJ). Whole genome profiling of three different SIRT6 knockout (Sirt6-/-) MM cell lines identified a restricted effect of SIRT6 silencing on transcription of DNA damage genes, which also represented the most down-regulated genes. Consistent with these data, GSEA algorithm revealed that gene set regulating DNA repair were prominently enriched in SIRT6 depleted cells (p<0.0001 and FDR=0.003), confirming the role of SIRT6 in this pathway. We next examined the therapeutic relevance of SIRT6 inhibition in MM by evaluating the effect of SIRT6 depletion on cytotoxicity induced by genotoxic agents. SIRT6 shRNA impaired DNA DSB repair pathways triggered by DNA damaging agents, thereby enhancing overall anti-MM activity of these agents. Finally, in concert with our in vitro data, studies using our human MM xenograft model confirmed that SIRT6 depletion enhanced anti-MM activity of DNA-damaging agents. Conclusion Collectively, our data provide basis for targeting SIRT6 as a novel therapeutic strategy in combination with genotoxic agents to enhance cytotoxicity and improve patient outcome in MM. Disclosures: Tai: Onyx: Consultancy. Hideshima:Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

scholarly journals KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma

2020 ◽  
Author(s):  
Kevin Christian M. Gulay ◽  
Keisuke Aoshima ◽  
Yuki Shibata ◽  
Hironobu Yasui ◽  
Qin Yan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Damage Response ◽  
Histone Demethylase ◽  
Tumor Bearing ◽  
Lysine Specific Demethylase ◽  
Damage Response ◽  
Epigenetic Regulators ◽  
Induced Apoptosis

AbstractEpigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we found that lysine-specific demethylase 2B (Kdm2b) was highly expressed in HSA cell lines compared to normal canine endothelial cells. Silencing of Kdm2b in HSA cells resulted to increased cell death in vitro compared to the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced Kdm2b silencing in tumor xenografts resulted to decreased tumor sizes compared to the scramble control. Furthermore, Kdm2b was also highly expressed in clinical cases of HSA, and its expression levels was higher than in hemangioma, a benign counterpart of HSA. Based on these results, we hypothesized that pharmacological Kdm2b inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treated HSA cells with GSK-J4, a histone demethylase inhibitor, and found that GSK-J4 treatment also induced apoptosis and cell death. On top of that, GSK-J4 treatment in HSA tumor-bearing mice decreased tumor sizes without any obvious side-effects. In this study, we demonstrated that Kdm2b acts as an oncogene in HSA by enhancing DNA damage response and can be used as a biomarker to differentiate HSA from hemangioma. Moreover, we indicated that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.

scholarly journals Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells

2020 ◽  
Author(s):  
Xiaolong Wei ◽  
Jiekun Yang ◽  
Sara J. Adair ◽  
Cem Kuscu ◽  
Kyung Yong Lee ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Genetic Alterations ◽  
Model System ◽  
Drug Combinations ◽  
Pancreatic Cancer Cells ◽  
Synergistic Drug Combinations

ABSTRACTPancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancer to treat. Due to the asymptomatic nature of the disease and ineffective drug treatment modalities, the survival rate of PDAC patients remains one of the lowest. The recurrent genetic alterations in PDAC are yet to be targeted; therefore, identifying effective therapeutic combinations is desperately needed. Here, we performed an in vivo CRISPR screening in a clinically relevant patient-derived xenograft (PDX) model system to identify synergistic drug combinations for PDAC treatment. Our approach revealed protein arginine methyltransferase gene 5 (PRMT5) as a promising druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to gemcitabine. Genetic and pharmacological inhibition results indicate that of PRMT5 depletion results in synergistic cytotoxicity with Gem due to depleted replication protein A (RPA) levels and an impaired non-homology end joining (NHEJ) DNA repair. Thus, the novel combination creates conditional lethality through the accumulation of excessive DNA damage and cell death, both in vitro and in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is an effective approach in identifying synthetic lethal drug combinations for cancer treatment.STATEMENT of SIGNIFICANCEIdentify synergistic drug combinations for PDAC is a significant unmet need. Through CRISPR screening, we discovered and validated that PRMT5 depletion creates synergistic vulnerability of PDAC cells to gemcitabine. Mechanistically, the combination impairs DNA repair, synergistic accumulation of DNA damage and cell death in vitro and in vivo.

scholarly journals The Major Tegument Protein of Bovine Herpesvirus 1, VP8, Interacts with DNA Damage Response Proteins and Induces Apoptosis

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Sharmin Afroz ◽  
Ravendra Garg ◽  
Michel Fodje ◽  
Sylvia van Drunen Littel-van den Hurk
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Bovine Herpesvirus 1 ◽  
Damage Response ◽  
Infected Cells ◽  
Herpesvirus 1 ◽  
Induced Apoptosis

ABSTRACTVP8, theUL47gene product in bovine herpesvirus-1 (BoHV-1), is a major tegument protein that is essential for virus replicationin vivo. The major DNA damage response protein, ataxia telangiectasia mutated (ATM), phosphorylates Nijmegen breakage syndrome (NBS1) and structural maintenance of chromosome-1 (SMC1) proteins during the DNA damage response. VP8 was found to interact with ATM and NBS1 during transfection and BoHV-1 infection. However, VP8 did not interfere with phosphorylation of ATM in transfected or BoHV-1-infected cells. In contrast, VP8 inhibited phosphorylation of both NBS1 and SMC1 in transfected cells, as well as in BoHV-1-infected cells, but not in cells infected with a VP8 deletion mutant (BoHV-1ΔUL47). Inhibition of NBS1 and SMC1 phosphorylation was observed at 4 h postinfection by nuclear VP8. Furthermore, UV light-induced cyclobutane pyrimidine dimer (CPD) repair was reduced in the presence of VP8, and VP8 in fact enhanced etoposide or UV-induced apoptosis. This suggests that VP8 blocks the ATM/NBS1/SMC1 pathway and inhibits DNA repair. VP8 induced apoptosis in VP8-transfected cells through caspase-3 activation. The fact that BoHV-1 is known to induce apoptosis through caspase-3 activation is in agreement with this observation. The role of VP8 was confirmed by the observation that BoHV-1 induced significantly more apoptosis than BoHV-1ΔUL47. These data reveal a potential role of VP8 in the modulation of the DNA damage response pathway and induction of apoptosis during BoHV-1 infection.IMPORTANCETo our knowledge, the effect of BoHV-1 infection on the DNA damage response has not been characterized. Since BoHV-1ΔUL47 was previously shown to be avirulentin vivo, VP8 is critical for the progression of viral infection. We demonstrated that VP8 interacts with DNA damage response proteins and disrupts the ATM-NBS1-SMC1 pathway by inhibiting phosphorylation of DNA repair proteins NBS1 and SMC1. Furthermore, interference of VP8 with DNA repair was correlated with decreased cell viability and increased DNA damage-induced apoptosis. These data show that BoHV-1 VP8 developed a novel strategy to interrupt the ATM signaling pathway and to promote apoptosis. These results further enhance our understanding of the functions of VP8 during BoHV-1 infection and provide an additional explanation for the reduced virulence of BoHV-1ΔUL47.

Sign in / Sign up

Export Citation Format

Share Document