Panobinostat (LBH589) a Promising New Partner for Combination with Doxorubicin in Acute Myeloid Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1638-1638 ◽  
Author(s):  
Patricia Maiso ◽  
Enrique Colado ◽  
Enrique M Ocio ◽  
Mercedes Garayoa ◽  
Peter Atadja ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Cell Population ◽  
Mechanism Of Action ◽  
Drug Combination ◽  
Ex Vivo ◽  
Annexin V ◽  
Hel Cells ◽  
Acute Myeloid

Abstract Background and Aims: The combination of cytarabine with an anthracyclin has been the gold standard for the induction treatment of acute myeloid leukaemia (AML) for the last three decades. Nevertheless, 20–50% of patients fail to respond to this scheme and among those who achieve complete response (CR) the relapse rate come close to 50%. In the present study, we have investigated the potential value of panobinostat (LBH589) for the treatment of AML. This drug has already demonstrated antileukemic activity. Nevertheless, since there is a large body of evidence indicating that AML treatment requires drug combination, we analysed the potential synergism of panobinostat with other well established anti-AML agents. Material and Methods: The efficacy of panobinostat and of its combination with each one of three other agents (cytarabine, doxorubicin and fludarabine) was analyzed both in vitro, (in four AML cell lines: HEL, HL-60, KG-1 and MV4.11, by MTT) and ex vivo (in freshly isolated cells from 6 AML patients by flow cytometry). In addition the toxicity in normal hematopoietic cells was analyzed. The mechanism of action was investigated by Annexin V, cell cycle profile, DioC6 staining, Western Blot and gene expression profile (GEP) analysis by microarrays. Results: Panobinostat potently suppressed the viability of AML cells. IC50 values were 9 nM (HEL), 7 nM (HL-60), 17 nM (KG1), and 6 nM (MV4-11). Comparison of the IC50 of panobinostat with other drugs commonly used in AML, indicated that panobinostat was more potent than cytarabine, fludarabine, and doxorubicin (IC50 = 740 nM, 362 nM and 220 nM respectively, for HEL cells). Panobinostat increased the anti-AML effect of cytarabine and fludarabine. Nevertheless, the most significant effect was observed for the combination with doxorubicin. The CI range values were 0.05–0.41 in all cell lines, and accordingly, remaining experiments focused on this combination. This efficacy was also confirmed in ex vivo experiments. Using quadruple staining (annexin V-FITC/CD33-PE/CD45-PerCP/CD34-APC), we identify and distinguish the blast cell population (CD34−/+, CD33−/+, CD45dim) from the normal residual lymphocytes (CD45+, SSClo) and quantify the number of apoptotic cells in each cell population. In all six cases a potentiation was observed with P+D. Interestingly no effect was observed in terms of toxicity on non leukemic residual hematopoietic cells from the same patients’ samples. An important question to be asked, upon using drug combinations, is whether the genes deregulated by the combination just represent the sum of those targeted by each of the drugs or if the drug combination is inducting new targeting pathways. In order to answer this question we compared the GEP of HEL cells exposed to the P+D with those as single agents. While there were 285 genes deregulated with panobinostat and 43 with doxorubicin after 24 hours with each drug; 12 hours of treatment with P+D resulted in the deregulation of 841 genes. Accordingly, 588 genes were exclusively deregulated after P+D treatment, indicating that panobinostat and doxorubicin affect different groups of genes and pathways. The two most significantly deregulated functional categories were genes involved in the control of cell cycle and apoptosis. Treatment with P+D down-regulated Cyclin B1 (−3.04), AVEN (−2.58), Bcl-X (−2.96), TNFRSF25 (−3.48) or HSPA5 (−3.87), and up-regulated the levels of Cyclin G2 (5.49), BTG1 (5.47), or BNIP3L (2.17). c-jun was upregulated after treatment with panobinostat (3.77) and particularly with doxorubicin (26.30), and the upregulation was even higher upon treatment the AML cells with the combination of both drugs (58.38). Mechanistic experiments showed that P+D activated apoptosis (Annexin V staining and PARP- and caspases-cleavage by Western Blot) at concentrations that did not induce any cytotoxic effect when panobinostat and doxorubicin were used as single agents. P+D activated the intrinsic pathway of apoptosis with loss of mitochondrial membrane potential and subsequent release of Cytochrome C to the cytoplasm. A decrease in MCL-1 and BCL-X cleavage was also observed with the combination while not with the single agents. Interestingly, P+D also induced cell cycle arrest. Conclusion: Panobinostat + doxorubicin show a marked synergistic activity against AML cells, with unique mechanism of action, and represent a most attractive combination for clinical investigation.

Expression and Role of APP Gene in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4691-4691
Author(s):  
Fanyi Meng ◽  
Wei Wang ◽  
Zoufang Huang ◽  
Ming Huang ◽  
Lixiang Liu
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Western Blot ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
App Gene ◽  
Acute Myeloid

Abstract Abstract 4691 Introduction Amyloid precursor protein(APP) gene was increasingly expressed in solid tumors, promoted the proliferation of tumor cells and the overexpression of APP was a bad prognostic factor to oral squamous cell carcinoma. However, little has been known about the clinical significance and role of APP in acute myeloid leukemia(AML). Methods The expressions of APP mRNA in 85 AML patients and 20 nonmalignant hematological diseases that worked as control were measured by real-time PCR and the expressions of APP in AML cell lines were examined by real-time PCR and western blot. Small interfering RNAs(siRNAs) targeting APP gene were synthesized and transfected into HL60 cell by lipofectamine2000, after RNAi 24h, 48h and 72h, cell growth of HL60 was measured by trypan blue dye exclusion method and MTT, differentiation was observed by Wright-Giemsa staining, cell cycle was examined by PI/RNase staining, apoptosis induction was analyzed by Annexin V/PI and Hoechst33342 staining; apoptosis-related proteins NF-κB, bcl-2 and Caspase-3 were detected by Western blot after RNAi 48h; sensitivity of HL60 to adrimycin was measured by MTT. Results The expression of APP mRNA among AML subtypes was significantly different(P=0.019), M2 with t(8;21) was the highest expression subtype and M5b was the lowest. APP expression had no significant effect on AML clinical characteristic excepting AML subtypes. kasumi-1 was the highest expression cell in AML cell lines and U937 was the lowest(P<0.05), and the expression of APP in HL60/ADM was significantly lower than HL60(P<0.05). The APP expressions in AML cell lines was in agreement with its expressions in primary AML subtypes. After RNAi 24h, 48h, and 72h, no significant differences in proliferation, differentiation, apoptosis, cell cycle and sensitivity of HL60 to adriamycin were detected between interfering group and control groups. Conclusions The APP mRNA expression in M2 with t(8;21) was high and M5b was low. Down-regulation of APP expression had no significant effect on biological behaviour of HL60 and APP was not tightly related to pathogenesis of AML. Disclosures: No relevant conflicts of interest to declare.

Activation of the EfnB1/EphB1 Forward Signaling Promotes Cell Cycle Arrest in Acute Myeloid Leukemia Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4719-4719
Author(s):  
Kim R Kampen ◽  
Arja ter Elst ◽  
Evelina S. De Bont
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Annexin V ◽  
Cycle Arrest ◽  
Apoptosis Assay ◽  
Ligand Stimulation ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Abstract 4719 Ephrin signaling has been shown to contribute to the pathogenesis of many solid tumors with respect to tumor growth, tumor cell survival, angiogenesis, and metastasizing capacity (Cytokine Growth Factor Rev. Dec;15(6):419-33, et al. Neuro Oncol. 2012). Recently, an aberrant DNA methylation status of ephrin receptors and ligands was described to be associated with outcome in acute lymphoblastic leukemia (Blood.2010 Mar 25;115(12):2412-9). In acute myeloid leukemia (AML), we found an intriguing heterogeneity in membrane receptor expression levels of EphB1. Therefore, we challenged to evaluate the role of EphB1 receptor forward signaling in AML. We investigated the influence of the EphB1 receptor forward signaling in THP-1 (EphB1high), HL60 (EphB1int), and MOLM13 (EphB1low) AML cell lines through exogenous stimulation with the EphB1 ligand; EfnB1. EfnB1 stimulation of the AML cell lines demonstrated to reduce AML growth solely in the EphB1high and EphB1int cell lines (Fig. 1A,P = 0.001 and P =.056). In addition, EfnB1 stimulation induced apoptosis most pronounced in the EphB1high cells (Fig. 1B). Interestingly, MGG stained cytospins of EfnB1 treated THP-1 showed multinucleation of AML cells (Fig. 1C). We hypothesized that these phenotypic effects could be assigned to cell cycle arrest in THP-1 cells. Additionally, cell cycle regulatory proteins CDC2 and CyclinB1 were evaluated by immunoblotting of EfnB1 stimulated THP-1 cells. Phosphorylation of the inactivating CDC2 Tyr15-site demonstrated to be up-regulated in EfnB1 stimulated THP-1 cells, which might be initiated by the increased total CDC2 protein levels that we found (Fig. 1D). CyclinB1 displays enhanced protein expression in EfnB1 treated THP-1 cells. Moreover, quantitative RT-PCR analysis showed that the expression of cell cycle inhibitor p21 is significantly induced by 3-fold in EfnB1 stimulated THP-1 cells, via increasing levels of p53 (Fig. 1E, both P = <0.001). To verify whether the EfnB1 induced cell cycle arrest is EphB1 specific, we enforced EphB1 expression in HL60 EphB1int and MOLM13 EphB1low AML cells by introducing a GFP fused EphB1 overexpression construct. EphB1 overexpression increased EphB1 protein expression levels sufficiently in both AML cell lines, as confirmed by flowcytometric analysis and immunoblots. Exogenous EfnB1 stimulation further increased the apoptosis in EphB1 overexpressing cells in both AML cell lines (Fig. 1F). Again, we found increasing levels of phospho-CDC2Tyr15 and CyclinB1 by immunoblots. From this study, we conclude that AML cells with high EphB1 expression can be forced into a cell cycle arrest upon ligand binding in vitro, while AML cells lacking EphB1 expression have a proliferative and anti-apoptotic survival advantage. The clinical significance and exploitation of EphB1 induced cell cycle arrest in AML will be analyzed in the near future. Figure 1. EfnB1 induced activation of the EphB1 in AML cell lines promotes cell cycle arrest and apoptosis Figure 1. EfnB1 induced activation of the EphB1 in AML cell lines promotes cell cycle arrest and apoptosis (A) Absolute cell counts represent the growth inhibitory effects of EfnB1 ligand stimulation in AML cell lines THP-1 and HL60. (B) Flowcytometric Annexin V/PI apoptosis assay displayed the induction of apoptosis as a result of EfnB1 ligand stimulation in THP-1 and MOLM-13 AML cells. (C) MGG stained cytospins of EfnB1 treated THP-1 cells promotes the induction of multinucleated cells due to cell cycle arrest. (D) Immunoblots showed an enhanced apoptotic BAX/BCL2 ratio, in synergy with an upregulation of cell cycle inactivating checkpoint kinase CDC2Tyr15 upon EfnB1 stimulation in THP-1. (E) qRT-PCR confirms cell cycle inhibition by a 3-fold upregulation of p21 and a 1.5-fold induced expression of p53 in THP-1 EfnB1 stimulated cells. (F) The flowcytometric Annexin V/PI apoptosis assay showed that EfnB1 ligand induced apoptosis is even further induced in EphB1 overexpressing HL60 and MOLM-13 cells. Disclosures: No relevant conflicts of interest to declare.

Prima-1Met Combined with Bortezomib Has Synergistic Anti-Myeloma Activity By Modulation of Apoptosis and Cell Cycle Regulating Genes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4213-4213
Author(s):  
Priya Khoral ◽  
Robert J Guo ◽  
Jahangir Abdi ◽  
Hong Chang
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Western Blot ◽  
Real Time ◽  
Cell Lines ◽  
Blot Analysis ◽  
Drug Combination ◽  
Western Blot Analysis ◽  
Rt Pcr ◽  
Novel Therapeutic

Abstract INTRODUCTION Multiple Myeloma (MM) is a plasma-cell malignancy characterized by dismal prognosis and a high level of relapse, thus novel therapeutic approaches are needed. PRIMA-1Met is a novel small molecule showing anti-tumour activity and currently in clinical phase I-II trials. We recently demonstrated that PRIMA-1Met has potent anti-MM activity in vitro and in vivo. Bortezomib (BTZ) is a proteasome inhibitor that has been successfully used for treating some cases of relapsed MM. The aim of the current study is to determine whether PRIMA-1Met could be used in combination with BTZ to enhance the cytotoxic effects in myeloma cells. METHODS Using three different MM cell lines (LP1, U266 and 8226), we established dose response curves for both PRIMA-1Met and BTZ, and tested drug cytotoxicity using MTT assays. We then tested drug cytotoxicity of a range of concentrations of the drugs in combination. The Chou Talay method was used to determine whether or not the drug combinations were synergistic. A gene expression array was used to investigate the mechanism of the drug combination's effects. Total RNA was isolated from MM cell pellets, then synthesized cDNAs were applied to real time RT-PCR gene expression arrays containing 84 genes of interest. The genes selected were involved in apoptotic as well as cell growth and proliferation pathways. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in both drug treated and control samples using the 2-ΔΔCt algorithm. Western blot analysis was used to further investigate proteins of interest. RESULTS Cell viability of 8226, LP1 and U266 cells treated with individual concentrations of PRIMA-1Met (10uM) and BTZ (10nM) was on average 65%, 45% and 72.5%, respectively. However, combination of above doses reduced viability to 20% in 8226 and LP1, and to 40% in U266. The Chou Talay method identified this drug combination as synergistic in 2 out of the three tested cell lines, with Combination Index (CI) values of 0.72 in 8226 and 0.582 in U266. The gene expression analysis in real time RT-PCR indicated that the drug combination resulted in downregulation of genes involved in cell cycle and proliferation (CCND1, CDK4, CDK6, CDK2, IGFIR), genes from the Bcl-2 family of apoptosis regulation (Bcl-2, Bcl-XL, Mcl-1), as well as MDM2 from the p53 signalling pathway, and MYC, which is involved in both apoptosis and cell cycle progression. Western blot analysis revealed up-regulation of cleaved caspase-3 and -9, implying involvement of the intrinsic apoptotic pathway in the drug combination's activity. CONCLUSION Our results reveal that PRIMA-1Met synergistically enhances the anti-MM effect of BTZ, leading to a significantly higher level of MM cell death. Real time RT-PCR gene array analysis offers some insight into the mechanism of this combination's effect, implicating apoptotic, cell cycle and growth regulating genes. Our study provides framework for further evaluation of this drug combination as a novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinical Impact and Therapeutic Opportunity of Insulin Receptor Substrates 1/2 in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1512-1512
Author(s):  
Juan Luiz Coelho-Silva ◽  
Diego Antonio Pereira-Martins ◽  
Josiane Lilian Schiavinato ◽  
Eduardo Magalhães Rego ◽  
João Agostinho Machado-Neto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Cell Viability ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Annexin V ◽  
Ros Production ◽  
Insulin Receptor Substrates ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Background: The identification of biological and clinical prognostic factors in acute myeloid leukemia (AML) allowed the definition of patient subgroups and the realization of risk-adapted and targeted treatment strategies. Insulin-like growth factor 1 receptor/Insulin Receptor Substrates (IGF1R/IRS) pathway plays an important role in the development of neoplasia. IRS1/2 activates AKT/mTOR and MAPK pathways, through their interaction with PI3K and GRB2, culminating in increasing cell proliferation. NT157 is an allosteric inhibitor of IGF1R-IRS1/2 signaling that showed antineoplastic effects in preclinical studies of solid tumors. However, IRS1/2 clinical function and NT157 effects were not assessed in AML. Aims: To investigate IRS1 and IRS2 mRNA expression in AML patients and their impact in clinical outcomes, and to analyze the effects of the NT157 in AML cell lines. Material and methods: Comparison of IRS1 (probe nº 204686) and IRS2 (probe nº209184_s) expression from 581 AML patients and 8 CD34+ cells from healthy subjects were analyzed using data from Amazonia! Platform. For survival analysis, IRS1 and IRS2 mRNA expression levels from 173 AML patients (92 male - median age 58 years [range: 18-65]) were obtained from TCGA AML study available online on CBioPortal for Cancer Genomics. NB4, NB4-R2, Kasumi-1 and THP1 cell lines were submitted to NT157 (0.5, 1.0, 2.0, 4.0, 8.0 or 16 µM) 72 hours and evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), cell cycle (PI), ROS production (DCFDA), mitochondria staining (MitoTracker), and protein expression/activation (western blot). Bone marrow mononuclear cells (BMMC) were obtained from 4 AML patients at diagnosis and submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA, Mann-Whitney or Kruskal-Wallis and Spearman correlation tests, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with the log-rank test. Cox regression analysis was also applied. Results: IRS1 expression, but not IRS2, predicted outcomes. Reduced IRS1 expression showed poorer disease-free survival (DFS) (survival median time [MT]: 10.1 months [mos] vs. 28.4 mos, P<0.001; Hazard ratio [HR]: 0.51 [CI95:0.32 - 0.79]) and overall survival (OS) (MT: 14.5 mos vs. 27.4 mos, P=0.009; HR: 0.61 [CI95:0.42 - 0.88]). IRS1 expression independently predicted poorer DFS (HR: 0.59 [CI95: 0.36 - 0.79]; P= 0.03) using cytogenetic risk stratification, age and leukocytes as confounders. Of note, IRS1 level was positively correlated with proapoptotic CD27 (r=0.51; P<0.001) and with IL17RA (r=0.62; P<0.001) related to CD34 cell differentiation. IRS2 expression was upregulated in AML harboring t(15;17) (n=36; P<0.01) and inv(16) (n=37; P=0.01) in comparison to CD34+. In NB4, NB4-R2 and Kasumi-1 cells, NT157≥0.5µM reduced cell viability (P<0.05) and increased apoptosis (P<0.05). The mean percentage of annexin V+ cells for control, NT157 2.5, 5 and 10µM were 11, 47, 73 and 75% for NB4, 11, 41, 69 and 75% for NB4-R2 and 17, 45, 61 and 64% for Kasumi-1, respectively. In TP53-null cell line THP1, NT157 reduced cell viability at doses higher than 2µM (P<0.05) and induced apoptosis at 10µM (9.1 vs. 25%; P<0.05). NT157 induced ROS production in NB4 (fold-increase of mean fluorescence intensity [MFI]: 25.8 and 24.8), NB4-R2 (MFI: 26.7 and 31.4), Kasumi-1 (MFI: 5.8 and 6.6) and THP1 (MFI: 1.8 and 4.1) at 5 and 10µM (all P<0.05) and increased mitochondrial mass in NB4 (MFI: 3.9 and 3.7), NB4-R2 (MFI: 2.6 and 2.9), Kasumi-1 (MFI: 3.2 and 4.7) and THP-1 (MFI: 2.6 and 2.2) (all P<0.05). NT157 also modulated cell cycle progression, as evidenced by G2/M arrest in THP-1 and sub-G0/G1 in other cell lines (P<0.05). The IGF1R-IRS1/2 inhibitor NT157 reduced activation/expression of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70), ERK1/2 (Thr185/Tyr187) and induced DNA damage (increased γH2AX). NT157 did not induce autophagy, as demonstrated by non-degradation of p62 and lack of conversion of LC3BI into LC3BII in cell lines tested. NT157≥0.5 µM reduced cell viability and induced apoptosis in BMMC from 4 AML patients in a dose dependent manner (P<0.05). Conclusions: In AML, downregulation of IRS1 predicted dismal prognosis and the IGF1R-IRS1/2 inhibitor NT157 exerted an antineoplastic activity, downregulated PI3K/AKT and MAPK signaling. IRS1/2 arises as a promising therapeutic target for AML patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals BCL-2 Inhibitor Venetoclax Enhances Temozolomide Sensitivity in AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2646-2646
Author(s):  
Joseph M Brandwein ◽  
Asmaa Basonbul
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Drug Combination ◽  
Research Funding ◽  
Annexin V ◽  
Complete Response ◽  
Strand Break ◽  
Single Strand Break ◽  
Mgmt Expression

Introduction: Many older patients with acute myeloid leukemia (AML) are ineligible for intensive chemotherapy due to frailty and co-morbidities; for such patients, existing treatments are often ineffective and new treatments are needed. Temozolomide (TMZ) is an alkylating agent that causes DNA methylation at O6 guanine, generating single strand break leading to apoptosis. However, the efficacy of TMZ depends on the DNA repair protein O6-methylguanine methyltransferase (MGMT), that maintains the genomic integrity by removing the O6-methyl group and restoring guanine nucleobase, thereby enhancing resistance to TMZ. Previous clinical trials in AML found that responses to TMZ correlated with low MGMT expression; however, even in those with low MGMT expression complete response rates were only in the 25% range. BCL-2, an anti-apoptotic protein, is overexpressed in AML cells. Direct inhibition by the selective BCL-2 inhibitor venetoclax (Venet) promotes apoptosis. This study evaluated the ability of Venet to enhance TMZ sensitivity in AML cells, including those with MGMT overexpression. Methods: KG1, MV4-11 and MOLM13 AML cell lines were studied, as well as bone marrow blast cells collected from AML patients. Western blot was used to measure MGMT and BCL-2 expression. The cells were incubated with TMZ at varying concentrations in combination with a fixed concentration of Venet. After 48 hours, cell viability and apoptosis assays were performed using spectrophotometry and flow cytometry, respectively. Synergy was evaluated by the Chou-Talalay method. Cleaved-PARP was measured by Western blot in selected combination doses after 3 hours in MV4-11 and MOLM13 and after 6 hours in KG1. Results: KG1 cells expressing high MGMT demonstrated strong resistance to TMZ; however, co-incubation with 1 uM Venet resulted in a marked enhancement of sensitivity to TMZ. Similarly, in MV4-11 and MOLM13 cell lines, which demonstrated very low or absent MGMT expression. Venet 2.5 nM in combination with TMZ markedly increased the cytotoxicity to TMZ. A synergistic effect was demonstrated in all cell lines with combination index (CI) < 1. Cells overexpressed annexin V and propidium iodide (PI) apoptotic marker after drug combination in all cell lines. Apoptotic effect with the drug combination was verified by cleaved-PARP expression. Most (6/8) AML patient samples which were resistant to TMZ in vitro became sensitized to TMZ in combination with 1 uM Venet, including those with moderate to high MGMT expression. Conclusion: Venetoclax synergizes with TMZ and induces cytotoxicity in all AML cell lines and in most AML patient samples, including those in whom MGMT was highly expressed, by activating apoptotic pathways to trigger cell death. This combination represents a potentially promising new treatment. Further studies evaluating this combination in animal models are in progress. Disclosures Brandwein: Roche: Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria; Jazz Pharma: Consultancy, Honoraria. OffLabel Disclosure: Temozolomide for AML

Raf-1 Is Over Expressed in Chronic Lymphocytic Leukemia and Promotes Cell Survival by Phosphorylation of ERK and BAD

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3120-3120 ◽  
Author(s):  
Miao Wang ◽  
P.M. Kluin ◽  
Stefano Rosati ◽  
Marjan Luinge ◽  
Simon M. G. J. Daenen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Western Blot ◽  
Cell Survival ◽  
Cell Lines ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Cyclin D3 ◽  
Qrt Pcr ◽  
Mrna Ratio

Abstract Introduction: The serine threonine kinase Raf-1 plays a protective role in many cell types, but its expression and function in CLL cells has not been studied in detail. In the present study, we analyzed Raf-1 expression and tested the hypothesis that Raf-1 is critical for CLL cell survival. Materials and Methods: By using qRT-PCR and western blot, we compared the expression of Raf-1 of mRNA and protein levels in purified B cells from 45 CLL cases and CD38 negative B cells from 5 reactive tonsils. By western blot, we analyzed the activity of phospho-Raf-1 (ser338) and its downstream targets (phospho-ERK1/2, phospho-BAD) in 23 CLL cases and 4 CLL cell lines (JVM-3, MEC-1, MEC-2 and MO1043) before and after IgM stimulation. By immunoprecipitation, we analyzed if Raf-1 co-localizes with Bcl-2. We correlated the change in phosphorylation status (Raf-1, ERK and BAD) in response to IgM stimulation with the ZAP-70/SYK mRNA ratio, which was detected by qRT-PCR in purified B cells from CLL cases. After using specific inhibitors, including the Raf-1 inhibitor GW5074, the Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137, we investigated apoptosis by Annexin V flowcytometry in CLL cases and CLL cell lines as well as cell cycle changes in the 4 cell lines by flowcytometry. Results: In comparison to normal resting (CD38 negative) B cells, there was a strongoverexpression of Raf-1 in CLL cells, both at at the mRNA and protein level. Using qRT-PCR there was an almost linear correlation between Raf-1 and Bcl-2 expression. Moreover, the phosphorylation status of Raf-1 and ERK in response to IgM stimulation strongly correlated with the ZAP-70/SYK mRNA ratio. Using immunoprecipitation and confocal miscroscopy we found colocalization of Raf-1 with Bcl-2, which might account for the observed constitutive activation of BAD in CLL cells. The Raf-1 inhibitor GW5074, Raf-1 destabilizer Geldanamycin and Bcl-2 inhibitor YC137 all led to p-Raf-1 inhibition as well as downregulation of p-ERK and p-BAD. Additionally, all three inhibitors downregulated cyclin D3 and cyclin E, which are important for G0/G1 transition. We also found that GW5074 induced apoptosis in CLL cell lines and primary cells of CLL cases. Conclusion/Discussion: In conclusion, our study identifies Raf-1 as a critical anti-apoptotic and cell cycle regulating kinase in CLL cells.

The Hybrid Molecule, Edo-S101, Impairs Double Strand Breaks Repair in Multiple Myeloma and Synergizes with Bortezomib and Dexamethasone

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5354-5354 ◽  
Author(s):  
Ana Alicia López-Iglesias ◽  
Ana Belen Herrero ◽  
Laura San-Segundo ◽  
Susana Hernández-García ◽  
Lorena González-Méndez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Comet Assay ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Ex Vivo ◽  
Hybrid Molecule ◽  
Cycle Arrest

Abstract Introduction. EDO-S101 is a hybrid molecule of bendamustine plus vorinostat, new in its class. Our group has previously demonstrated that EDO-S101 is effective in vitro in MM cell lines independently of p53 state, and also in a murine plasmacytoma model where it decreases tumor growth and prolongs survival with respect to bendamustine and/or vorinostat treatment. The objective of this work was to gain further insights into the efficacy of EDO-S101, its mechanism of action and its combination with other drugs used in MM. Methods. The mechanism of action was assessed by western blot, comet assay, immunohistochemistry, and flow cytometry. Homologous recombination (HR) efficiency was calculated using chromosomally integrated green fluorescent protein reporter construct-based assay. The efficacy of different combinations was studied in vitro (HMCLs), in vivo (murine plasmacytoma model CB-17 SCID mice) and ex vivo (cells from patients). Results. In addition to the activity of EDO-S101 in MM cell lines we demonstrated that it was active ex vivo in cells isolated from 7 MM patients, with median IC50 of 5 µM (ranging from 1,8 to 8 µM), some of them previously exposed and resistant to alkylators such as melphalan. Interestingly, EDO-S101 could also overcome alkylators-resistance in vitro, as it was active in melphalan resistant cells (U266-LR7 and RPMI8226-LR5). EDO-S01 was also effective in the presence of factors that confer proliferative advantage to plasma cells, like IL-6, IGF or co-culture with mesenchimal cells hMSC-TERT. Regarding its mechanism of action, we found that the apoptosis induced by EDO-S101 was caspase-independent but calpain-dependent, since PD150606, an inhibitor of this protein could overcome EDO-S101-induced apoptosis, whereas the caspase inhibitor Z-VAD -FMK did not. This data was consistent with the finding that under treatment with EDO-S101, MM1S cells showed AIF (apoptotic inducing factor) translocation from the mitochondria into the nucleus. Interestingly, the release of this pro-apoptotic protein from the mitochondria could be mediated by calpains, as it has been described in literature. We subsequently demonstrated that EDO-S101 causes DNA damage, as revealed by the phosphorylation and subsequent activation of several components of the DNA Damage Response (DDR) such as ATM, H2AX, chk1, chk2 or p53, and the induction of DNA fragmentation, that was detected by the comet assay. EDO-S101 was also found to induce cell cycle arrest in different phases depending on the dose and cell line. It has previously been suggested that DACi may impair DNA repair by inhibiting homologous recombination (HR), a pathway related with genomic instability and progression, very active in MM. Therefore we next evaluated the efficiency of HR using a reported construct that was chromosomally integrated in two MM cell lines, JJN3 and U266. Treatment with EDO-S101 significantly reduced the efficiency of HR in both cell lines, by 50% and 20% of untreated controls respectively. Finally, we tested potential combinations with other antimyeloma agents like lenalidomide and thalidomide; and also with proteasome inhibitors (bortezomib, carfilzomib and oprozomib). EDO-S101 potentiated the activity of all these agents, but the most synergistic combination was that including Bortezomib + Dexamethasone (CI 0,4). This combination was also evaluated in vivo, where it significantly decreased tumor growth and prolonged survival compared to agents in monotherapy and in double combinations. We are currently deepening into the mechanism of action of this combination. Conclusions. EDO S101 is active ex vivo in cells isolated from patients and is able to overcome resistance to alkylators. It induces caspase-independent apoptosis, and cell cycle arrest in MM cell lines. These effects are due to the potent DNA damage which is enhanced by HR impairment induced by the hybrid molecule. Moreover, the combination with bortezomib and dexamethasone is especially attractive to be taken into the clinical setting. Disclosures Mehrling: 4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Mateos:Takeda: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Onyx: Consultancy.

Antitumor Effect of Pomolic Acid in Acute Myeloid Leukemia Cells Involves Cell Death, Decreased Cell Growth and Topoisomerases Inhibition

2019 ◽  
Vol 18 (10) ◽  
pp. 1457-1468
Author(s):  
Michelle X.G. Pereira ◽  
Amanda S.O. Hammes ◽  
Flavia C. Vasconcelos ◽  
Aline R. Pozzo ◽  
Thaís H. Pereira ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Natural Compound ◽  
Dna Topoisomerases ◽  
Human Dna ◽  
Pomolic Acid ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. Objectives: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. Methods: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. Results: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. Conclusion: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.

scholarly journals Glioblastoma Multiforme Stem Cell Cycle Arrest by Alkylaminophenol through the Modulation of EGFR and CSC Signaling Pathways

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 681 ◽  
Author(s):  
Phuong Doan ◽  
Aliyu Musa ◽  
Akshaya Murugesan ◽  
Vili Sipilä ◽  
Nuno R. Candeias ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Glioblastoma Multiforme ◽  
Cell Cycle Arrest ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Cell Population ◽  
Significant Role ◽  
Dependent Manner ◽  
Cycle Arrest

Cancer stem cells (CSCs), a small subpopulation of cells existing in the tumor microenvironment promoting cell proliferation and growth. Targeting the stemness of the CSC population would offer a vital therapeutic opportunity. 3,4-Dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), a small synthetic phenol compound, is proposed to play a significant role in controlling the CSC proliferation and survival. We assessed the potential therapeutic effects of THTMP on glioblastoma multiforme (GBM) and its underlying mechanism in various signaling pathways. To fully comprehend the effect of THTMP on the CSCs, CD133+ GBM stem cell (GSC) and CD133- GBM Non-stem cancer cells (NSCC) population from LN229 and SNB19 cell lines was used. Cell cycle arrest, apoptosis assay and transcriptome analysis were performed for individual cell population. THTMP strongly inhibited NSCC and in a subtle way for GSC in a time-dependent manner and inhibit the resistance variants better than that of temozolomide (TMZ). THTMP arrest the CSC cell population at both G1/S and G2/M phase and induce ROS-mediated apoptosis. Gene expression profiling characterize THTMP as an inhibitor of the p53 signaling pathway causing DNA damage and cell cycle arrest in CSC population. We show that the THTMP majorly affects the EGFR and CSC signaling pathways. Specifically, modulation of key genes involved in Wnt, Notch and Hedgehog, revealed the significant role of THTMP in disrupting the CSCs’ stemness and functions. Moreover, THTMP inhibited cell growth, proliferation and metastasis of multiple mesenchymal patient-tissue derived GBM-cell lines. THTMP arrests GBM stem cell cycle through the modulation of EGFR and CSC signaling pathways.

MicroRNA-126 attenuates cell apoptosis by targeting TRAF7 in acute myeloid leukemia cells

2018 ◽  
Vol 96 (6) ◽  
pp. 840-846 ◽  
Author(s):  
Qian Ding ◽  
Qing Wang ◽  
Yi Ren ◽  
Hong Qian Zhu ◽  
ZhuYun Huang
Keyword(s):  
Acute Myeloid Leukemia ◽  
Western Blot ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Myeloid Leukemia ◽  
Flow Cytometric Analysis ◽  
Tumor Necrosis Factor Receptor ◽  
Luciferase Assay ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Acute myeloid leukemia (AML) has a 5-year survival rate of only about 30%–40% due to the self-renewal and differentiation ability of leukemia stem-like cells (LSCs). To address the potential for novel therapeutic targets in LSCs, we investigated the roles of miRNA-126 and tumor necrosis factor receptor-associated factor 7 (TRAF7) in AML. We used qRT-PCR and Western blot to investigate the expression levels of miRNA-126 and TRAF7 in AML cell lines. Then, we uncovered the effect of miRNA-126 on AML cell proliferation and apoptosis by MTT assay and flow cytometric analysis, respectively. Furthermore, dual-luciferase assay and Western blot were used to determine the target of miRNA-126 in AML and the potential mechanism by which cell apoptosis is suppressed by miRNA-126. We found that miRNA-126 was highly expressed in all of the AML cell lines, and that inhibition of miRNA-126 significantly induced cell death through apoptosis. The suppression of apoptosis in AML with high expression of miRNA-126 was caused by down-regulating TRAF7, which blocked the c-FLIP pathway. The role of miRNA-126 in AML makes it a potential therapeutic target to improve clinical outcomes for patients with AML.

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