Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1137-1144 ◽  
Author(s):  
Yiwen Li ◽  
Hongli Li ◽  
Mei-Nai Wang ◽  
Dan Lu ◽  
Rajiv Bassi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Neutralizing Antibody ◽  
Myelogenous Leukemia ◽  
Mitogen Activated Protein Kinase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Leukemia ◽  
Wild Type ◽  
Antibody Treatment

AbstractFMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, is expressed at high levels in the blasts of approximately 90% of patients with acute myelogenous leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and point mutations in the kinase domain of FLT3 are found in approximately 37% of AML patients and are associated with a poor prognosis. We report here the development and characterization of a fully human anti-FLT3 neutralizing antibody (IMC-EB10) isolated from a human Fab phage display library. IMCEB10 (immunoglobulin G1 [IgG1], κ) binds with high affinity (KD = 158 pM) to soluble FLT3 in enzyme-linked immunosorbent assay (ELISA) and to FLT3 receptor expressed on the surfaces of human leukemia cell lines. IMC-EB10 blocks the binding of FLT3 ligand (FL) to soluble FLT3 in ELISA and competes with FL for binding to cell-surface FLT3 receptor. IMC-EB10 treatment inhibits FL-induced phosphorylation of FLT3 in EOL-1 and EM3 leukemia cells and FL-independent constitutive activation of ITD-mutant FLT3 in BaF3-ITD and MV4;11 cells. Activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and AKT is also inhibited in these cell lines by antibody treatment. The antibody inhibits FL-stimulated proliferation of EOL-1 cells and ligand-independent proliferation of BaF3-ITD cells. In both EOL-1 xenograft and BaF3-ITD leukemia models, treatment with IMC-EB10 significantly prolongs the survival of leukemia-bearing mice. No overt toxicity is observed with IMC-EB10 treatment. Taken together, these data demonstrate that IMC-EB10 is a specific and potent inhibitor of wild-type and ITD-mutant FLT3 and that it deserves further study for targeted therapy of human AML. (Blood. 2004;104:1137-1144)

Aberrant DNA Methylation of the Src Tyrosine Kinase Hck Is a Frequent Event in Human Leukemia and May Predict for Poor Prognosis in Adult Acute Lymphocytic Leukemia (ALL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1542-1542
Author(s):  
Koyu Hoshino ◽  
Hui Yang ◽  
Claritsa Santos-Malave ◽  
Blanca Sanchez-Gonzalez ◽  
Guillermo Garcia-Manero
Keyword(s):  
Dna Methylation ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Human Leukemia ◽  
Expression Levels ◽  
Leukemia Cell Lines ◽  
Aberrant Dna Methylation

Abstract Aberrant DNA methylation of promoter-associated CpG islands is a frequent phenomenon in human leukemias, and in particular in adult ALL. Hck is a member of the Src family of tyrosine kinases, and functionally is located downstream of BCR-ABL signaling in chronic myelogenous leukemia (CML). Hck expression is limitedly to myeloid cells and B cell lymphocytes. Although some evidence indicates that Hck is required for malignant transformation and apoptosis, its role in leukemia is not fully understood. Here we analyze the role of aberrant DNA methylation of Hck in leukemia cell lines and patients. Using BLAT, we first identified the presence of a canonical CpG island in the near proximity of the transcription start site of HcK. To detect and measure DNA methylation, we designed a combined bisulfite restriction PCR assay. Using this assay, we found that Hck was methylated in 13 out of 23 hematopoietic and 8 out of 10 non-hematopoietic cell lines, but not in the bone marrow from 6 healthy individuals. We subsequently studied Hck expression by real-time PCR using GAPDH expression as an internal control. Hck expression was lower (dCT = −14.2± 3.6) in 7 Hck methylated cell lines than in 8 Hck unmethylated ones (dCT= −9.0± 3.5), p=0.017. All the cell lines studied were of myeloid or B cell origin. We then treated the Raji cell line with the hypomethylating agent 5-aza-2-deoxycytidine (DAC). DAC treatment resulted in partial hypomethylation of Hck and in an increment of Hck expression (dCT: −19.37 to −8.47). Subsequently, the effects of DAC treatment on Hck protein expression levels were analyzed using Western blot. These experiments showed a strong correlation between hypomethylation, gene re-expression and protein expression levels. These data therefore indicates that DNA methylation is an important aberrant regulator of Hck expression in leukemia cell lines. Based on the relevance of these findings, we then analyzed the frequency of Hck methylation in patients with leukemia. Using a cut-off of 10%, Hck was found to be methylated in 15 out of 44 (34%) patients with ALL, 9 out 23 pts (39%) with CML, and 3 out 10 pts (30%) with AML. Of importance, the density of Hck methylation was significantly higher in patients with ALL (mean 11.3%; range 0–76) compared to those with CML(5.2%; range 0–12) or AML ( 7.5%, range 0–14), p=0.02. Hck methylation was not associated with a B cell phenotype or the presence of the Philadelphia chromosome in patients with ALL. Nine ALL pts out of 15 with Hck methylation had died compared to 7 out 29 unmethylated (total ALL group n=34). Median survival had not been reached for the group of patients with no Hck methylation (n=29) compared to 116 weeks for those with Hck methylation (n=15) (p=0.08). All pts had been treated with hyperCVAD based chemotherapy. These data indicates that Hck methylation is a frequent phenomenon in human leukemia that maybe associated with a worse prognosis in ALL and suggests that Hck has a tumor suppressor like function in these disorders.

scholarly journals Inhibition of growth of human leukemia cell lines by retrovirally expressed wild-type p16INK4A

Leukemia ◽  
1997 ◽  
Vol 11 (10) ◽  
pp. 1673-1680 ◽  
Author(s):  
AF Gombart ◽  
R Yang ◽  
MJ Campbell ◽  
JD Berman ◽  
HP Koeffler
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Wild Type ◽  
Human Leukemia Cell ◽  
Inhibition Of Growth ◽  
Leukemia Cell Lines

Expression of tie receptor tyrosine kinase in human leukemia cell lines

1997 ◽  
Vol 21 (7) ◽  
pp. 595-601 ◽  
Author(s):  
Catherine Bredoux ◽  
Cord C. Uphoff ◽  
Hans G. Drexler
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Human Leukemia Cell ◽  
Leukemia Cell Lines

MEIS1 Is a Required Transcription Factor for AML Cell Proliferation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1944-1944
Author(s):  
Stefan Heinrichs ◽  
Chad J. Brenner ◽  
Rima V. Kulkarni ◽  
A. Thomas Look
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Leukemia Cell ◽  
Tyrosine Kinase Receptor ◽  
Mouse Bone Marrow ◽  
Human Leukemia ◽  
Knock Down ◽  
Low Levels

Abstract MEIS proteins constitute an important subgroup of three amino acid loop extension (TALE) class transcription factors, which are characterized by an atypical homeodomain. MEIS1 has been shown to act as an important cofactor in HOXA9-mediated leukemogenesis in bone marrow transduction experiments. In human leukemia, MEIS1 was identified as a gene consistently upregulated in leukemias with MLL translocations. However, MEIS1 overexpression is also frequently found in AMLs with normal or complex karyotypes. To investigate the role of MEIS1 in AML pathogenesis, we measured the expression of MEIS1 and MEIS2 in 26 different established human AML cell lines, and documented high levels of expression of one or both genes in 20 of these lines. Despite the prevalence of MEIS1 expression in AML blasts, the downstream pathways and their contribution to the proliferation or self-renewal of the malignant clone are not well understood. To identify gene expression programs controlled by MEIS1, we targeted MEIS1 expression by RNAi in two AML cell lines that express HOXA9 and MEIS1, but only low levels of MEIS2. In comparison to a control shRNA, cells expressing two different MEIS1-specific shRNAs were markedly deficient in thymidine incorporation indicating very low levels of cellular proliferation. The RNAi-mediated knock-down of MEIS1 expression resulted in down-regulation of the tyrosine kinase receptor FLT3 by quantitative RT-PCR. Thus our data in human AML is consistent with results of Wang et al. (Blood, 106(1):254–64) implicating MEIS1 in the regulation of FLT3 tyrosine kinase expression, based on overexpression studies in mouse bone marrow progenitors. Our results in human AML suggest that MEIS1 is required for high levels of FLT3 expression, which is significant because FLT3 is mutated and activated in these two myeloid leukemia cell lines, suggesting the hypothesis that FLT3 is an important component of the pathway through which MEIS1 promotes proliferation. Programmed re-expression of activated FLT3 in AML lines with MEIS knock-down is underway to test whether FLT3 is the essential component acting downstream of MEIS1 expression in AML pathogenesis.

Study Of Activity Of E6201, a Dual FLT3 and MEK Inhibitor, In Acute Myelogenous Leukemia With FLT3 Or RAS Mutation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2683-2683 ◽  
Author(s):  
Weiguo Zhang ◽  
Gautam Borthakur ◽  
Chen Gao ◽  
Ye Chen ◽  
Yong S. Lan ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Inhibitory Activity ◽  
Point Mutations ◽  
Myelogenous Leukemia ◽  
Vehicle Group ◽  
Wild Type ◽  
Ras Mutation ◽  
Induced Apoptosis ◽  
Resistant Cells

Abstract Internal tandem duplication (ITD) or point mutation of Fms-like tyrosine kinase 3 (FLT3) and N/KRAS mutations in patients with acute myeloid leukemia (AML) lead to aberrant activation of FLT3 and/or RAS–mitogen-activated protein kinase (MAPK) pathways and are associated with poor prognosis (Kottaridis et al, Leuk Lymphoma Vol. 44:905, 2003; Thiede et al, Blood Vol. 99;4326, 2002). Therapy with inhibitors targeting these pathways individually may at best result in short lasting responses in the appropriate mutational context (Borthakur et al, Haematologica Vol. 96:62, 2011; Cortes et al, Blood Vol. 114:636a, 2009). Persistent activation of MEK/ERK signaling pathway is seen in cells resistant to FLT3-ITD inhibitor sorafenib that harbor acquired point mutations of FLT3 in tyrosine kinase domains in addition to ITD mutation (Moore et al, Leukemia Vol. 26:1462, 2012). E6201 is a MEK1/FLT3 dual inhibitor with inhibitory activity in low nanomolar concentrations against both targets. We tested E6201 against AML cells including FLT3-inhibitor resistant cells, AML patient samples and investigated its efficacy in murine AML model. E6201 inhibited cell growth and induced apoptosis in AML cells with FLT3 ITD mutations (including sorafenib-resistant cells harboring ITD plus N676D/Y842C point mutations) at nanomolar levels, and showed 600 to 1000-fold more selective activity against cells with FLT3-ITD mutations than those with FLT3-WT (IC50s 0.003µM, 0.005 and 0.002µM, respectively, in Ba/F3-ITD and FLT3-ITD mutant MOLM13 and MV4-11cells compared to 3.18µM in Ba/F3-FLT3-WT cells). In addition, OCI/AML3 cells [FLT3 and RAS wild-type(WT)], which have high basal p-ERK level and are resistant to most of chemotherapeutic drugs, were sensitive to E6201 (IC50 = 0.037µM). Consistent with its MEK1 inhibitory activity, E6201 was more active against NRAS mutation carrying OCI/AML3 and MV4-11 cells than their NRAS-WT isogenic cells. E6201-induced apoptosis appears to be p53 dependent as p53-wild-type OCI/AML3 and MOLM13 cells were significantly more sensitive compared to their paired p53-knockdown cells. EC50 of E6201 was at sub-micromolar levels in all 5 FLT3-ITD mutant primary AML samples, which included one with FLT3-ITD/RAS dual mutation. NOG mice bearing xenografts of MOLM13-Luc-GFP (FLT3-ITD mutated) cells were treated with E6201 i.v. starting on day 5 after leukemia cell injection until day 21 on a twice-per-week schedule. Bioluminescence imaging revealed that the tumor burden (mean luminescence) was reduced (3.1 x 106 and 2.7 x 106vs. 5.6 x 106 Photons/sec, p< .01 in 20mg/kg and 40mg/kg groups compared with vehicle group)(Fig 1) and histologically leukemia cells infiltrations were profoundly reduced in the bone marrows, spleens, livers and lungs on Day 9 after first drug treatment. Additionally, the median survival was modestly extended from 16 days of vehicle-treated mice to 18 days of E6201-treated mice (P <0.01). Mechanistically, E6201 significantly suppressed p-FLT3 and p-ERK in all tested FLT3 mutant AML cell lines and p53 wild-type OCI/AML3 cells. In addition, decrease of Bcl-xL and Mcl-1 levels and increase of cleaved-caspase-3 was observed in all FLT3 mutant cell lines after treatment with E6201 for 24 hours. In conclusion, E6201 is active against AML cells with FLT3 and/or RAS mutation. A clinical trial is in development in FLT3 and/or RAS mutant AML. Disclosures: Borthakur: Eisai, Inc.: Research Funding. Nomoto:Eisai, Inc.: Employment. Zhao:Eisai, Inc.: Employment.

scholarly journals Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

1986 ◽  
Vol 6 (5) ◽  
pp. 1803-1811 ◽  
Author(s):  
L Naldini ◽  
A Stacchini ◽  
D M Cirillo ◽  
M Aglietta ◽  
F Gavosto ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Chronic Myelogenous Leukemia ◽  
Kinase Activity ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Kinase Assay ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Viral Oncogene

Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells

1986 ◽  
Vol 6 (5) ◽  
pp. 1803-1811
Author(s):  
L Naldini ◽  
A Stacchini ◽  
D M Cirillo ◽  
M Aglietta ◽  
F Gavosto ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Chronic Myelogenous Leukemia ◽  
Kinase Activity ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Kinase Assay ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Viral Oncogene

Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.

RNAi-induced down-regulation of FLT3 expression in AML cell lines increases sensitivity to MLN518

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2952-2954 ◽  
Author(s):  
Denise K. Walters ◽  
Eric P. Stoffregen ◽  
Michael C. Heinrich ◽  
Michael W. Deininger ◽  
Brian J. Druker
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Present Report ◽  
Phase 1 ◽  
Myelogenous Leukemia ◽  
Human Leukemia ◽  
Down Regulation ◽  
Flt3 Inhibitor ◽  
Acute Myelogenous ◽  
Flt3 Expression

AbstractFMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in approximately 30% of acute myelogenous leukemia (AML) patients and appears to confer an adverse prognosis. Thus, development of inhibitors and/or antibodies that specifically target FLT3 has been of substantial interest. In this regard, phase 1 and 2 trials involving FLT3 inhibitors have recently reported FLT3 inhibition and leukemic blast reduction in some patients. Despite this, issues such as specificity and resistance need to be addressed. Consequently, the development of alternative approaches for targeting FLT3 would be of great consequence. In the present report, we demonstrate that FLT3 siRNA effectively down-regulates FLT3 expression in Ba/F3 cells transfected with FLT3 containing an activating internal tandem duplication (ITD) in the juxtamembrane domain and FLT3-ITD–positive Molm-14 human leukemia cells. Treatment with the FLT3 siRNA results in growth inhibition and apoptosis of these cells. Furthermore, siRNA-induced down-regulation of FLT3 increased the sensitivity of both cell lines to treatment with the FLT3 inhibitor MLN518. This illustrates the potential benefit of combined therapeutic approaches.

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