HDAC and LSD1 Inhibitors Synergize to Induce Cell Death in Acute Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1427-1427 ◽  
Author(s):  
Lorimar Ramirez ◽  
Melissa Singh ◽  
Joya Chandra
Keyword(s):  
Cell Death ◽  
Monoamine Oxidase ◽  
Acute Leukemia ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines

Abstract Abstract 1427 Histone deacetylase inhibitors (HDACi) are a class of emerging epigenetic therapies which are being used to treat cancer. Two HDACi (vorinostat and romidepsin) are FDA approved for cutaneous T-cell lymphoma. HDACi have been employed in clinical trials for acute leukemia, but single agent activity has been limited. Improved efficacy is observed when combined with other anticancer agents. In the current study we addressed acute leukemia models using vorinostat, a pan-HDACi that inhibits HDAC class I, II, and IV and entinostat, a newer HDACi that inhibits HDAC class I more specifically. These HDACi were combined with inhibition of another histone modifying enzyme: lysine specific demethylase 1 (LSD1). The LSD1 gene encodes a favin-dependent monoamine oxidase, which demethylates mono- and di-methylated lysines, specifically lysines 4 and 9 on histone 3 (H3K4 and H3K9), thus it is also involved in gene regulation through post-translational histone modification. LSD1 overexpression has been linked to human carcinogenesis in bladder carcinomas, lung cancer, and poorly differentiated neuroblastoma. However, it has not been studied in hematologic malignancies. Because LSD1 is structurally similar to monoamine oxidase (MAO), it has been shown that nonselective MAO inhibitors also inhibit LSD1. Here we employed tranylcypromine, a monoamine oxidase inhibitor (MAOi), as an irreversible LSD1 inhibitor. Recently published work from our laboratory has shown synergistic effects of combined HDAC and LSD1 inhibition in brain tumors (glioblastoma multiforme). Similar results have been published in breast cancer cells, but no work has been done in hematological malignancies. The objective of this study was to investigate the possible synergy of HDAC and LSD1 inhibitors in acute leukemia cells. LSD1 protein expression in several leukemia cells lines was analyzed by Western blot analysis. LSD1 was expressed in all leukemia cell lines tested, which included T-cell ALL (Jurkat, Sub-T1, MOLT4), B-cell ALL (JM-1,697), and Philadelphia chromosome positive ALL (Z33, Z119, Z181). To determine whether synergy exists between HDACi and LSD1 inhibitors, Jurkat cells were exposed to different concentrations of tranylcypromine and vorinostat or entinostat. After 24 hr, DNA fragmentation was assessed by propidium iodide (PI) staining followed by flow cytometric analysis. A combination index (CI) less than 1.0 is representative of synergism as measured by Calcusyn software. Results showed a synergistic effect on DNA fragmentation when combining the 2.5 μM dose of vorinostat with a range of tranylcypromine doses (1 mM CI= 0.78, 1.5 mM CI= 0.49, and 2 mM CI= 0.39). The same effect was observed with the combination of 2.5 μM entinostat with 2 mM tranylcypromine (CI=0.52). Viability studies performed with the same drug concentrations in conbination also showed statistically significant cell death. Additional acute leukemia cell lines, 697 and MOLT-4, also demonstrated significantly increased cell death with the combination relative to treatment with either agent alone. Since these agents inhibit histone deacetylation and lysine demethylation, we tested whether these histone modifications were promoted by combination treatment. Jurkat cell lysates were generated by acid extraction of histones and Western blot analysis was conducted. We demonstrated that in fact histone acetylation was increased with combination treatment, indicating that these modifications coordinately regulate each other in acute leukemia cells. A molecular target for LSD1 is p53, a tumor suppressor protein whose activity is regulated by lysine methylation and demethylation. Western blot analysis showed that p53 is downregulated in leukemia cells after exposure to the combination of HDAC and LSD1 inhibitors. Future studies will address if p53 downregulation is a trigger for the synergistic cell death. Taken together, our data shows the efficacy of combining LSD1 inhibitors with HDAC inhibitors in multiple acute leukemia models. Since tranylcypromine is also a FDA-approved agent, these results urge the design of a feasible and effective clinical trial combining LSD1 and HDAC inhibitors for acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Combination of Farnesyl Transferase Inhibitor (Tipifarnib) with Perifosine Induces Apoptosis through phos-PDK1 in Human Lymphoma and Leukemia Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1488-1488 ◽  
Author(s):  
Ebenezer David ◽  
Rajni Sinha ◽  
Claire Torre ◽  
Jonathan L. Kaufman ◽  
Sagar Lonial
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Targeted Agents ◽  
Leukemia Cell Lines ◽  
Targeted Agent ◽  
The Impact

Abstract Introduction: Novel agents as anti-cancer therapy are used in the setting of specific molecular abnormalities that provide a survival advantage for malignant cells. One such agent, tipifarnib, is theoretically targeted at Ras mutations which are present in a number of different human cancers. Our previous experience with the FTIs (David et al, in press Blood) has demonstrated that they are ideal agents to combine with other targeted agents. We have investigated the combination of the AKT inhibitor perifosine with tipifarnib in human leukemia and lymphoma cell lines with the hypothesis that the combination of 2 targeted agents will disrupt separate survival pathways and ultimately result in synergistic tumor cell death. Methods: In this study we used the human leukemia cell lines HL-60, Jurkat, and the lymphoma cell line HT. Western blot analysis was used to assess for the effect of either single agent perifosine, tipifarnib, or the combination on AKT, p-AKT, PDK-1, and caspase cleavage. Flow cytometry was utilized to assess for Annexin V staining following combination therapy. Results:Dose escalation studies demonstrated that doses of tipifarnib up to 5μm demonstrated a significant cell death in HL-60 and HT cells. Perifosine doses of 1–5uM also induced cell death in both HL-60 and HT cells. When apoptosis was assessed using western blot analysis of caspase 3 activity and cleavage, the combination of perifosine and tipifarnib demonstrated significant apoptosis using low doses of both agents. The apoptosis was associated with downregulation of phos-PDK1, with a resultant downregulation in p-AKT. The level of phos-PDK1 was completely inhibited in less than 24 hrs in both the HL-60 and HT cell lines in combination than when either agent was given alone. Conclusion: The combination of perifosine, and AKT targeted agent, with tipifarnib, a Ras targeted agent, appear to induce significant cell death in lymphoma and leukemia cell lines with rapid downregulation of p-AKT via the PDK-1 pathway. This apoptosis occurs in vitro using concentrations well below those that have been achieved in current clinical trials using these agents. Additional studies are being carried out to further delineate the mechanism of synergy as well as to further explore the impact of sequence of administration using this combination. Further studies are also planned to xplore the impact of the combination on primary human leukemia and lymphoma cells from the blood and bone marrow.

Uncovering Cardioprotective Strategies For Anthracycline Therapy By Analysis Of Redox and Apoptotic Effects

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1293-1293
Author(s):  
Daniela E. Egas Bejar ◽  
Joy M. Fulbright ◽  
Fernando F. Corrales-Medina ◽  
Mary E. Irwin ◽  
Blake Johnson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Vitamin E ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Cell Types ◽  
Leukemia Cells ◽  
Soluble Form ◽  
Leukemia Cell Lines

Abstract Anthracyclines are among the most powerful drugs used for the treatment of leukemia, however their use has been associated with cardiotoxicity. Reactive oxygen species (ROS) are generated in both cancer and normal cells after anthracycline exposure and have been implicated in both early and late onset cardiotoxicity. Counteracting this ROS generation are intracellular antioxidants such as the ubiquitous antioxidant glutathione (GSH), levels of which are depleted upon anthracycline exposure. Basal expression of GSH pathway components and other antioxidants vary greatly between different cell types. Due to this differential expression of cellular antioxidants in cardiomyocytes versus leukemia cells, we posit that anthracyclines exert distinct effects on oxidative stress and consequent apoptosis induction in leukemia cells and nontransformed hematopoietic cells (PBMC) relative to cardiomyocytes. As a result, we expect potentially varied mechanisms of cell death induction in these cell lines after anthracycline treatment. To test this hypothesis, the acute leukemia cell lines Jurkat and ML-1 and the cardiomyocyte line H9C2 were used. Dose responses with the anthracyclines, doxorubicin and daunorubicin, were carried out and trypan blue exclusion and propidium iodide staining followed by flow cytometry were used to assess viability and DNA fragmentation respectively. Cardiomyocytes had a 25-150 fold higher IC50 value than the acute leukemia cell lines, indicating selectivity. To assess whether apoptosis was induced by anthracyclines, caspase 3 activity was measured and found to be increased at 24 hours in Jurkat cells which preceded decreases in viability, supporting an apoptotic mechanism of cell death. GSH levels also decreased markedly after 24 hours of treatment with anthracyclines in this cell line, however, a pan-caspase inhibitor did not block GSH depletion, indicating that these events occur independent of each other. To evaluate whether antioxidants conferred protection against loss of viability in all cell types, cells were pretreated for at least 30 minutes with antioxidants and then treated with doxorubicin and daunorubicin for 24 hours. Antioxidants used were N-acetylcysteine (NAC, a GSH precursor and amino acid source), GSH ethyl ester (cell permeable form of GSH), tiron (free radical scavenger) and trolox (a water soluble form of vitamin E). GSH ethylester did not prevent cytotoxicity of anthracyclines in acute leukemia lines or cardiomyocytes. Therefore boosting GSH levels in leukemia cells does not reverse cytotoxicity. Trolox, however, did block anthracycline induced cell death in ML-1 cells, suggesting that vitamin E supplementation would counteract leukemia cell specific effects of anthracyclines on AML cells. Tiron protected PBMC from doxorubicin cytotoxicity but did not protect leukemia cells or cardiomyocytes, hinting at a protective strategy for normal non-leukemia blood cells. Interestingly, NAC did not interfere with the cytotoxic effects of anthracyclines on acute leukemia cells or PBMC, but protected H9C2 cells from daunorubicin cytotoxicity. Taken together, these data reveal differential protective effects of antioxidants in cardiomyocytes and PBMCs relative to ALL and AML cells. Our work indicates that NAC can protect cardiomyocytes without interfering with anthracycline cytotoxicity in acute leukemia cells. In humans, one randomized control trial tested the addition of NAC to doxorubicin therapy, detecting no evidence of cardioprotective activity by chronic administration of NAC. However, the schedule used for administration of NAC in that study may not have been optimal, and biomarkers for oxidative stress reduction by NAC were not incorporated into the trial. Previously, other antioxidants have been used with very limited clinical success and possible contributing factors include inadequate sample size, choice of agent, dose used, duration of intervention and the lack of biomarker endpoints. Designing a cardioprotective and antioxidant strategy with attention to these factors may prove to be efficacious in protecting cardiac cells without interfering with the antitumoral effect of anthracyclines. To this end, our data suggests that trolox and vitamin E analogues should not be used in acute leukemia as they may interfere with the cytotoxic action of anthracyclines but NAC or cysteine may be used as cardioprotectants. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel SAHA-Bendamustine Hybrid Induces Apoptosis of Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2227-2227
Author(s):  
Jing Yu ◽  
Shaowei Qiu ◽  
Qiufu Ge ◽  
Ying Wang ◽  
Hui Wei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Western Blot ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Nb4 Cells ◽  
Leukemia Cell Lines ◽  
Primary Leukemia

Abstract Introduction Hybrid anticancer drugs are of great therapeutic interests as they can potentially overcome the flaws of conventional chemotherapy drugs and improve their efficacy. Histone deacetylase inhibitors (HDACi) and DNA damaging agents have showed synergistic effects in recent studies. In this study, we reported a novel hybrid NL-101 that combines chemo-active groups from suberoylanilide hydroxamic acid (SAHA) and bendamustine, the typical HDACi and alkylating agent respectively.The anticancer effect of NL-101 and its possible mechanisms were investigated in human leukemia cell lines and primary leukemia cells. Methods MTT assay was performed to determine the proliferation of Kasumi-1 and NB4 cells treated with NL-101. Cell cycle distribution and apoptosis rate were detected by flow cytometry. Western-blot analysis was used to analyze the level of acetylated H3 as well as apoptotic-related proteins including γ-H2AX, PARP, caspase-3, Bax, Bcl-2 and Bcl-xL. Bone marrow mononuclear cells of AML patients were isolated by density gradient centrifugation. Wright staining and Western blot were performed to determine the inducing apoptosis effect. Results NL-101 inhibited the proliferation of leukemia cell lines Kasumi-1 and NB4 cells with similar IC50 to that of SAHA. Cell cycle analysis indicated that NL-101 induced S phase arrest. As expected, apoptotic cell death was observed in response to NL-101 treatment. After treatment with 2 µmol/L NL-101 for 48 hours, the apoptosis rate of Kasumi-1 and NB4 cells were (60.19±12.01)% and (49.43±11.61)%, respectively. Western blot analysis showed that NL-101 exposure could induce the accumulation of acetylated Histone H3 and γ-H2AX as the biomarker of DNA double-strand breaks. Anti-apoptotic protein Bcl-xL involved in mitochondrial death pathway was also decreased. Moreover, NL-101 induced apoptosis with a low micromolar IC50 in various leukemia cell lines but not in nonmalignant cell line HEK293. The efficacy of NL-101 was also tested in human primary leukemia cells and all the treated samples exhibited apoptosis confirmed by the morphological examination and expression of apoptotic markers. Conclusions The novel SAHA-bendamustine hybrid NL-101 inhibited the proliferation and induced apoptotic cell death of leukemia cell lines and primary leukemia cells. It presented the properties of both HDAC inhibition and DNA damaging. Down-regulation of Bcl-xL was also involved in the apoptosis induction. These results indicated that NL-101 might be a potential compound for the treatment of leukemia. Disclosures Wang: Bristol Myers Squibb: Consultancy; Novartis: Consultancy.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

Nutlin-3A, an MDM2 Antagonist, Induces p53-Dependent Cell Cycle Arrest and Apoptosis in Hodgkin Lymphoma (HL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2506-2506
Author(s):  
Elias Drakos ◽  
Athanasios Thomaides ◽  
Jiang Li ◽  
Marina Konopleva ◽  
L. Jeffrey Medeiros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Small Molecule ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
P53 Gene ◽  
Cycle Arrest

Abstract p53 is the most frequently mutated tumor suppressor gene in human cancer. However, in Hodgkin lymphoma (HL) p53 is mutated only in a small subset of cases suggesting that modulation of wild-type-p53 (wt-p53) levels in Hodgkin and Reed-Sternberg (HRS) cells may have therapeutic implications in these patients. MDM2 (HDM2 in humans) is a physiologic negative regulator of p53 levels through a well-established auto-regulatory feedback loop. Nutlin-3A is a recently developed small molecule, which antagonizes mdm2 through disruption of p53-MDM2 interaction resulting in p53 stabilization. We hypothesized that nutlin 3A may stabilize p53 in HRS cells carrying wt-p53 gene, thus leading to p53-dependent apoptosis and G1-S cell cycle arrest. We used two novel classical HL cell lines recently established in our Institution, MDA-V and MDA-E, which have been shown to carry wt-p53 gene. As a control, we used a HL cell line L-428 harboring a mutant p53 (mt-p53) gene product (deletion at exon 4). We investigated effects on apoptosis and cell cycle arrest after treatment of cultured HRS cells with nutlin-3A or a 150-fold less active enantiomere, nutlin-3B. Treatment with nutlin-3A resulted in substantial cell death (up to 65%) in a concentration-dependent manner associated with increased apoptosis as shown by apoptotic morphology (DAPI immunofluorescence), annexin V binding (flow cytometry) and caspase activation (Western blot analysis) in MDA-V and MDA-E cells, but not in L-428 cells. Nutlin-3A-induced apoptotic cell death was accompanied by stabilization of p53 protein as detected by western blot analysis and immunofluorescence and up-regulation of pro-apoptotic Bax, a known target of p53. Inhibition of nuclear export by leptomycin B stabilized p53 at a similar level as compared to nutlin-3A treatment in these cells, suggesting that nutlin-3A stabilized p53 through inhibition of MDM2-mediated degradation of the protein. By contrast, no changes in cell viability, growth or apoptosis were seen after treatment with the inactive nutlin-3B small molecule. Treatment with nutlin-3A also resulted in a significant decrease (up to 85%) of cells in S-phase and a dose-dependent increase of cells in G1 phase of cell cycle as detected by flow cytometry, in MDA-V and MDA-E cells, but not in L-428 cells. Cell cycle arrest was associated with up-regulation of the cyclin-dependent kinase inhibitor p21, a transcriptional target of p53. In contrast, treatment of HRS cells with nutlin-3B had no effects on the cell cycle irrespective of p53 mutation status. Furthermore, combined treatment with nutlin-3A and doxorubicin revealed synergistic effects and enhanced cytotoxicity in HRS cells with wt-p53 gene. Targeting MDM2 with the specific antagonist nutlin-3A that leads to non-genotoxic p53 activation, apoptosis induction and cell cycle inhibition may provide a new therapeutic approach for patients with HL.

Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3571-3571
Author(s):  
Matthew F. Clarkson ◽  
Aru Narendran ◽  
Randal N. Johnston
Keyword(s):  
Cell Death ◽  
High Risk ◽  
Cell Viability ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Treatment Strategies ◽  
Leukemia Cells ◽  
Western Blots ◽  
Pediatric Leukemia ◽  
Leukemia Cell Lines

Abstract Abstract 3571 Purpose: Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children. Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design: A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results: Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions: In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures: No relevant conflicts of interest to declare.

Phenotypic, Genotypic, and Functional Characterization Of AML-Derived Endothelial Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1411-1411
Author(s):  
Russell J Pizzo ◽  
Myra Coppage ◽  
Karen Rosell ◽  
Kimberly Morse ◽  
Jane L. Liesveld
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Leukemia Cell ◽  
Normal Subjects ◽  
Rt Pcr ◽  
Altered Expression ◽  
Normal Subject

Abstract Background In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to regulation of hematopoiesis with effects on cell proliferation and survival. Characteristics of marrow—derived endothelial cells from normal subjects have been described (Blood 1994; 84: 10-19), but characterization of endothelial cells in leukemia states is incomplete. Angiogenesis is known to be increased in AML marrows, and circulating endothelial progenitors are increased and correlate with disease status and response to treatment. Furthermore, cytokines secreted by endothelial cells such as vascular endothelial growth factor (VEGF) have been found to serve as growth factors for leukemia, sometimes in a paracrine or autocrine fashion. Despite these findings, inhibition of VEGF with agents such as bevacizumab has not demonstrated clinical anti-leukemia activity. Since our group and others have shown that endothelial cells from multiple vascular beds (human umbilical vein endothelial cells—HUVECs), human microvascular endothelial cells derived from skin (HMEC-1 cell line), and normal subject—derived endothelial cells are able to prevent spontaneous or therapy-induced apoptosis in AML blasts, it is important to understand the phenotype and characteristics of endothelial cells isolated from AML patients to understand their functional roles and to see if they might have an angiogenic gene expression profile as has been described in multiple myeloma (Clin Cancer Res 2009 15:5369). Methods Endothelial cells were purified from marrow aspirates obtained with consent from normal subjects or from newly diagnosed AML patients. Cells were isolated using anti-CD105-PE (BD Bioscience) followed by anti-PE microbead selection (Miltenyi™) or after disruption of marrow spicules with subsequent selection for endothelial cells in endothelial cell selective medium (EGM-2, Lonza). Cells between 2nd and 4th passage were utilized for analysis. Protein expression was determined by flow cytometry, Western blotting, or RT-PCR. Matrigel™ tubule formation and acetyl-LDL expression were determined as per previously published methods, as were adhesion, CFU-L, and transmigration assays. RNASeq was performed by the Functional Genomics Core at the University of Rochester after extraction of polyadenylated RNA from purified total RNA. Conversion to cDNA occurred with the Illumina TruSeq™ preparation kit, and sequencing was accomplished with the Illumina Genome Analyzer IIx. CASAVA software was utilized for analysis. Results Marrow derived endothelial cells from normal and AML subjects express CD105 (endoglin), CD31(PECAM), CD106 (VCAM), CD146 (MCAM), CD54 (ICAM), and CD34. They do not express CD14 nor CD45, and they demonstrate low level expression of CD144 (VE-cadherin). By RT-PCR, they express Tie-2, VEGF, and eNOS (endothelial nitric oxide synthase). They express acetyl-LDL and form tubular structures in Matrigel™. Phosphorylated components of the mTOR and PI3K/Akt pathways were also expressed by Western blot analysis. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and CFU-L outgrowth, but no significant differences were noted in these functions between normal and AML—derived endothelial cells in vitro assays. RNASeq analysis revealed 130 genes significantly up—or down—regulated in AML derived endothelial cells as compared with those derived from normal marrow. Endothelial cells from both sources had a distinct signature from marrow—derived fibroblasts. The genes differentially expressed (p<0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos, and this upregulation in AML vs. normal subject derived endothelial cells was confirmed by Western blot analysis. Genes involved in chemotaxis such as CXCL16 were also upregulated. Conclusions AML—derived endothelial cells exhibit similar phenotype and function as their normal marrow—derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

JQ1, a Potential Therapeutic Molecule for Myeloid Leukemia with PTEN Deficiency

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5899-5899 ◽  
Author(s):  
Nicholas J Baltz ◽  
Natalia C Colorado ◽  
Yan Yan ◽  
Shelly Lensing ◽  
Delli Robinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Colony Formation ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Jak Inhibitors ◽  
Facs Analysis ◽  
Leukemia Cell Lines

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy that continues to have high relapse and treatment-related mortality rates, despite recent advances in clinical management and therapy. Janus kinase (JAK) inhibitors inhibit the activity of the JAK/STAT pathway and have demonstrated some clinical responses in AML patients. However, survival analysis suggests that more than half of AML patients do not benefit from treatment with JAK inhibitors. Furthermore, PTEN deficiency is frequently found in patients in the late stages of cancer, which causes hyperactivated AKT and MAPK pathways. However, emerging data suggests that leukemia cells with PTEN deficiency are resistant to MAPK inhibitors. Over the past decade, it has been demonstrated that dysregulated epigenetics play an important role in myeloid leukemogenesis. The bromodomain and extraterminal domain (BET) family includes adaptor proteins Brd2, Brd3, Brd4, and Brdt that regulate gene expression via binding to acetylated chromatin and subsequently activating RNA Polymerase II driven transcriptional elongation, resulting in the promotion of gene expression. BRD4 is a BET protein required for disease maintenance in AML. JQ1 is a small molecule that interferes with transcriptional regulators, such as BRD4, by preventing them from interacting with acetylated regions of the genome and thus inhibiting the transcriptional activation of BRD4 target genes. Prior research in lymphocytic leukemia cell lines suggests that JQ1 also decreases STAT5-dependent gene transcriptional activities. We hypothesize that the inhibition of BET proteins may correct the over-activated transcriptional activities in myeloid leukemia cells and induce disease regression. We tested our hypothesis in PTEN deficient myeloid leukemia cell lines, TF-1a and K562, and used human cord blood mononuclear cells (CB) for normal cell comparison. Methods: 1) To test whether JQ1 can inhibit colony formation, we seeded cells on 0.3% agar and McCoys' 5A medium supplemented with nutrients and 15% fetal bovine serum, without cytokines, and added JQ1 diluents to the cultures at concentrations of 32.5-1000nM overnight after the cultures were established. 2) To test whether JQ1 can inhibit leukemia cell proliferation, we cultured cells in liquid medium with JQ1 for 48-72 hours, and quantified the viable cells using alamarBlue® assay. 3) To investigate whether JAK/STAT5 activity is altered by JQ1 in leukemia cells, we quantified phosphorylated STAT5 (pSTAT5) in cells via flow cytometry and western blot. We treated the cells with JQ1 at various concentrations for 2 hours and then stimulated the cells for 15 minutes in medium with 0.5% BSA and 10ng/mL GM-CSF prior to staining the cells with anti-pStat5 (pY694) antibody conjugated with Alexa Fluor® 647 for FACS analysis or lysing the cells for western blot analysis. Results: In the colony formation assay, we found that TF-1a cells were more sensitive to JQ1 than the CB cells and K562, with an IC50 of 62.5-125 nM for TF-1a cells (p<0.0001), and 250-500nM for both CB and K562 cells, respectively. Proliferation assay results also supported that TF1a cells are sensitive to JQ1 with an IC50 of 125-250nM, whereas neither CB nor K562 reached the IC50 in the tested concentration range. This suggests that the IC50 of JQ1 for TF1a cells is achievable at concentrations that are mostly nontoxic to normal CB cells, but K562 cells are not sensitive to JQ1. FACS analysis revealed that pSTAT5 is constitutively activated in K562 cells but not in TF-1a cells. Interestingly, the levels of pSTAT5 in both TF-1a and K562 cells were not altered by JQ1 treatment at tested concentrations, which was confirmed by western blot. Conclusions: Our data suggest: 1) JQ1 and other bromodomain inhibitors could be potential therapeutic molecules for selected myeloid leukemias; 2) JQ1 inhibition on colony formation and proliferation in TF-1a cells is not pSTAT5 related. Further studies are underway to test whether JQ1 is effective in primary mouse leukemia cells with Pten deficiency. Disclosures No relevant conflicts of interest to declare.

C-jun N-terminal kinase dependent autophagic cell death in cancer cells induced by Zanthoxylum fruit extract from Japanese pepper.

2017 ◽  
Vol 35 (4_suppl) ◽  
pp. 653-653
Author(s):  
Toru Kono ◽  
Reo Nozaki ◽  
Hiroki Bochimoto ◽  
Tsuyoshi Watanabe ◽  
Kaori Oketani ◽  
...  
Keyword(s):  
Cell Death ◽  
Drug Development ◽  
Western Blot ◽  
Anticancer Activity ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Fruit Extract ◽  
Cytoplasmic Vacuolization

653 Background: Natural products constitute a promising resource for drug development including an anticancer drug. Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) is an important component of Daikenchuto, which is a form of Japanese traditional medicine. Recently, we have reported that Daikenchuto has an anticancer activity in vivo, however precise mechanism is still unclear. Therefore, we investigated the potential anticancer activity of ZFE as an inducer of autophagic cell death (ACD). Methods: ZFE powder was provided by Tsumura (Japan). We investigated the effect of ZFE on the morphology of six types of human cancer cells and normal cells by using phase contrast microscopy and electron microscopy. Knockdown of autophagy-related gene 5 (ATG5), which is an essential gene for autophagy, by transfecting small interfering RNA was performed and confirmed by quantitative RT-qPCR and Western blot analysis. Effect of bafilomycin A1 (Baf A1), an inhibitor of vacuolar type H+-ATPases, on the anticancer activity of ZFE was investigated. Western blot analysis revealed LC3-II levels, a marker of autophagy. Results: ZFE caused remarkable autophagy-like cytoplasmic vacuolization with the inhibition of cell proliferation and subsequent induction of cell death in human cancer cell lines, DLD-1, HepG2 and Caco-2 cells but not in A549, MCF-7 or WiDr cells. ZFE increased LC3-II protein levels. Suppression of an ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, ZFE increased the phosphorylation of c-jun N-terminal kinase (JNK) in cancer cells which can be induced cell death by ZFE and JNK inhibitor SP600125 attenuated both vacuolization and cell death induced by ZFE. Instead, ZFE-induced cell death was neither apoptosis nor necrosis according to the morphological perspective and the marker of apoptosis or necrosis. And normal intestinal cell was not affected by ZFE. Conclusions: ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

Sign in / Sign up

Export Citation Format

Share Document