A Small Molecule Stat Inhibitor Blocks Stat3 and Stat5 Phosphorylation and Demonstrates Cytotoxicity In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2904-2904
Author(s):  
Robyn M. Dennis ◽  
Brandon Ballard ◽  
David John Tweardy ◽  
Karen Rabin
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Positive Control ◽  
Wild Type ◽  
Atp Assay ◽  
Antibody Staining

Abstract Abstract 2904 Survival has improved dramatically in acute lymphoblastic leukemia (ALL), but further gains are unlikely using conventional chemotherapy alone. Several recently discovered, novel cytogenetic lesions with adverse prognostic impact, JAK2 activating mutations and CRLF2 rearrangements, occur in up to 15% of adult and pediatric ALL. These lesions are associated with activation of Jak2 and Stat5, and hold promise as targets for novel therapies affecting these signaling pathways. We performed in vitro testing of a novel small molecule Stat inhibitor, C188-9, in B-lineage ALL cell lines and patient samples with and without JAK2/CRLF2 alterations. C188-9 treatment for one hour at 10 μM inhibited Stat3 and Stat5 phosphorylation in ALL cell lines with JAK2 and CRLF2 alterations, but not in cell lines with wild-type JAK2 and CRLF2, as measured by phospho-flow cytometry (Fig. 1A). Only the cell lines with JAK2 and CRLF2 alterations demonstrated basal Stat5 phosphorylation on Western blot analysis, and this was inhibited by C188-9 treatment (Fig. 1B). C188-9 demonstrated cytotoxicity in ALL cell lines regardless of JAK2/CRLF2 status, with IC50s in the low micromolar concentration range (Fig. 1C). While C188-9 is undergoing investigation currently as a potent inhibitor of Stat3 in acute myeloid leukemia (AML), it also merits further investigation as an agent with Stat5 inhibitory activity and cytotoxicity in ALL. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 883 An anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human acute myeloid leukemia (AML) cell lines in vitro

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A925-A925
Author(s):  
Alessandra Romano ◽  
Nunziatina Parrinello ◽  
Sara Marino ◽  
Enrico La Spina ◽  
Massimo Fantini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Functional Analysis ◽  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Phase 1 ◽  
Adcc Activity ◽  
Phenotypic Analysis

BackgroundNEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 and has demonstrated tumor sensitivity and specificity in epithelial cells. Functional analysis has revealed that NEO-201 can engage innate immune effector mechanisms including ADCC and CDC to directly kill tumor cells expressing its target. A recent Phase 1 clinical trial at the NCI has determined both safety and recommended Phase 2 dosing. We have also seen the expression of the NEO-201 target on hematologic cells, specifically Tregs and neutrophils. Due to epitope being expressed both on malignant epithelial cells as well as several hematologic cells, we designed this study to explore the reactivity of NEO-201 against hematological neoplastic cells in vitro.MethodsPhenotypic analysis was conducted by flow cytometry. Cell lines used were six AML (HL60, U937, MOLM13, AML2, IMS-M2 and OCL-AML3), two multiple myelomas (MM) (OPM2, MM1.S), two acute lymphoblastic leukemia (ALL) (SUP-B15, RPMI8402) and four mantle cell lymphoma (MCL) (Jeko-1, Z138, JVM2 and JVM13). Markers used for flow cytometry analysis were CD15, CD45, CD38, CD138, CD14, CD19 and NEO-201. Functional analysis was performed by evaluating the ability of NEO-201 to mediate ADCC activity against AML cell lines using human NK cells as effector cells.Results5 of 6 AML cell lines tested bind to NEO-201 and the% of positive cells were 47%, 99.5%,100%,100% and 97.8% for HL60, U937, MOLM13, AML3 and IMS-M2, respectively. The% of positive cells in the two MM cell line were 99% and 18% for OPM2 and MM1.S, respectively. NEO-201 binding was not detected in the two ALL and the four MCL cell lines tested. Functional analysis has demonstrated that NEO-201 can mediate ADCC activity against the AML cell line (HL60) tested.ConclusionsThis study demonstrates that NEO-201 mAb’s target is expressed in most of the AML cell lines tested in vitro. In addition, we have shown it can mediate ADCC activity against HL60 cells (AML). Together, these findings provide a rationale for further investigation of the role of NEO-201 in AML as well as MM, further exploring patient PBMCs and bone marrow samples.

Dramatic Down-Regulation Of MNDA Expression In CP-CML Cells From The LSC Compartment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2717-2717
Author(s):  
Céline Bourgne ◽  
Alexandre Janel ◽  
Juliette Berger ◽  
Agnès Guerci ◽  
Caroline Jamot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Differentiation ◽  
Cell Line ◽  
Cell Lines ◽  
Fold Increase ◽  
Positive Control ◽  
Polymorphonuclear Cells ◽  
Primary Cells ◽  
Down Regulation ◽  
Cd34 Cells

Abstract Introduction Chronic Phase - Chronic Myeloid Leukemia (CP-CML) is a myeloproliferative disorder characterized by malignant proliferation of the granulocytic lineage without the arrest of cell differentiation. Tyrosine Kinase Inhibitors (TKI) have revolutionized CML treatment but several studies showed that a combination of TKI and Interferon alpha (IFNα) provides better clinical response. Myeloid Nuclear Differentiation Antigen (MNDA), which belongs to the hematopoietic interferon-inducible nuclear proteins with the 200-amino-acid repeat (HIN200) gene family, encodes a protein expressed in myeloid cells but whose function remains poorly understood. Because of its high expression in polymorphonuclear cells, its involvement in cell differentiation and apoptosis, and its induction by IFNα, we evaluated MNDA expression in CML cells and its modulation after incubation with IFNα. Material and methods We tested MNDA expression in several cell lines (K562, KCL22, LAMA84, TF1 and U937 (positive control)), in polymorphonuclear cells from healthy donors (HD-PMN, n=13) and in primary cells from patients with CP-CML at diagnosis (CP-CML; n=17). The relative expression of the MNDA transcript was analyzed using the 2-ΔΔCt method and was normalized to the endogenous reference gene GAPDH. HD-PMN were used as calibrator. We developed a multiparametric flow cytometry assay (CD45-V500/CD14-APC-H7/CD15-PerCpCy5.5/CD34-PC7/CD38-V450/MNDA-FITC) to detect MNDA protein in the different cell subsets, particularly in CD34+cells. Results As previously described, MNDA was poorly expressed in the K562 cell line. Similarly, mRNA was detected at low levels in two other CML cell lines (KCL22, LAMA84) and in TF1 cells, but at a high level in the U937 cell line, used as a positive control. In each cell line, the transcript expression was correlated to the protein level, as evaluated by flow cytometry (MFI ratio: 2.04±0.21, 2.36±0.24, 1.59±0.14, 1.88±0.11 and 8.77±0.54 for K562, KCL22, LAMA84, TF1 and U937, respectively (n=3)). In CP-CML primary cells, MNDA expression was greatly diminished as compared with HD-PMN in both mRNA (0.20±0.08 (n=17) vs. 1.32±0.21 (n=10); p=1.52x10-6) and protein (MFI ratio: 6.9±0.98 vs. 16.31±1.25, p=0.001). After having verified that IFNα (2000 U/ml, 16 hours) induced MNDA expression in HD mononuclear cells but not in PMN, we observed that induction of MNDA was moderate in CML cell lines K562 and LAMA84 (2-fold increase, n=3) whereas the level of MNDA mRNA was significantly increased in TF1 cells (28-fold increase, n=4). Induction in primary CML cells was variable (3/5 patients). Aiming to evaluate the expression of MNDA in leukemic stem cells (LSC), we first analyzed MNDA expression in CD34+ and CD34+/CD38- cells from HD. We observed that MNDA is down-regulated in healthy CD34+ and CD34+/CD38- cells compared to mature cells (mRNA: about 4 logs, protein: 8-10 fold lower, n=4), but we always detected a significant signal in CD34+cells (MFI ratio: 2.76±0.46, n=3). However, MNDA was not expressed by CML cells from the LSC compartment (n=4). This inhibition does not seem to be antagonized by nilotinib or IFNα (n=2). Discussion/Conclusion MNDA expression appears to be clearly down-regulated in CP-CML cells and dramatically so in the LSC compartment. In some patients, we observed sustained sensitivity to IFNα, but only in the compartment of more mature cells. This suggests early deregulation of MNDA expression which seems to be only partially dependant on differentiation. The mechanisms involved in this down-regulation remain to be elucidated but could be independent to TK activity of BCR-ABL protein and resistant to IFNα in the LSC compartment. This marked deregulation of MNDA in the LSC compartment is an additional argument in favor of intrinsic changes specific to primitive cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fingolimod Is Cytotoxic in Acute Myeloid Leukemia Independent of Additional Chemotherapeutic Agents

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5126-5126
Author(s):  
Carter Thomas Davis ◽  
Arati V. Rao ◽  
Eross Guadalupe ◽  
Dale J. Christensen ◽  
J. Brice Weinberg
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Flow Cytometric Analysis ◽  
Chemotherapeutic Agents ◽  
Flow Cytometric ◽  
Acute Myeloid

Abstract INTRODUCTION: Conventional treatment of acute myeloid leukemia (AML) remains largely unchanged for over thirty years. With poor overall survival and disease cure rates, novel therapies are needed. The SET oncoprotein has been implicated in AML as essential for proliferation through inhibition of the tumor suppressor protein phosphatase 2A (PP2A). Interaction between SET and PP2A leads to inactivation of PP2A, leaving cell survival and proliferation signals unchecked. PP2A has been postulated to be an important target in AML. Fingolimod (FTY720), an FDA approved drug for relapsing-remitting multiple sclerosis, is a sphingosine-1 phosphate receptor agonist that has off-target activity to activate PP2A. In this work, we show evidence of FTY720's efficacy in AML cells derived from cell lines and patients, and provide preliminary data regarding SET expression in AML cell lines. METHODS: Cytotoxicity experiments were performed using HL-60, THP-1, MV-4, and Kasumi-3 cell lines, as well as patient-derived samples of AML, obtained through an IRB-approved protocol. Cells were incubated overnight with varied concentrations of FTY720, azacitidine, idarubicin, cytarabine, or drugs in combination. After incubation, cells were analyzed by colorimetric assay. Percent cytotoxicity was estimated as a proportion of light absorbance compared with blank media and untreated control cells. Inhibitory concentration of 50% of cells (IC50) was estimated using GraphPad Prism software, version 6.0. Flow cytometry experiments for confirmation of cytotoxicity were also performed with antibodies against Annexin V and propidium iodide. For estimation of SET expression, we performed ELISA with antibodies against SETα and SETß and quantified measurements by light absorption. RESULTS: FTY720inhibits growth of AML cells independently in both cell lines and patient-derived samples. In the THP-1 cell line, we estimated the IC50 of FTY720 to be 3.4 μM (Figure 1). In the HL-60 cell line, we estimated the IC50 to be 2.5 μM. In patient-derived samples of AML, we had similar findings. The mean IC50 was 3.24 μM (SD = 1.32, n = 8). Flow cytometry of tested samples confirmed induction of both apoptosis and cell death within a 3-hour time frame (Figure 2). Samples were also incubated with combination of FTY720 and conventional cytotoxic chemotherapeutic agents used in AML (Table 1). In the HL-60 cell line, the following IC50s were estimated for these drugs: idarubicin (0.02 μM); cytarabine (0.6 μM); azacitidine (5.7 μM). In combination with FTY720, there was no appreciable change. Results of ELISA showed measurable but low SETα and SETß levels, when compared to a known positive control, the Ramos cell line for Burkitt's lymphoma (Table 2). In the MV-4 AML cell line, the SETα/ß ratio was 0.096. In Kasumi-3 cells, the α/ß ratio was measured at 0.063. DISCUSSION: These data support the assertion that FTY720 is a cytotoxic agent in AML. This effect is independent of other cytotoxic agents, as no additive or synergistic effect was demonstrated when drugs were combined. The micromolar cytotoxicity poses challenges to the adoption of this agent as an active drug in AML, as serum concentrations from currently prescribed doses in multiple sclerosis have been shown to achieve only nanomolar concentrations. It is notable that the volume of distribution of FTY720 is very high and over 90% is concentrated in blood cells, so actual cell concentrations may be substantially higher. Our work has not yielded the same results others have reported with increased SET α/ß ratios in AML cells. In other tumor types, high SET alpha ratios have been associated with higher SET activity; thus, these results would not be suggestive of such a role in AML. Despite our findings, the activity of FTY720 in these cells merits further investigation into SET expression in AML. We have recently a flow cytometric assay for SETα and SETß that can be used to quantify SET levels, and we plan to analyze patient samples used in cytotoxicity experiments to help identify the SET α/ß ratio in AML. We hope that these experiments will establish SET and PP2A as targets for drug development in AML. Figure 1 Cytotoxicity curve of FTY720 in THP-1 cells (n=3) Figure 1. Cytotoxicity curve of FTY720 in THP-1 cells (n=3) Figure 2 Flow cytometric analysis of FTY720 cytotoxicity in HL-60 cells. Figure 2. Flow cytometric analysis of FTY720 cytotoxicity in HL-60 cells. Disclosures Rao: Gilead, Inc.: Employment.

ARF Loss, a Negative Prognostic Factor in Philadelphia-Positive Acute Lymphoblastic Leukemia, May Be Efficiently Overcome by the Small Molecule MDM2 Antagonist RG7112

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2574-2574
Author(s):  
Ilaria Iacobucci ◽  
Federica Cattina ◽  
Silvia Pomella ◽  
Annalisa Lonetti ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Prognostic Impact ◽  
Wild Type ◽  
Mdm2 Antagonist ◽  
Wild Type P53

Abstract Abstract 2574 Recently, using genome-wide single nucleotide polymorphism arrays and gene candidate deep exon sequencing, we identified lesions in CDKN2A gene, encoding p16/INK4A and p14/ARF tumor suppressors, in 27% (32/117) adult newly diagnosed Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients and in 47% (14/30) relapsed cases. Clinically, in our cohort CDKN2A deletions were associated by univariate analysis to a worse outcome in terms of overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) (OS: 27.7 vs 38.2 months, p = 0.0206; DFS: 10.1 vs. 56.1 months, p = 0.0010; CIR: 73.3 vs 38.1, p = 0.0014). Noteworthy, the negative prognostic impact of CDKN2A deletion on DFS was also confirmed by the multivariate analysis (p = 0.0051). These results showed that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation and tumor cell growth by the action of MDM2, a negative regulator of p53, we investigated the preclinical activity of the MDM2 antagonist RG7112 in primary B-ALL patient samples and leukemic cell line models. BV-173, SUPB-15 and K562 Ph+ cell lines were incubated with increasing concentration of RG7112 (0.5–10 μM) and its inactive enantiomer for 24, 48 and 72 hours (hrs). MDM2 inhibition by RG7112 resulted in a dose and time-dependent cytotoxicity with IC50 (24 hrs) of 2 μM for BV-173 and SUPB-15 which harbor homozygous deletion of CDKN2A but wild-type p53. No significant changes in cell viability were observed in K562 p53-null cell line after incubation with RG7112. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/Propidium Iodide analysis showed a significant increase in apoptosis after 24 hrs in BV-173, SUPB-15 and in primary leukemia blasts, whereas no apoptosis was observed in K562 cells. To examine the possible mechanisms underlying RG7112-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased upon treatment with RG7112 after 24 hrs of incubation with concentrations equal to the IC50. These data demonstrate the ability of RG7112 to activate the intrinsic apoptotic pathway by a p53-dependent mechanism. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was next performed, comparing sensitive cell lines (BV-173 and SUPB-15) after 24 hrs exposure to 2 μM RG7112 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). They include genes involved in cell cycle and apoptosis control (e.g. Histone H1, TOP2, GAS41, H2AFZ) and in the down-regulation of the Hedgehog signaling (e.g. BMI1, BMP7, CDKN1C, POU3F1, CTNNB1, PTCH2) with a strong repression of stemness genes and re-activation of INK4/ARF as illustrated in Figure 1. Actually, both GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. In our data they were strongly down-regulated (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells, suggesting that these genes have a potential as new biomarkers of activity. In conclusion, inhibition of the p53–MDM2 interaction by RG7112 can activate the p53 pathway, resulting in apoptosis and inhibition of stemness genes in B-ALL with wild-type p53. Our findings provide a strong rational for further clinical investigation of RG7112 in Ph+ ALL. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione–Università 2007–2009. Disclosures: Baccarani: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

MLN4924, an Investigational Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Inhibits NF-κB Activity and Induces G1 Cell Cycle Arrest and Apoptosis in Pre-Clinical Models of Hodgkin Lymphoma (HL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3717-3717
Author(s):  
Matthew J. Barth ◽  
Cory Mavis ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Myron S. Czuczman
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Viability ◽  
Tumor Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Wild Type ◽  
Cycle Arrest ◽  
Molecule Inhibitor

Abstract Abstract 3717 The incorporation of combined-modality therapy, risk-stratified chemotherapy selection, high-dose chemotherapy and autologous stem cell support (HDC-ASCS), and monitoring treatment response by functional imaging are factors that have contributed to the improvement in clinical outcomes in HL patients. Unfortunately, those patients not eligible for or that have failed HDC-ASCS remain a challenge for the treating oncologist, stressing the need for novel therapeutic strategies. Significant improvements in the understanding of the biology of HL have been achieved, including cellular pathways altered in HL (e.g. the ubiquitin-proteasome system) and the role of the tumor microenvironment. MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an enzyme responsible for activating NEDD8, an ubiquitin-like molecule in the neddylation cascade that is responsible for cullin-ring ligase (CRL) mediated polyubiquitination of proteins targeting them for proteasomal degradation. In order to better understand the activity of MLN4924 in HL, we performed pre-clinical testing in IkB wild type (L-1236), IkB mutated (KM-H2 and L-428) HL cell lines, and in primary tumor cells derived from a HL patient. Malignant cells were exposed to escalating doses of MLN4924 and changes in cell viability were quantified at different time periods by alamar Blue reduction assay. Patient tumor cells were incubated with MLN4924 for 48 hrs and cell viability was determined using the CellTiterGlo assay. Induction of apoptosis in HL cell lines following exposure to MLN4924 was determined by flow cytometry for Annexin-V and propidium iodide (PI) staining and western blot for caspase-3 and PARP cleavage. Cell cycle analysis was performed by flow cytometry using PI staining. Inhibition of NAE by MLN4924 in HL cell lines was measured by western blot for NEDD8-cullin. Finally, changes in NF-kB activity following MLN4924 exposure were determined by p65 nuclear localization using Image stream technology. MLN4924 exhibited a dose- and time-dependent decrease in cell viability in all HL cell lines at nM concentrations. No differences in anti-tumor activity were observed between IkB-wild type (L-1236 IC50 = 250nM) and IkB–mutated HL cell lines (KM-H2 IC50 = 250nM and L-428 IC50 = 300nM). MLN4924 induced apoptosis in a dose-dependent manner in all cell lines tested. In addition, MLN4924 induced cell cycle arrest in G1 phase and inhibition of NAE was demonstrated by a decrease in NEDD8 conjugated CRL. L1236 cells exposed to MLN4924 also demonstrated a decrease in degradation of IκBα as evidenced by increased levels of p-IκBα following exposure to MLN4924 with a corresponding decrease in p65 nuclear translocation. Surprisingly KMH-2 cells, which carry a mutated IκBα protein that is truncated and non-functional, had a decrease in nuclear p65 following exposure to MLN4924, suggesting an alternative mechanism of NF-kB inhibitory activity by MLN4924. In summary, MLN4924 demonstrates activity against HL cells in vitro through inhibition of NF-kB, and is a promising novel agent for the treatment of HL. We continue to investigate the pre-clinical activity of MLN4924 both as a single-agent and in combination with traditional chemotherapy and other novel agents. Disclosures: No relevant conflicts of interest to declare.

Mer Receptor Tyrosine Kinase Is Over-Expressed In and Contributes to Oncogenesis In Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1390-1390
Author(s):  
Alisa B. Lee-Sherick ◽  
Kristen M. Eisenman ◽  
Susan Sather ◽  
Deborah DeRyckere ◽  
Jennifer Schlegel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Targeted Therapy ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Cell Survival ◽  
Cell Lines ◽  
Wild Type ◽  
Knock Down ◽  
Acute Myelogenous

Abstract Abstract 1390 The abnormal activation of tyrosine kinases in pediatric leukemias has been associated with a poor prognosis, and provides a potential focus for targeted therapy. Pediatric acute myelogenous leukemia (AML) is known to be particularly difficult to treat successfully. The development of therapy for AML targeted against a specific cancer-promoting signaling pathway would potentially allow for a more efficacious clinical response with less therapy-associated toxicity. The Mer Tyrosine Kinase (TK), a transmembrane receptor in the TAM family, is known to regulate intracellular pathways promoting cell survival and proliferation in a number of malignancies, but has not previously been explored in AML. We assessed the prevalence of Mer TK expression in AML. Western blot and flow cytometric analysis demonstrated aberrant expression of Mer TK in 80% (13 of 15) of AML cell lines. Similarly, greater than 85% (24 of 28) of samples from newly diagnosed pediatric AML patients expressed Mer TK on leukemic blasts. In addition, 5 of 6 pediatric patients with relapsed or refractory AML had increased or equivalent Mer expression by flow cytometry relative to diagnostic samples. To assess whether Mer plays a role in proliferation in AML, we investigated downstream signaling pathways in the Nomo-1 and Kasumi-1 AML cell lines. Phosphoarray and western blot analysis demonstrated increased phospho-Erk 1/2, phospho-Akt, phospho-mTOR and phospho-MSK1 following treatment with Gas6, the Mer ligand. These data demonstrate activation of pathways which are known to aid in malignant cell survival. To assess the effect of Mer TK inhibition on myeloblast phenotype, we used two different shRNA constructs to decrease expression of Mer by >50% in the Nomo-1 and Kasumi-1 cell lines. The ability of these cell lines to evade apoptosis was determined by flow cytometry following staining with propidium iodide and Yo-Pro-1-iodide. Compared to wild-type Nomo-1 and Kasumi-1, the cell lines expressing decreased levels of Mer demonstrated two to four times more apoptosis in response to serum starvation (p<0.5). Additionally, myeloblast proliferative capacity was assessed using methylcellulose colony forming assays. Compared to wild-type, the AML cell lines expressing reduced levels of Mer demonstrated a 40–70% decrease in total colony forming units (p<0.5). To explore how knockdown of Mer affects myeloblast survival in vivo, we used a mouse xenograft model. Sub-lethally irradiated NSG mice were injected intravenously with wild-type Nomo-1 or Mer knock-down Nomo-1 lines and tumor-free survival was determined. Kaplan-Meier curves were generated and demonstrated a statistically significant difference in survival between mice injected with wild-type Nomo-1 cells and those injected with a Nomo-1 Mer knock-down cell line (20 versus 43 days, p<0.1). These data demonstrate a role for Mer in acute myelogenous leukemogenesis in vivo and suggest that inhibition of Mer TK may have a clinically significant effect in patients as a targeted therapy in the treatment of human AML. Disclosures: No relevant conflicts of interest to declare.

Selected Adaptor Proteins NTAL, LAT, PAG, LIME Function in Proximal Signaling, Corticosteroid Driven Apoptosis and Expression of IKAROS Isoforms in the T-Leukemic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5034-5034
Author(s):  
Karel Svojgr ◽  
Tomas Kalina ◽  
Tomas Brdicka ◽  
Tereza Kacerova ◽  
Jana Volejnikova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Cell Lines ◽  
Jurkat Cell ◽  
Adaptor Proteins ◽  
Leukemic Cells ◽  
Wild Type ◽  
Tcr Signaling ◽  
Proximal Signaling

Abstract Abstract 5034 Transmembrane adaptor proteins NTAL, LAT, PAG and LIME have an important role in proximal signaling of T-lymphocytes. They lack enzymatic or kinase function, but when tyrosine-phosphorylated they bind other signaling molecules and mediate signaling from T-cell receptor (TCR) to nucleus. In our previous studies, we showed that in childhood T-cell acute lymphoblastic leukemia (T-ALL) the level of NTAL significantly correlates with early response to treatment. Patients with high NTAL levels responded favorably (prednisone good-responders) whereas patients with low NTAL were prednisone poor responders (p=0.05). We confirmed this clinical data also in in-vitro experiment - derivative Jurkat cell line (human T-ALL) transfected with NTAL expression construct (Jurkat/NTAL+) was more sensitive to corticosteroid treatment compared to wild-type Jurkat cell line (Jurkat/wt, NTAL negative). In the present study, we performed a more detailed analysis of a relationship between adaptor proteins, TCR signaling and apoptosis in T-ALL. Monoclonal IgM antibody C305 binding the Jurkat TCR with high affinity was used to stimulate TCR signalization in Jurkat/wt and Jurkat/NTAL+ cell lines. To assess the influence of TCR signaling on corticosteroid-driven apoptosis, we treated both the Jurkat cell lines with the C305 antibody and/or with methylprednisolone. At 24 hours we detected higher percentage of cells with the stimulation marker CD69 in Jurkat/NTAL+ cells compared to the Jurkat/wt (median 70% vs. 60%, p<0.05). More cells treated with C305 and methylprednisolone together expressed higher CD69 than cells treated by C305 alone (median 75% vs. 65%, p<0.004). At 24 and 48 hours we determined the level of apoptosis. At each time point the number of living cells in the untreated control was set to 100%. Jurkat/NTAL+ cell line was more prone to apoptosis than the wild-type cell line in all settings. At 24 hours, the percentage of surviving cells Jurkat/NTAL+ vs. Jurkat/wt was 44% vs. 50% when treated with methylprednisolone alone (p<0.1), 24% vs. 42% (C305 alone, p<0.1) and 15% vs. 26% (methylprednisolone + C305, p<0.1). Similar differences were detected after 48hours of treatment. Using flow cytometry, we further determined phosphorylation status of key downstream kinases ERK, p38 and JNK after TCR stimulation by the C305 antibody. We detected hyperphosphorylation of ERK higher in Jurkat/NTAL+ compared to Jurkat/wt (1.57, 1.53 and 1.46 fold at 5, 15 and 30 minutes after stimulation, respectively). Levels of phosphorylated P38 and JNK did not differ from the unstimulated controls or between the two cell lines. Our data show that the differences in apoptosis are not driven by an upregulation of FAS or FAS ligand as any significant increase in the levels of FAS/FASL was not detected by flow cytometry throughout the above described experiments. It was shown previously that aberrant TCR signaling affects the incidence of dominant-negative isoforms of IKAROS. Thus, to further elucidate the role of the signaling in T-ALL we analyzed presence and expression profile of IKAROS splicing variants (using on-chip electrophoresis) in a cohort of 29 pediatric T-ALL patients. While we did not observe any link between NTAL or LAT levels and expression of different IKAROS isoforms, we found a close correlation between IKAROS isoforms IK1 and IK4a and another adaptors, PAG (major inhibitor of Src kinases in lymphocytes) and LIME (adaptor involved in signalization via CD4 and CD8). The two patients expressing IK1 and IK4A as dominant transcripts had >2 logs lower PAG (p=0.02) and LIME (p<0.1) expression levels compared to the rest of the cohort (n=27) showing IK2 as a dominant transcript. Based on this data, we propose that NTAL acts as a tumor suppressor enhancing proximal signaling of lymphocytes. Via phosphorylation of ERK, the NTAL sensitizes T-leukemic cells to corticosteroid induced apoptosis that is not mediated by FAS or FASL. The role of IKAROS isoforms and its connection with adaptor proteins is under investigation. The work was supported by grants MSM0021620813 and GAUK35607. Disclosures No relevant conflicts of interest to declare.

High Pro-Apoptotic Effects of 17-Allylamino-17-Demethoxy Geldanamycin (17-AAG) In Philadelphia Acute Lymphoblastic Leukemia (Ph+ ALL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3226-3226
Author(s):  
Emmanuelle Tavernier-Tardy ◽  
Pascale Flandrin ◽  
Francoise Solly ◽  
Jérôme Cornillon ◽  
Karine Augeul-Meunier ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Apoptotic Proteins ◽  
Hsp 90

Abstract Abstract 3226 Heat Shock Protein 90 (HSP 90), a 90 Kda molecular weight protein, is one of the most abundant cytosolic chaperone, acting in protein folding, refolding and degradation and of particular importance in cell survival. HSP 90 is overexpressed in acute leukemia cells and this high expression is related with a poor prognosis. This ATP-dependent chaperone could be an interesting potential drug target. 17-AAG, a Geldanamycin derivates, is a HSP 90 inhibitor with a promising antitumor activity. Very few reports have evaluated the effects of 17-AAG in acute lymphoblastic leukemia (ALL). The aim of this work is to study the cytotoxicity of 17-AAG and to compare Philadelphia positive (Ph+) ALL to common B-cell ALL. Two human leukemia cell lines, Reh (common B-cell ALL) and SUPB15 (Ph+ ALL) were used in this study. We identified 63 consecutive patients treated in our institution for ALL (44 common B-cell ALL and 19 Ph+ ALL) and leukemic samples were collected at diagnosis from bone marrow aspiration after patient's consent. The expression of HSP 90, pro-apoptotic BAX protein, anti-apoptotic bcl-2 and bcl-xl proteins was studied by flow cytometry. Cell lines and ALL patient's cells were cultured in RPMI 1640 and exposed to various concentrations of 17-AAG. Apoptosis was evaluated by an Annexin V and an activated caspase-3 staining by flow cytometry. Results were expressed as percentage of positive cells. Pro-apoptotic effect of 17-AAG in Ph+ ALL cells was superior when compared to common B-cells with a 100% mortality rate after exposure to [10μM] 17-AAG for 24 hours for SUPB15 cell line whereas 24% of cell surviving was noted for Reh cell line. Similar results were observed with patient's leukemic samples (as shown in figure). The susceptibility of Ph+ ALL cells to 17-AAG was confirmed by the assessment of the IC50, estimated at 1.1 μM for SUPB15 cells versus 2.9 μM for ReH cells. As assessed by Annexin V binding and activated caspase-3 staining, 17-AAG induced apoptosis in ALL cells. As regard Ph+ ALL, the median percentage of Annexin-V positive cells and caspase-3 positive cells after exposure to [5μM] 17 AAG for 24 hours was 54% and 57% respectively and increased up to 99% and 99,5% after exposure for 48 hours. Spontaneous apoptosis in control cell culture was measured in 1% at 24 hours and 3% at 48 hours. The pattern of expression of HSP90 and apoptotic proteins was different between common B-cell ALL and Ph+ ALL cells. The percentage of HSP90 positive cells was 62% for Reh cell line whereas it reached 100% for SUP B15 cell line. As shown in the Table, the percentage of HSP90-positive cells and anti-apoptotic bcl-2 and bcl-xl proteins expression were higher for Ph+ ALL samples when compared to common B-cell ALL samples. HSP90 Bcl-2 Bcl-xl Bax Common B-cell ALL samples n=44 32.5% (±19.8) 33% (± 21.4) 28.5% (±19.5) 33% (±17) Ph+ ALL samples n = 19 79% (±7.5) 82% (±8.4) 84% (±7.8) 12% (±4.3) When ALL cells were exposed in culture to various concentrations of 17-AAG, there was no change in HSP90 expression but we observed a down-regulation in bcl-2 and bcl-xl expression and an up-regulation in bax expression. In summary, we showed that HSP90 and anti-apoptotic proteins were expressed at a higher level on Ph+ ALL cells when compared to common B-cell ALL. High percentage of HSP90-positive cells was associated with high sensitivity to 17-AAG. 17-AAG is a new targeted therapy that induces the apoptotic death of leukemic cells via a caspase-3 dependant way. It would be interesting to test its antileukemic activity in combination with chemotherapeutic agents to study additional or synergistic effects. Despite therapeutic improvement with the development of tyrosine kinase inhibitors (TKI) in the treatment of Ph+ ALL, relapse remains a major problem. Considering that Bcr-Abl constitutes HSP 90 substrates and depends on this chaperone for its maturation and conformational maintenance, 17-AAG could be of particular interest for Ph+ ALL disease, in combination with TKI. We can hypothesise that this drug could restore the sensitivity to TKI treatment for patients with Bcr-Abl mutation. Disclosures: No relevant conflicts of interest to declare.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

scholarly journals Sirtuin 2 in Endometrial Cancer: A Potential Regulator for Cell Proliferation, Apoptosis and RAS/ERK Pathway

2020 ◽  
Vol 19 ◽  
pp. 153303382098078
Author(s):  
Yanjuan Guo ◽  
Nannan Zhao ◽  
Jianli Zhou ◽  
Jianxin Dong ◽  
Xing Wang
Keyword(s):  
Cell Proliferation ◽  
Endometrial Cancer ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Erk Pathway ◽  
Uterine Epithelial Cell ◽  
Knock Down ◽  
Ishikawa Cells ◽  
And Control

Objective: The present study aimed to explore the function of sirtuin 2 (SIRT2) on cell proliferation, apoptosis, rat sarcoma virus (RAS)/ extracellular signal-regulated kinase (ERK) pathway in endometrial cancer (EC). Methods: SIRT2 expression in human EC cell lines and human endometrial (uterine) epithelial cell (HEEC) line was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. SIRT2 knock-down and control knock-down plasmids were transfected into HEC1A cells, respectively; SIRT2 overexpression and control overexpression plasmids were transfected into Ishikawa cells, respectively. After transfection, SIRT2, HRas proto-oncogene, GTPase (HRAS) expressions were evaluated by RT-qPCR and western blot. ERK and phosphorylated ERK (pERK) expressions were evaluated by western blot. Meanwhile, cell proliferation and cell apoptosis were measured. Results: Compared to normal HEEC cell line, SIRT2 mRNA and protein expressions were increased in most human EC cell lines (including HEC1A, RL952 and AN3CA), while were similar in Ishikawa cell line. In HEC1A cells, SIRT2 knock-down decreased cell proliferation but increased apoptosis. In Ishikawa cells, SIRT2 overexpression induced cell proliferation but inhibited apoptosis. For RAS/ERK pathway, SIRT2 knock-down reduced HRAS and inactivated pERK in HEC1A cells, whereas SIRT2 overexpression increased HRAS and activated pERK in Ishikawa cells, suggesting that SIRT2 was implicated in the regulation of RAS/ERK pathway in EC cells. Conclusion: SIRT2 contributes to the EC tumorigenesis, which appears as a potential therapeutic target.

Sign in / Sign up

Export Citation Format

Share Document