Molecular targeting in acute myeloid leukemia (AML) therapy: APcK110 is a new inhibitor of c-Kit activity in AML blasts

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7019-7019
Author(s):  
S. Faderl ◽  
W. Bornmann ◽  
D. Maxwell ◽  
Z. Peng ◽  
Q. Van ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Potent Inhibitor ◽  
Kinase Assay ◽  
Inhibition Of Proliferation ◽  
Kit Mutation ◽  
Downstream Signaling ◽  
Dose Dependent Fashion ◽  
Dose Dependent

7019 Background: Tyrosine kinases (TK) are important for most cellular functions and require stringent control and regulation. C- Kit, a TK and transmembrane receptor for stem cell factor, plays a crucial role in hematopoiesis. Mutations of c-Kit can contribute to AML pathophysiology and have been associated with shorter survival and higher relapse risk following standard AML therapy. On the other hand, responses have been observed following TK inhibitor therapy such as imatinib. Identification of c-Kit or its pathways in AML leukemogenesis and development of new compounds targeting these mutations may therefore hold promise for therapy. Methods: APcK110 is the result of a structure-based design of c-Kit inhibitors and was derived of a set of compounds with favorable IC50 values in a c-Kit kinase assay. Here we present results of the activity and mechanism of action of APcK110. We used the mastocytosis cell line HMC1.1 carrying an activating c-Kit mutation and two AML lines (OCIM2 and OCI/AML3) as well as primary samples from patients with AML. Results: We demonstrate that: (1) APcK110 inhibits proliferation of all three cell lines in a dose dependent fashion using an MTT assay. Inhibition of proliferation is most significant in the SCF-dependent cell line OCI/AML3; (2) inhibition of OCIM2 cells (SCF-responsive) by APcK110 can be enhanced when adding SCF suggesting recruitment of c-Kit-dependent signaling components and increased activity of the c-Kit inhibitor under these circumstances; (3) APcK110 is a more potent inhibitor of OCI/AML3 proliferation than imatinib and dasatininb; (4) using Western immunoblotting, APcK110 decreases levels of phospho-Akt, phospho-Stat3 and 5 in a time- and dose-dependent fashion demonstrating activity of APcK110 on c-Kit downstream signaling pathways; (5) APcK110 induces apoptosis by cleavage of caspase 3 and PARP; (6) APcK110 inhibits proliferation of primary AML cells in a clonogenic assay, but does not affect proliferation of normal controls. Conclusions: APcK110 is a potent inhibitor of AML cell lines and primary samples from patients with AML. Activation of c-Kit or downstream pathways increases activity of APcK110. APcK110 and similar compounds should be evaluated in clinical trials of patients with AML. No significant financial relationships to disclose.

APCK110, a Novel and Potent Inhibitor of c-Kit, Blocks Phosphorylation of AKT and STAT3, Induces Apoptosis, and Inhibits Proliferation of Acute Myeloid Leukemia (AML) Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 153-153 ◽  
Author(s):  
Stefan Faderl ◽  
William Bornmann ◽  
David Maxwell ◽  
Ashutosh Pal ◽  
Zheng-Hong Peng ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cell Lines ◽  
Caspase 3 ◽  
Potent Inhibitor ◽  
Antiproliferative Effect ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Western Immunoblotting ◽  
Inhibition Of Proliferation

Abstract Tight control of protein tyrosine kinase (TK) activity is crucial for the regulation and maintenance of vital cellular functions such as proliferation, differentiation, and apoptosis. c-KIT is a TK and transmembrane receptor for stem cell factor (SCF). Binding of SCF to c-KIT results in activation of marrow precursors and other blood cells. Activating mutations of c-KIT associated with amino acid Asp-816 (D816) have been identified in leukemic cells of patients with AML and are thought to play an important pathophysiologic role in leukemogenesis. Identification of activating c-KIT mutations and development of novel compounds targeting these mutations may therefore be of therapeutic benefit in AML. Based on the 3-dimensional structure of c-KIT we have generated a number of compounds with activity against c-KIT mutated cells. Here we present initial results of the activity and mechanism of action of the novel c-KIT inhibitor APCK110 in AML cell lines and primary samples from patients with AML. Using an MTT assay, we first studied the antiproliferative effect of APCK110 in the AML cell lines OCI/AML3 and the SCF-responsive cell line OCIM2. Cells were incubated for 72 hours without or with APCK110 at concentrations of 50, 100, 250, and 500 nM, then harvested and their metabolic activity and viability determined as optical density. Next we determined expression of phospho-AKT and -STAT3 in the mastocytosis cell line HMC1.1 and phospho-c-KIT in the AML cell line OCI/AML3 by Western Immunoblotting. We further analyzed induction of caspase 3 and PARP cleavage in OCI/AML3 cells at APCK110 concentrations of 50, 100, 250, and 500 nM using Western Immunoblotting. To demonstrate the effect of APCK110 on primary AML cells, we incubated diagnostic marrow cells from 3 patients with AML with increasing concentrations of APCK110 and used the blast colony assay to measure inhibition of proliferation. We then compared the antiproliferative effect of APCK110 with that of dasatinib and imatinib in OCI/AML3 cells. We show that 1) APCK110 strongly inhibits proliferation of AML cells with 80% inhibition at 500 nM; 2) similar to cell lines, APCK110 also inhibits AML colony growth of primary samples in a dose-dependent manner of up to 80% at 500 nM concentration; 3) APCK110 blocks activation of phospho-AKT, phospho-STAT3, and phospho-c-KIT; 4) APCK110 induces apoptosis by cleavage of caspase 3 and PARP; and 5) APCK110 demonstrates more potent inhibition (up to 100% at 500 nM) of AML cell proliferation than dasatinib (60% at 500 nM) and dasatinib (none at 500 nM). In summary, APCK110 is a novel and potent inhibitor of mutated c-KIT that inhibits AML cell proliferation, blocks activation of intracellular signaling molecules, and induces caspase-dependent apoptosis. Further development of APCK110 for clinical trials of patients with AML should be pursued.

Antioxidant effects and in vitro cytotoxicity on human cancer cell lines of flavonoid-rich Flamboyant (Delonix regia (Bojer) Raf.) flower extract

2020 ◽  
Vol 21 ◽  
Author(s):  
Putthiporn Khongkaew ◽  
Phanphen Wattanaarsakit ◽  
Konstantinos I. Papadopoulos ◽  
Watcharaphong Chaemsawang
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Flower Extract ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Murine Leukemia Cell ◽  
Dose Dependent

Background: Cancer is a noncommunicable disease with increasing incidence and mortality rates both worldwide and in Thailand. Its apparent lack of effective treatments is posing challenging public health issues. Introduction: Encouraging research results indicating probable anti-cancer properties of the Delonix regia flower extract (DRE) have prompted us to evaluate the feasibility of developing a type of product for future cancer prevention or treatment. Methods and Results: In the present report, using High Performance Liquid Chromatography (HPLC), we demonstrate in the DRE, the presence of high concentrations of three identifiable flavonoids, namely rutin 4.15±0.30 % w/w, isoquercitrin 3.04±0.02 %w/w, and myricetin 2.61±0.01 % w/w respectively while the IC50 of DPPH and ABTS assay antioxidation activity was 66.88±6.30 µg/ml and 53.65±7.24 µg/ml respectively. Discussion: Our cancer cell line studies using the MTT assay demonstrated DREs potent and dose dependent inhibition of murine leukemia cell line (P-388: 35.28±4.07% of cell viability remaining), as well as of human breast adenocarcinoma (MCF-7), human cervical carcinoma (HeLa), human oral cavity carcinoma (KB), and human colon carcinoma (HT-29) cell lines in that order of magnitude. Conclusion: Three identifiable flavonoids (rutin, isoquercitrin and myricetin) with high antioxidation activity and potent and dose dependent inhibition of murine leukemia cell line and five other cancer cell lines were documented in the DRE. The extract’s lack of cytotoxicity in 3 normal cell lines is a rare advantage not usually seen in current antineoplastic agents. Yet another challenge of the DRE was its low dissolution rate and long-term storage stability, issues to be resolved before a future product can be formulated.

Design, Synthesis, Molecular Docking, and Anticancer Evaluation of Pyrazole Linked Pyrazoline Derivatives with Carbothioamide Tail as EGFR Kinase Inhibitors

2020 ◽  
Vol 21 (1) ◽  
pp. 42-60
Author(s):  
Farah Nawaz ◽  
Ozair Alam ◽  
Ahmad Perwez ◽  
Moshahid A. Rizvi ◽  
Mohd. Javed Naim ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Cancer Cell Lines ◽  
Kinase Assay ◽  
Cervix Uteri ◽  
Egfr Kinase

Background: The Epidermal Growth Factor Receptor (known as EGFR) induces cell differentiation and proliferation upon activation through the binding of its ligands. Since EGFR is thought to be involved in the development of cancer, the identification of new target inhibitors is the most viable approach, which recently gained momentum as a potential anticancer therapy. Objective: To assess various pyrazole linked pyrazoline derivatives with carbothioamide for EGFR kinase inhibitory as well as anti-proliferative activity against human cancer cell lines viz. A549 (non-small cell lung tumor), MCF-7 (breast cancer cell line), SiHa (cancerous tissues of the cervix uteri), and HCT-116 (colon cancer cell line). Methods: In vitro EGFR kinase assay, in vitro MTT assay, Lactate dehydrogenase release, nuclear staining (DAPI), and flow cytometry cell analysis. Results: Compounds 6h and 6j inhibited EGFR kinase at concentrations of 1.66μM and 1.9μM, respectively. Furthermore, compounds 6h and 6j showed the most potent anti-proliferative results against the A549 KRAS mutation cell line (IC50 = 9.3 & 10.2μM). Through DAPI staining and phase contrast microscopy, it was established that compounds 6h and 6j also induced apoptotic activity in A549 cells. This activity was further confirmed by FACS using Annexin-V-FITC and Propidium Iodide (PI) labeling. Molecular docking studies performed on 6h and 6j suggested that the compounds can bind to the hinge region of ATP binding site of EGFR tyrosine kinase in a similar pose as that of the standard drug gefitinib. Conclusion: The potential anticancer activity of compounds 6h and 6j was confirmed and need further exploration in cancer cell lines of different tissue origin and signaling pathways, as well as in animal models of cancer development.

scholarly journals Proton-irradiated breast cells: molecular points of view

2019 ◽  
Vol 60 (4) ◽  
pp. 451-465 ◽  
Author(s):  
Valentina Bravatà ◽  
Francesco P Cammarata ◽  
Luigi Minafra ◽  
Pietro Pisciotta ◽  
Concetta Scazzone ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Fate ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Proton Irradiation ◽  
Expression Profiles ◽  
Treatment Plan ◽  
Mcf10a Cell ◽  
Specific Cell ◽  
Dose Dependent

Abstract Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome

scholarly journals Transcriptome of Two Canine Prostate Cancer Cells Treated With Toceranib Phosphate Reveals Distinct Antitumor Profiles Associated With the PDGFR Pathway

2020 ◽  
Vol 7 ◽  
Author(s):  
Priscila E. Kobayashi ◽  
Patrícia F. Lainetti ◽  
Antonio F. Leis-Filho ◽  
Flávia K. Delella ◽  
Marcio Carvalho ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Growth Factor ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Dependent Manner ◽  
Upregulated Genes

Canine prostate cancer (PC) presents a poor antitumor response, usually late diagnosis and prognosis. Toceranib phosphate (TP) is a nonspecific inhibitor of receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and c-KIT. This study aimed to evaluate VEGFR2, PDGFR-β, and c-KIT protein expression in two established canine PC cell lines (PC1 and PC2) and the transcriptome profile of the cells after treatment with TP. Immunofluorescence (IF) analysis revealed VEGFR2 and PDGFR-β protein expression and the absence of c-KIT protein expression in both cell lines. After TP treatment, only the viability of PC1 cells decreased in a dose-dependent manner. Transcriptome and enrichment analyses of treated PC1 cells revealed 181 upregulated genes, which were related to decreased angiogenesis and cell proliferation. In addition, we found upregulated PDGFR-A, PDGFR-β, and PDGF-D expression in PC1 cells, and the upregulation of PDGFR-β was also observed in treated PC1 cells by qPCR. PC2 cells had fewer protein-protein interactions (PPIs), with 18 upregulated and 22 downregulated genes; the upregulated genes were involved in the regulation of parallel pathways and mechanisms related to proliferation, which could be associated with the resistance observed after treatment. The canine PC1 cell line but not the PC2 cell line showed decreased viability after treatment with TP, although both cell lines expressed PDGFR and VEGFR receptors. Further studies could explain the mechanism of resistance in PC2 cells and provide a basis for personalized treatment for dogs with PC.

NaCl but not urea activates p38 and jun kinase in mIMCD3 murine inner medullary cells

1996 ◽  
Vol 271 (6) ◽  
pp. F1234-F1238 ◽  
Author(s):  
Z. Zhang ◽  
D. M. Cohen
Keyword(s):  
Cell Line ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Duct Cell ◽  
Kinase Assay ◽  
Cellular Protection ◽  
Jun Kinase ◽  
Mitogen Activated Protein ◽  
Dose Dependent ◽  
In Cells

The mitogen-activated protein kinases (MAPKs), p38 and jun kinase (JNK), are activated by diverse stressors in cells of nonrenal medullary origin. Epithelial cells of the renal medulla are among the very few cells of higher eukaryotes routinely subjected to hyperosmotic stress, composed of principally NaCl and urea. Hyperosmotic NaCl activated p38 and JNK in a time- and dose-dependent fashion in cells of the murine terminal inner medullary collecting duct cell line (mIMCD3) as determined by immune complex kinase assay. Hyperosmotic urea exerted a minimal effect upon only p38 activation, which was evident only at 5 min. The NaCl effect was dose dependent to 800 mosmol/kgH2O; 800 mosmol/kgH2O urea, in contrast, exerted no effect. Consistent with these observations, NaCl (800 mosmol/kgH2O) but not urea (800 mosmol/kgH2O) increased tyrosine phosphorylation of p38 and JNK at 10 min. Therefore, even in the extremely osmotolerant renal medullary mIMCD3 cell line, derived from a tissue adapted for routine exposure to elevated osmolality, hypertonic NaCl activated two stress-responsive MAPKs. Urea, in contrast, exerted virtually no effect; therefore, cellular protection from urea stress operates through a mechanism distinct from the stress-responsive MAPKs.

WP-1034, a Novel Jak-Stat Inhibitor, Has Proapoptotic and Antileukemic Activity in Acute Myeloid Leukemia (AML) Cell Lines and AML Patient Samples.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2528-2528
Author(s):  
Stefan Faderl ◽  
Alessandra Ferrajoli ◽  
David Harris ◽  
Quin Van ◽  
Waldemar Priebe ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinases ◽  
Caspase 3 ◽  
Potent Inhibitor ◽  
Annexin V ◽  
Parp Cleavage ◽  
Western Immunoblotting ◽  
Inhibition Of Proliferation ◽  
G0 Phase ◽  
Marrow Cells

Abstract Proliferation and growth of AML cells result from stimulation by cytokines and high levels of cytokines are associated with poor prognosis in AML. Cytokines act through cellular receptors that are associated with members of the Jak family of protein tyrosine kinases. Upon phosphorylation and activation of Jak, proteins bound to Jak initiate signaling pathways including those regulated by Stat proteins. Since constitutive activation of Jak-Stat has been associated with leukemogenesis, we hypothesize that inhibition of Jak-Stat inhibits proliferation of AML cells. To do this, we studied the effects of WP-1034, a novel and potent inhibitor of Jak-Stat, in the OCIM2 AML cell line and fresh samples from AML patients. OCIM2 cells were deprived of serum for 2 hours and then incubated with 1 to 5 μM WP-1034 to investigate its effect on OCIM2 cell proliferation. After incubation of the cells without and with 1, 2.5, 5, 7.5, and 10 μM WP-1034 for 1 hour, and at 5 μM for 0, 20 min, 40 min, and 1, 2, 3, and 4 hours, we determined expression of Stat 1, 3, and 5, as well as Phospho-stat 1, 3, and 5 in the cells by Western Immunoblotting. In addition, we analyzed cell cycle status by PI staining and flow cytometry. We further evaluated induction of apoptosis of OCIM2 cells following incubation with WP-1034 at 3, 5, and 6 μM using the annexin V-CY5 assay and analyzed caspase 3 and PARP cleavage using Western Immunoblotting. To demonstrate the effect of WP-1034 on marrow cells from AML patients and healthy volunteers we incubated marrow cells with WP-1034 at increasing concentrations and used the blast colony assay to measure inhibition of proliferation. Our results show that: i) WP-1034 effectively inhibits proliferation of OCIM2 cells and AML blast proliferation from patient samples; ii) WP-1034 blocks activation of Stat 3 and 5 by decreasing the amount of Phospho-stat 3 and 5 in OCIM2 cells; iii) WP-1034 causes cell cycle arrest in sub-G0 phase and is able to induce apoptosis in OCIM2 cells; and iv) WP-1034 induces apoptosis involving cleavage of caspase 3 and PARP. Our data suggest that WP-1034, a potent inhibitor of Jak-Stat, inhibits proliferation of AML cells by inhibition of Stat 3 and 5 and induction of caspase-dependent apoptosis.

Effects of AMN107, a Novel Aminopyrimidine Tyrosine Kinase Inhibitor, on Human Mast Cells Bearing Wild-Type or Mutated Codon 816 c-kit.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3528-3528 ◽  
Author(s):  
Srdan Verstovsek ◽  
Cem Akin ◽  
Giles J. Francis ◽  
Manshouri Taghi ◽  
Ly Huynh ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Kit Mutation

Abstract Background. Majority of adult patients with systemic mastocytosis (SM) have activating mutation in codon 816 of c-kit (CD117), a receptor on the surface of mast cells. This abnormality is responsible for the pathogenesis of the disease. Methods. We investigated the effects of a newly designed tyrosine kinase inhibitor, AMN107, by comparing its in vitro inhibitory potency on c-kit mutated mast cell lines and patient samples with that of imatinib mesylate, another tyrosine kinase inhibitor, effective in some patients with SM. Two cell lines, subclones of HMC-1 cells, were used: HMC-1560 carrying juxtamembrane domain mutation in codon 560 of c-kit, and HMC-1560, 816 carrying both codon 560 mutation and tyrosine kinase domain mutation in codon 816 of c-kit. Results. In HMC-1560 mast cell line carrying wild-type codon 816, AMN107 was as potent as imatinib in inhibiting cellular proliferation, with IC50 values of 108 and 74 nM respectively, while in HMC-1560, 816 cell line carrying 816 mutation, neither medication had an effect. AMN107 was also as effective as imatinib in inhibiting phosphorylation of c-kit tyrosine kinase in HMC-1560 cells. The inhibition of cellular proliferation was associated with induction of apoptosis in HMC-1560 cells. AMN107 in concentrations up to 1 uM had no effect on bone marrow mast cells carrying D816V c-kit mutation obtained from patients with mastocytosis. Conclusions. Our results suggest similar potency of AMN107 and imatinib in mast cells that carry wild-type codon 816, but no activity against codon 816 mutation carrying cells.

AC220 Is a Uniquely Potent and Selective Second-Generation FLT3 Inhibitor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 859-859 ◽  
Author(s):  
Patrick P Zarrinkar ◽  
Robert C Armstrong ◽  
Ruwanthi N Gunawardane ◽  
Joyce James ◽  
Mazen W Karaman ◽  
...  
Keyword(s):  
Cell Line ◽  
Tyrosine Kinases ◽  
Second Generation ◽  
Receptor Tyrosine Kinases ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Inhibition Of Proliferation ◽  
Flt3 Inhibitor ◽  
Pharmacokinetic Properties ◽  
Flt3 Inhibitors

Abstract Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30 % of acute myeloid leukemia (AML) patients, implicating FLT3 as a potential target for kinase inhibitor therapy. While several compounds have been evaluated in the clinic as FLT3 inhibitors, including CEP-701 (lestaurtinib), PKC-412 (midostaurin), MLN-518 (tandutinib) and most recently sorafenib, none of these was originally developed as a FLT3 inhibitor. We report here the characterization of AC220, a novel compound that has been expressly optimized as a selective FLT3 inhibitor. We demonstrate that this second generation FLT3 inhibitor has a unique combination of high potency, exceptional selectivity, bioavailability, and pharmacokinetic properties compatible with once a day oral dosing. In an in vitro binding assay AC220 interacted with FLT3 with high affinity (Kd = 1.6 nM). In the FLT3 dependent human leukemia cell line MV4-11, which harbors a homozygous activating FLT3 internal tandem duplication (ITD) mutation, AC220 inhibited FLT3 autophosphorylation and cellular proliferation with subnanomolar potency. Inhibition of proliferation of a FLT3 independent cell line was several hundred-fold less potent, demonstrating cellular selectivity for FLT3. A biochemical screen against a panel of 402 kinase assays representing almost 80 % of human protein kinases revealed a highly focused and selective interaction pattern. The only targets with affinity for AC220 within 10-fold that for FLT3 were closely related class III receptor tyrosine kinases (KIT, PDGFR, RET, CSF1R), and the only targets with affinity within 100-fold that for FLT3 were four additional receptor tyrosine kinases (FLT1, FLT4, DDR1, VEGFR2). When orally administered to mice at a dose of 10 mg/kg, AC220 achieved a peak plasma concentration (Cmax) of 3.8 μM (2,100 ng/mL) within two hours of dosing. When corrected for plasma protein binding, the concentration of AC220 in plasma remained above the cellular IC50 for FLT3 inhibition 24 hours after dosing. Total exposure (AUC0-24 h) as well as Cmax increased proportionally with the administered dose from 0.1 to approximately 30 mg/kg. At higher doses, both Cmax and AUC0-24 h continued to increase, approaching a plateau above 100 mg/kg. In a FLT3-ITD-dependent MV4-11 tumor xenograft model, AC220 showed substantial, dose dependent efficacy when dosed at 1, 3 and 10 mg/kg orally once a day for 28 days. Tumors regressed at 3 and 10 mg/kg, and remained static at 1 mg/kg. In a follow-on study at the 10 mg/kg oral dose, tumor size was monitored for an additional 60 days after dosing was discontinued. By the end of the study eight complete responses and two partial responses were observed in the ten animals treated with AC220. AC220 also had activity in a leukemia tumor model at doses as low as 1 mg/kg given orally once a day. A direct comparison of AC220 with the first generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib and sunitinib revealed that the combination of potency, selectivity and pharmacokinetic properties is unique to AC220. AC220 is a second generation FLT3 inhibitor that has been explicitly optimized for the combination of properties believed to be required for the successful treatment of FLT3-dependent AML, and specifically to test the hypothesis that selective FLT3 inhibition will result in clinical benefit. AC220 is currently being evaluated in a phase I clinical trial in relapsed or refractory AML patients.

Evidence for Cooperation of Receptor Tyrosine Kinases and Activating NOTCH Mutations to Hyperactivate mTOR in T-Cell Leukemia: A Rationale Basis for Targeted Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1381-1381
Author(s):  
Adrian Schwarzer ◽  
Johann Meyer ◽  
Martijn Brugman ◽  
Axel Schambach ◽  
Martin Stanulla ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Microarray Analysis ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Akt Activation ◽  
Rtk Signaling

Abstract Abstract 1381 T-cell acute lymphoblastic leukemia (T-ALL) remains a therapeutic challenge. T-ALLs are characterized by recurring chromosomal rearrangements causing aberrant expression of transcription factors (Myb; TAL/SCL; HOX) dividing patients into different subgroups. Activating mutations in NOTCH, the master regulator of T-cell development, are found in more than 60% of T-ALLs independently of subtype. Most T-ALLs display a hyperactivation of the PI3K-AKT-mTOR pathway, a potential target for therapeutic intervention. The master regulator of PI3K-AKT signalling is PTEN, which is frequently inactivated in cancer. Recent data suggests that complete PTEN loss due to mutation is rare in primary human T-ALL, whereas PTEN-inhibiting posttranslational modifications are more common (Barata et al., J. Clin. Invest. 2008, 118). As these modifications decrease, but do not abolish the phosphatase activity of PTEN, we hypothesized that further input from tyrosine kinases, particularly receptor tyrosine kinases (RTK), may be needed to sustain PI3K-AKT-mTOR activation. In order to investigate how RTK-signaling may contribute to the pathogenesis of T-ALL we used an established murine bone marrow transplantation model (Li et al. Blood 2009, 113). To mimic tyrosine-kinase signaling we expressed δTrkA, a constitutively active TRKA receptor tyrosine kinase (TRK =tropomyosin-related kinase) from gammaretroviral or lentiviral vectors in c-kit+ Sca-1+ Lin− (KSL) cells. Intravenous injection of δTrkA-transduced hematopoietic cells in C57BL6 mice (n=10) induced transplantable T-ALL with a latency of about 120 days. The resulting T-ALLs could be propagated in culture as clonal cell lines. Signaling studies showed that δTRKA activates predominantly ERK upon expression in murine hematopoietic cell lines. However, the obtained δTRKA+ T-ALL lines (n=7) showed a profound shift in the use of downstream signaling cascades, displaying a very high activation of AKT-mTOR and absent ERK phosphorylation, resembling human T-ALL. High AKT activation was uniformly detected regardless of PTEN protein expression in all but one T-ALL (#003). To understand the rewired signaling network we looked for a potential contribution of insertional mutagenesis and chromosomal aberrations. Array-CGH showed homozygous deletions on chr14c2 involving the T-cell receptor alpha and delta genes in 3/3 cell lines and heterozygous deletions in Ikzf1 in 2/3 cell lines. Viral integration sites showed no common insertion pattern and no insertion in genes implicated in RTK-signaling. The expression of genes in proximity to viral integrations (±500 kb) appeared unaltered as determined by cDNA-microarray analysis of the T-ALL cell line #483 against wild type CD4+CD8+ thymocytes. Microarray analysis revealed enrichment of Notch1 target genes in the T-ALL cell line #483. Sequencing of Notch1 revealed both, PEST domain mutations and the recently described (Aster et al, Blood 2010, 116) RAG mediated 5'-deletions in cis, in all but one investigated T-ALL. Northern and Western Blots confirmed the expression of truncated Notch1 transcripts and protein, respectively. The one cell line (#003) which retained the original δTrkA signaling pattern had no Notch mutation and could only be cultured on OP9-Delta-like-1 stroma cells, highlighting the importance of Notch signaling. As this cell line was established from a mouse displaying an enlarged thymus, but no full manifestation of T-ALL, our data suggests that acquisition of Notch mutations is a late, but necessary step required for overt leukemia, whereas the initiating events may arise in kinase signaling pathways of prethymic progenitors. All T-ALL cell lines were sensitive to mTOR or Notch inhibition with Rapamycin or Compound E, respectively. Finally, we used phosphoprotein-arrays to monitor the phosphorylation of 42 RTK in childhood T-ALL samples with different activating NOTCH mutations (n=5) and detected several activated RTK (e.g. MSPR, FGFR, ErbB4, VEGFR) in the patient samples. Taken together, our findings suggest a cooperation of RTK and activating NOTCH mutations in mTOR activation seen in T-ALL and encourage further investigation of 1) aberrant RTK-signaling in T-ALL 2) the role of RTK activation in creating a preleukemic cell clone, 3) evaluation of combined therapy targeting RTKs and NOTCH, and 4) the role of activated NOTCH on mTORC2-AKT activation independently of PTEN. Disclosures: Baum: Patent office: Patents & Royalties.

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