Inhibition of Cellular Aminopeptidases as Novel Therapy for AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2588-2588
Author(s):  
Christopher Jenkins ◽  
Chris Pepper ◽  
Ken Mills ◽  
Alan Burnett
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Human Cancer ◽  
Myeloid Cells ◽  
Chemotherapeutic Agents ◽  
Molecular Therapy ◽  
Leukemic Cells ◽  
Therapeutic Window ◽  
Acute Myeloid

Abstract CHR 2797 is one of a new class of enzyme inhibitors with a pleiotropic effect against a number of human cancer cells. It is thought to inhibit the M1 family of metalloenzymes that include aminopeptidases, and is under investigation for the treatment of acute myeloid leukemia. Aminopeptidases catalyse the hydrolysis of the terminal amino acids from short chain polypeptides and they are involved in the continuous cycle of protein formation and degradation in cells. As malignant cells are thought to be more highly dependant on this protein cycling, interrupting this pathway is therefore a potential therapeutic target for novel agents. The effects of the aminopeptidase inhibitor CHR 2797 were investigated in AML cells in-vitro. Leukemic cells and cell lines were treated with CHR 2797 at a range of 0.0002 – 20μM and IC50 values were calculated from the WST-1 proliferation experiments. The AML cell lines HL60, KG1, K562 and U937 had an average IC50 of 1μM with a range between 0.01 and 10μM. Primary diagnostic AML samples (n=40) were analysed and an IC50 range of between 0.01 and >40μM were detected, with a median of 0.8μM. The effects of CHR 2797 were also analysed on normal bone marrow samples (n=10). The IC50 range was between 6.2 and >40μM with a median of 15μM, demonstrating a potential therapeutic window between the treatment of the leukemic cells and toxicity to the normal samples. The level of synergy or antagonism with conventional therapeutic agents was calculated using a combination index. Synergy was demonstrated in 70% of cell samples in combination with ARA-C, and 80% with Velcade. Synergy was also shown in 60% of cells samples with ATRA, even in non-promyelocytic leukemia types. Annexin V and cell cycle analysis confirmed apoptosis after treatment with CHR 2797 in many cases. A degree of differentiation of acute promyelocytic cells to mature myeloid cells was also stimulated with the treatment. The effects of CHR 2797 on cellular aminopeptidases were also measured. CD13 is a cell surface protein which is expressed selectively on myeloid cells and is also classified as an aminopeptidase N. Its activity can be measured by the conversion of the substrate ala-MCA to the protein MCA that can be detected on a fluorometric plate reader. CHR 2797 was demonstrated to reduce CD13 activity in a time and dose responsive manner. The reduction in activity was demonstrated immediately following addition of the drug, and a persistent effect was shown over four days of cell culture. A reduction in CD13 activity was also shown with a concentration of CHR 2797 of under 0.5μM; and with a 10μM dose the activity in many AML samples was reduced by >90%. New treatments are needed for acute myeloid leukemia to improve survival and reduce the toxicity of conventional therapy. This study demonstrates that CHR 2797 might be an effective molecular therapy for AML, either alone or in combination with other chemotherapeutic agents.

scholarly journals All-trans retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell promoting activity of EVI1 in acute myeloid leukemia

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Chi Huu Nguyen ◽  
Katharina Bauer ◽  
Hubert Hackl ◽  
Angela Schlerka ◽  
Elisabeth Koller ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Acute Myeloid

AbstractEcotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1high AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1high AML.

Cellular Aminopeptidase Inhibition as a Target for the Therapy of AML by the Novel Agent CHR 2797.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1608-1608
Author(s):  
Christopher Jenkins ◽  
Ken Mills ◽  
Chris Pepper ◽  
Burnett Alan
Keyword(s):  
Intact Cell ◽  
Myeloid Cells ◽  
Culture Conditions ◽  
Chemotherapeutic Agents ◽  
Molecular Therapy ◽  
Leukemic Cells ◽  
Therapeutic Window ◽  
Aminopeptidase Activity ◽  
Dependent Manner ◽  
Normal Cells

Abstract CHR 2797 is one of a new class of enzyme inhibitors with pleiotropic effects against cancer cells which is currently in Phase II clinical trials. It inhibits a number of the M1 family of metalloenzymes that include the Zn++-dependent aminopeptidases. Aminopeptidases catalyze the hydrolysis of the terminal amino acids from short chain polypeptides, and are involved in the continuous cycle of protein formation and degradation in cells. As malignant cells may be more dependent on protein cycling than normal cells, interrupting this pathway is, therefore, a potential therapeutic target for novel agents. The effect of CHR 2797 on AML cells in-vitro was investigated to determine its effectiveness and mode of action. Treatment of leukemic cells with CHR 2797 was performed under standard culture conditions at a concentration range of 0.0002 - 20μM. The WST-1 reagent was used to determine cell viability at the end of the culture and the IC50 values were calculated. Primary diagnostic AML samples (n=52) were analyzed and an IC50 range of between 0.01 and >40μM was established, with a median of 1.2μM. There were no significant differences between the different classes of AML, although there was a trend towards a better response in the monocytic leukemias and the good prognostic cytogenetic groups. The comparative effects of CHR 2797 on normal bone marrow samples (n=10) were also analyzed. Under identical culture conditions, an IC50 range between 6.2 and >40μM was found, with a median of 15μM. This 13-fold higher result demonstrates a significant potential therapeutic window for leukemic versus normal cells. The level of synergy or antagonism with conventional therapeutic agents was calculated using a combination index. Synergy was demonstrated in 69% of cell samples in combination with cytarabine, and 78% with bortezomib. Synergy was also shown in 62% of cells samples with ATRA, even in non-promyelocytic leukemia types. Annexin V staining and cell cycle analysis demonstrated apoptosis after treatment with CHR 2797 in many cases. A degree of differentiation of acute promyelocytic cells to mature myeloid cells was also stimulated with the treatment. To determine the mode of drug action, the effect of CHR 2797 on intact cell aminopeptidase activity was also measured. The activity of these enzymes can be measured by their conversion of the membrane-permeant substrate ala-MCA to MCA, which can be fluorimetrically detected. CHR 2797 was demonstrated to reduce intact cell aminopeptidase activity in a time- and dose-dependent manner. A reduction in aminopeptidase activity was shown with a concentration of CHR 2797 of less than 0.5μM; with 10μM drug the activity in many AML samples was reduced by >90%. CD13 is a cell surface protein which is expressed selectively on myeloid cells and is also classified as an aminopeptidase N. By comparison with the results obtained with the CD13 blocking antibody WM15, CHR 2797 was demonstrated to fully inhibit CD13 activity. Further investigations proved that other extracellular, cytoplasmic and nuclear aminopeptidase enzymes were also inhibited. In conclusion new treatments are needed for acute myeloid leukemia to improve survival and circumvent the toxicity of conventional therapy. This study demonstrates that CHR 2797 might be an effective molecular therapy for AML, either alone or in combination with other chemotherapeutic agents.

Nampt/SIRT2 Hyperactivation Lead to Activation of ß-Catenin: a Possible Mechanism of Leukomogenic Transformation In CN Patients.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1486-1486
Author(s):  
Lan Dan ◽  
Ana Gigina ◽  
Karl Welte ◽  
Julia Skokowa
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Myeloid Cells ◽  
Nuclear Accumulation ◽  
Healthy Individuals ◽  
Leukemia Cell Lines ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell

Abstract Abstract 1486 Recently we demonstrated that nicotinamide phosphoribosyltransferase (NAMPT) is an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis via activation of NAD+/sirtuins/C/EBPs signaling cascade. Nampt levels were significantly elevated in plasma and in myeloid cells of patients with severe congenital neutropenia (CN). CN is characterized by a “maturation arrest” of granulopoiesis on the promyelocytic stage of differentiation and by leukemogenic tansformation of hematopoiesis in ca. 20 % of patients. The mechanism of the leukemic transformation is still unclear. Previously, we reported elevated levels of activated oncogene ß-catenin in nuclei of myeloid progenitor cells of CN patients. The activity and nuclear translocation of ß-catenin is regulated by glycogen synthase kinase-3 ß (GSK3ß), which activates ß-catenin degradation complex. In the present study we found that in myeloid cells of CN patients GSK3ß was inhibited by phosphorylation on Ser9, as compared to healthy individuals. Therefore, we assume that GSK3ß-ß-catenin pathway could be involved in the leukemogenic transformation of hematopoiesis. Since, Nampt was also elevated in CN patients, we aimed to investigate the connection between hyperactivated Nampt and ß-catenin in leukemogenesis. The Nampt functions in hematopoiesis are dependent on the dose of Nampt and NAD+. Thus, in vitro stimulation of CD34+ cells with Nampt led to granulocytic differentiation via activation of sirtuin/C/EBP-dependent pathway. At the same time, inhibitors of NAMPT have been identified as therapeutical targets for some cancers including leukemia. This suggested that different mechanisms are operating downstream of NAMPT in the “normal” and leukemogenic myeloid cells. Screening of the different sirtuins in primary acute myeloid leukemia (AML) blasts revealed significant upregulation of SIRT2 mRNA and protein levels, as compared to CD34+ and CD33+ hematopoietic cells of healthy individuals. SIRT2 levels were also elevated in myeloid cells of CN patients treated with G-CSF. Specific inhibition of NAMPT (using 10 nMol of FK866) or SIRT2 (using 100nMol of AC93253) significantly reduced proliferation and induced apoptosis in human myeloid leukemia cell lines (NB4, HL60 and U937). We further tested if inhibition of Nampt or SIRT2 has an effect on GSK3ß/ß-catenin pathway. GSK3ß is known to be inhibited by Akt and treatment of the acute myeloid leukemia cell lines NB4 and HL60 with FK866 or AC93253 resulted in the activation of Akt via phosphorylation on Thr308 and Ser473 and inactivation of GSK-3ß via inhibition of phosphorylation on Ser9. Moreover, activated ß-catenin protein was almost completely disappeared from the nucleus of cells treated with FK866. Taken together, our results provide strong evidence that NAMPT and SIRT2 participate in leukemogenic transformation via inactivation of GSK3ß leading to nuclear accumulation of oncogenic ß-catenin. 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.

scholarly journals Expression of isoforms of the human receptor tyrosine kinase c-kit in leukemic cell lines and acute myeloid leukemia

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1151-1158 ◽  
Author(s):  
PS Crosier ◽  
ST Ricciardi ◽  
LR Hall ◽  
MR Vitas ◽  
SC Clark ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Cellular Transformation ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Rnase Protection ◽  
Acute Myeloid

Abstract Because mutations in receptor tyrosine kinases may contribute to cellular transformation, studies were undertaken to examine c-kit in human leukemia. Isoforms of c-kit have been characterized in the human megakaryoblastic leukemia cell line M-07. Deletion of the four amino acids Gly-Asn-Asn-Lys in the extracellular domain represents an alternatively spliced isoform that has been shown by others, in mice, to be associated with constitutive receptor autophosphorylation (Reith et al, EMBO J 10:2451, 1991). Additional isoforms differ in the inclusion or exclusion of a serine residue in the interkinase domain, a region that contains the binding site for phosphatidylinositol 3- kinase. By RNase protection analysis, we have shown coexpression of the Gly-Asn-Asn-Lys+ and Gly-Asn-Asn-Lys- isoforms, with dominance of the Gly-Asn-Asn-Lys- transcript, in normal human bone marrow, normal melanocytes, a range of tumor cell lines, and the blasts of 23 patients with acute myeloid leukemia. Analysis of transcripts for the Ser+ and Ser- isoforms also showed coexpression in all normal and leukemic cells examined. The ratios of isoform expression for both the Gly-Asn-Asn-Lys and Ser variants were relatively constant, providing no evidence in the tumors examined that upregulation of one isoform contributes to the neoplastic process.

Dual PI3K/Akt/mTOR Pathway Inhibitor Potentiate Cytarabine Cytotoxicity For Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1289-1289
Author(s):  
Eun Kyung Kim ◽  
Hyoung Jin Kang ◽  
Hyung Joon Kim ◽  
Ji Won Lee ◽  
Hyery Kim ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Signal Transmission ◽  
Mtor Pathway ◽  
Leukemic Cells ◽  
Combination Regimen ◽  
Dual Inhibitor ◽  
Acute Myeloid

Abstract Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway integrating signals from multiple receptor tyrosine kinases has been firmly established as a major determinant for cell growth, proliferation, and survival in a wide array of solid cancers. PI3K/Akt/mTOR pathway is frequently activated in acute myeloid leukemia (AML) cells and contributes to survival and drug-resistance of AML through various mechanisms. BEZ235 is one of the most promising dual inhibitor of PI3K and mTOR currently under clinical development in solid tumor area. In this study, the potential of BEZ235 was investigated as antileukemic agent using alone or with cytarabinearabinoside (AraC) as combination regimen. AML cell lines KG-1, MV 4-11, THP-1 and HL60 were treated with AraC, BEZ235 and combination regimen with various mixed ratio. BEZ235 effectively inhibited leukemic cell growth with similar range of half maximal inhibitory concentration (IC50) values among different cell lines. Apoptosis was induced gradually as BEZ235 concentration increased, but significant level of apoptosis was not shown even at higher concentration beyond IC50 value. Then, AraC-resistant MV4-11 and THP-1 cell lines were chosen to investigate interaction between BEZ235 and AraC. Using CalcuSyn software based on Chou and Talalay analysis, Combination Index (CI) value was calculated in each combination regimen. Moderate to strong synergism was shown and it was well maintained as combination ratio of AraC versus BEZ235 gradually decreased from 20:1 to 1,000:1. BEZ235 reduced resistance to AraC when it was added as combination regimen, and significance of combination effect changed according to AraC concentration. When antileukemic effect was compared among combination regimens with different schedules, synergism was maximized when BEZ235 was pretreated before AraC administration. This means BEZ235 sensitizes leukemic cells to apoptotic effect of AraC. Genetic alteration in PI3K/Akt/mTOR pathway is an attractive target to investigate and dual pathway inhibitor BEZ235 has potential to maximize AML treatment through sensitization of leukemic cells to conventional drug. The results of this experiment suggest the possibility of designing early clinical trials for the therapy - AraC administration after dual inhibitor BEZ administration. An effective control of a signal transmission system using innovate new drugs along with an accurate understanding of a signal transmission system which is the target of therapy will establish a foothold for the development of AML therapy. Disclosures: No relevant conflicts of interest to declare.

OncoHist®, an rh Histone 1.3, Is Cytotoxic to Acute Myeloid Leukemia Cells and Results in Altered Downstream Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3604-3604
Author(s):  
Suiyang Liu ◽  
Surender Kharbanda ◽  
Richard M. Stone
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Histone H1 ◽  
Chemotherapeutic Agents ◽  
P53 Mutation ◽  
Blue Staining ◽  
Downstream Signaling ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogeneous myeloid stem cell disorder; subtypes, which can be defined histologically, cytogenetically, and genetically, have varying prognoses and clinical characteristics. However, all but those younger patients with favorable biological features are inherently resistant to available cytotoxic chemotherapeutic agents; novel and less toxic therapies are required. Studies have shown that exposure of cancer cells, including leukemic blasts, to histone H1 produce cytotoxicity. The mechanism of cytotoxicity is believed to be an increase in focal membrane permeability induced by histone H1 binding to phosphotidyl serine moieties. However, the precise mechanism and functional effects of histone H1 on AML cells is currently unclear. The present study was aimed at investigating the effect of recombinant human Histone 1.3, OncoHist® (Xenetic Biosciences), on the proliferation of AML cell lines and on primary AML cells. We demonstrate that purified OncoHist® exerts growth inhibition and induces necrosis by flow cytometry assessment of staining for propidium iodide (PI) of AML cell lines (MOLM14, MV4-11, U937, HL60) as well as primary AML (n=3, including one with a p53 mutation) cells independent of lineage, stage and maturation with an IC50 of 2-5 mM. An AML cell line (MOLM13) with a FLT3 activating mutation (length or internal tandem duplication) which was developed to express resistance to the FLT3 inhibitor midostaurin/PKC412 was sensitive to OncoHist®-induced growth arrest measured by trypan blue staining and death (flow for PI) with about 70-80% necrosis at 5mM. Furthermore, 2uM OncoHist® treatment of MV4-11 and MOLM-14 cells for 15 minutes was associated with inhibition of the FLT3 downstream effectors phospho-AKT and phospho-extra cellular regulated kinase (phospho-ERK) as assessed by immunoblotting. Moreover, treatment of MOLM14 cells with OncoHist® in combination with cytarabine was associated with a significant synergistic inhibition of growth as measured by Alamar blue staining with a CI index of 0.35. Our findings support the development of OncoHist® alone and in combination with chemotherapy for the treatment of AML. A Phase I trial of OncoHist® for the treatment of refractory/relapsed AML is planned. Disclosures Kharbanda: Xenetic: Equity Ownership. Stone:Xenetic: Consultancy.

Polo-like kinase 1 is overexpressed in acute myeloid leukemia and its inhibition preferentially targets the proliferation of leukemic cells

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 659-662 ◽  
Author(s):  
Annelies G. Renner ◽  
Cédric Dos Santos ◽  
Christian Recher ◽  
Christian Bailly ◽  
Laurent Créancier ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Leukemic Cell Lines ◽  
Clonogenic Potential ◽  
Established Cell ◽  
Interfering Rna ◽  
Acute Myeloid

Abstract Polo-like kinase 1 (Plk1) is a major mitotic regulator overexpressed in many solid tumors. Its role in hematopoietic malignancies is still poorly characterized. In this study, we demonstrate that Plk1 is highly expressed in leukemic cell lines, and overexpressed in a majority of samples from patients with acute myeloid leukemia compared with normal progenitors. A pharmacologic inhibitor, BI2536, blocks proliferation in established cell lines, and dramatically inhibits the clonogenic potential of leukemic cells from patients. Plk1 knockdown by small interfering RNA also blocked proliferation of leukemic cell lines and the clonogenic potential of primary cells from patients. Interestingly, normal primary hematopoietic progenitors are less sensitive to Plk1 inhibition than leukemic cells, whose proliferation is dramatically decreased by the inhibitor. These results highlight Plk1 as a potentially interesting therapeutic target for the treatment of acute myeloid leukemia.

scholarly journals Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1648-1648 ◽  
Author(s):  
Yaser Heshmati ◽  
Gözde Turköz ◽  
Aditya Harisankar ◽  
Sten Linnarsson ◽  
Marios Dimitriou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Loss Of Function ◽  
Potential Therapeutic Target ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.

Anti-Leukemic Activity of PIK-75, a PI3-Kinase p110α Selective Inhibitor, In Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 659-659
Author(s):  
François Vergez ◽  
Jean-Emmanuel Sarry ◽  
Nathalie Gallay ◽  
Camille Fialin ◽  
Sarah Scotland ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Critical Role ◽  
Selective Inhibitor ◽  
Leukemic Cells ◽  
High Dose ◽  
Inhibition Of Proliferation ◽  
Hl60 Cells ◽  
Acute Myeloid

Abstract Abstract 659 The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays a critical role in a variety of tumor cells including hematological malignancies. Class IA PI3Ks are heterodimers that consist of a p85 regulatory and a p110 catalytic subunit. There are several isoforms of both the catalytic (p110α, p110β and p110δ) and regulatory subunits. The p110α isoform of the class IA PI3-Ks was recently genetically validated as a promising target for anticancer therapy. To date, only one compound (imidazo[1,2-a]pyridine, PIK-75) has been described as a very potent and selective inhibitor of this isoform (>100-fold selectivity over p110α and p110δ). In acute myeloid leukemia (AML), aberrant PI3-K activation is detectable in most of cases both in leukemic bulk and in the immature compartment of the leukemic clone. This activation contributes to cell growth, proliferation, survival, and drug-resistance. Furthermore, we have previously shown that the level of Akt phosphorylation on threonine 308, the major target of PI3-K, is correlated with poor outcome in AML patients (Gallay et al, Leukemia 2009, 23(6):1029-38). Therefore, effective targeting of this pathway with pharmacological inhibitors could improve therapeutic outcome in AML. Here, we studied the anti-neoplastic activity of several inhibitors of the PI3-K p110 subunits in AML cell lines and primary patient specimens. Treatment with PIK-75 led to a decrease of the proliferation in all cell lines at low dose (MTT assay, IC50: 62 nM, 144 nM, 173 nM in KG1, HL60 and KG1a cell lines, respectively). This inhibition of proliferation was due to massive apoptosis of KG1 and KG1a cells in both liquid culture but also after adhesion of leukemic cells on a fibronectin matrix. By contrast, p110ß (TGX221, 10 μM) and p110γ (AS252424, 10 μM) inhibitors only slightly decreased cell proliferation in KG1 and HL60 cells while p110δ inhibitor (IC87114) has no effect up to 10 μM. PIK-75 inhibited the phosphorylation of Akt on Thr308, and downstream effectors (4-EBP1 and RPS6) in these cells. These results strongly suggest a major role of p110α subunit which is highly express in AML cell lines and 19/19 patients samples. Next, we assessed the PIK-75 efficacy in 1 AML cell lines, 9 AML samples and 1 normal bone marrow CD34+ cells using clonogenic assays. PIK-75 inhibited AML-CFU in both KG1 cell line and all patient samples tested with an IC50 of 214 nM and 72 nM, respectively. Interestingly, PIK-75 has no effect on normal CFU-GM colonies, even at high dose (IC50 not reached at 1 μM), a result consistent with the normal haematopoiesis observed in p110αfnKO mice (Gritsman et al, Blood (ASH Annual Meeting Abstracts) 2009 114: Abstract 3620). Since leukemic subpopulation bearing the CD34+CD38-CD123+ phenotype is thought to be more resistant to chemotherapy than the leukemic bulk, we have assessed the apoptosis of 17 AML primary cells treated with increasing doses of cytarabine (Ara-C). We found significant differences of IC50 with 9.5 μM and 43 μM in bulk and CD34+38-123+ subpopulation, respectively. By contrast, PIK-75 demonstrated potent activity in both leukemic compartments of 42 AML samples with IC50 of 589 nM and 638 nM, respectively. Interestingly, the effect of PIK75 was not altered at relapse neither in bulk (IC50: 513 nM vs 492 nM at diagnosis and at relapse) nor in CD34+38-123+ subpopulation (IC50: 567 nM vs 254 nM at diagnosis and at relapse) in 8 AML samples. In NOD/SCID mice engrafted with HL60 cells, PIK-75 delivered at 1 and 10 mg/kg/d for 4 days induced a significant decrease in tumor burden after apoptosis induction detected ex vivo by annexin V staining. Further in vivo studies using NSG mice engrafted with primary AML specimens are ongoing. These results demonstrate that PIK-75 is the most potent inhibitor of PI3-K in leukemic cells suggesting that the selective inhibition of the p110α subunit could be a critical target in AML. Moreover, PIK-75 targets both leukemic bulk and chemoresistant leukemic subpopulations paving the way for clinical studies assessing the combination of selective p110α inhibitor with conventional chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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