ABSTRACT
PURPOSE: Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. Given the relevance of FLT3 mutations in AML, we investigated the activity of CHIR-258, an orally active, multitargeted small molecule, with potent activity against FLT3 kinase and class III, IV, and V RTKs involved in endothelial and tumor cell proliferation in AML models. EXPERIMENTAL DESIGN: CHIR-258 was tested on two human leukemic cell lines in vitro and in vivo with differing FLT3 mutational status [MV4;11 cells express FLT3 internal tandem duplications (ITD) versus RS4;11 cells with wild-type (WT) FLT3]. RESULTS: Antiproliferative activity of CHIR-258 against MV4;11 was approximately 24-fold greater compared with RS4;11, indicating more potent inhibition against cells with constitutively activated FLT3 ITD. Dose-dependent down modulation of receptor phosphorylation and downstream signaling [signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase] in MV4;11 cells with CHIR-258 confirmed the molecular mechanism of action. Target modulation of phospho-FLT3, phospho-STAT5, and phospho-ERK in MV4;11 tumors was achieved at biologically active doses of CHIR-258. Tumor regressions and eradication of AML cells from the bone marrow were shown in s.c. and bone marrow engraftment leukemic xenograft models. Tumor responses were characterized by decreased cellular proliferation and positive immunohistochemical staining for active caspase-3 and cleaved poly(ADP-ribose) polymerase, suggesting cell death was mediated in part via apoptosis. CONCLUSIONS: Our data indicate that CHIR-258 may be an effective therapy in FLT3-associated AML and warrants clinical trials.
Subject(s)
Benzimidazoles/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Proto-Oncogene Proteins/genetics , Quinolones/pharmacology , Receptor Protein-Tyrosine Kinases/genetics , Animals , Cell Proliferation , DNA Mutational Analysis , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Humans , Immunohistochemistry , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/veterinary , Mice , Mice, SCID , Neoplasm Transplantation , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Tandem Repeat Sequences , Transplantation, Heterologous , Tumor Cells, Cultured , fms-Like Tyrosine Kinase 3ABSTRACT
SU6668 is a small molecule inhibitor of the angiogenic receptor tyrosine kinases Flk-1/KDR, PDGFRbeta, and FGFR1. In mice, SU6668 treatment resulted in regression or growth arrest of all large established human tumor xenografts examined associated with loss of tumor cellularity. The events underlying loss of tumor cellularity were elucidated in detail in several tumor models. SU6668 treatment induced apoptosis in tumor microvessels within 6 h of the initiation of treatment. Dose-dependent decreases in tumor microvessel density were observed within 3 days of the first treatment. These changes were accompanied by decreased tumor cell proliferation and increased tumor cell apoptosis. Rapid increases in VEGF transcript levels were seen, consistent with the induction of tumor hypoxia. Using Western blot analyses, we determined that these in vivo antiangiogenic and proapoptotic effects of SU6668 occur at doses comparable to those required to inhibit Flk-1/KDR and PDGFRbeta phosphorylation in tumors. Potent, dose-dependent inhibition of Flk-1/KDR activity in vivo was independently demonstrated using vascular permeability as a readout. These data demonstrate that SU6668-induced inhibition of angiogenic receptor tyrosine kinase activity in vivo is associated with rapid vessel killing in tumors, leading to broad and potent antitumor effects.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Indoles/pharmacology , Neoplasms, Experimental/drug therapy , Pyrroles/pharmacology , Receptors, Growth Factor/antagonists & inhibitors , Angiogenesis Inhibitors/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Apoptosis , Blood Vessels/drug effects , Blood Vessels/pathology , Cell Division/drug effects , Dose-Response Relationship, Drug , Humans , Indoles/therapeutic use , Kinetics , Mice , Mice, Nude , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/pathology , Oxindoles , Propionates , Pyrroles/therapeutic use , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Obtaining direct and rapid proof of molecular activity in early clinical trials is critical for optimal clinical development of novel targeted therapies. SU11248 is an oral multitargeted kinase inhibitor with selectivity for fms-related tyrosine kinase 3/Flk2 (FLT3), platelet-derived growth factor receptor alpha/beta, vascular endothelial growth factor receptor 1/2, and KIT receptor tyrosine kinases. FLT3 is a promising candidate for targeted therapy in acute myeloid leukemia (AML), because activating mutations occur in up to 30% of patients. We conducted an innovative single-dose clinical study with a primary objective to demonstrate inhibition of FLT3 phosphorylation by SU11248 in AML. EXPERIMENTAL DESIGN: Twenty-nine AML patients each received a single dose of SU11248, escalated from 50 to 350 mg, in increments of 50 mg and cohorts of three to six patients. FLT3 phosphorylation and plasma pharmacokinetics were evaluated at seven time points over 48 h after SU11248 administration, and FLT3 genotype was determined. Study drug-related adverse events occurred in 31% of patients, mainly grade 1 or 2 diarrhea and nausea, at higher dose levels. RESULTS: Inhibition of FLT3 phosphorylation was apparent in 50% of FLT3-wild-type (WT) patients and in 100% of FLT3-mutant patients. FLT3 internal tandem duplication (ITD) mutants showed increased sensitivity relative to FLT3-WT, consistent with preclinical predictions. The primary end point, strong inhibition of FLT3 phosphorylation in >50% patients, was reached in 200 mg and higher dose cohorts. Downstream signaling pathways were also inhibited; signal transducer and activator of transcription 5 (STAT5) was reduced primarily in internal tandem duplication patients and at late time points in FLT3-WT patients, whereas extracellular signal-regulated kinase (ERK) activity was reduced in the majority of patients, independent of FLT3 inhibition. CONCLUSIONS: This novel translational study bridges preclinical models to the patient setting and provides the first evidence of anti-FLT3 activity in patients. Proof of target inhibition accomplishes a crucial milestone in the development of novel oncology therapeutics.
Subject(s)
Indoles/toxicity , Leukemia, Myeloid, Acute/drug therapy , Proto-Oncogene Proteins/antagonists & inhibitors , Pyrroles/toxicity , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Administration, Oral , Adult , Aged , Blast Crisis/pathology , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/blood , Enzyme Inhibitors/toxicity , Female , Genotype , Humans , Indoles/administration & dosage , Indoles/blood , Leukemia, Myeloid, Acute/pathology , Male , Metabolic Clearance Rate , Middle Aged , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Pyrroles/administration & dosage , Pyrroles/blood , Sunitinib , fms-Like Tyrosine Kinase 3ABSTRACT
One challenging aspect in the clinical development of molecularly targeted therapies, which represent a new and promising approach to treating cancers, has been the identification of a biologically active dose rather than a maximum tolerated dose. The goal of the present study was to identify a pharmacokinetic/pharmacodynamic relationship in preclinical models that could be used to help guide selection of a clinical dose. SU11248, a novel small molecule receptor tyrosine kinase inhibitor with direct antitumor as well as antiangiogenic activity via targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases, was used as the pharmacological agent in these studies. In mouse xenograft models, SU11248 exhibited broad and potent antitumor activity causing regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. To predict the target SU11248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU11248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU11248 selectively inhibited Flk-1/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-100 ng/ml. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for 12 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU11248 in these preclinical studies has aided in the design, selection, and evaluation of dosing regimens being tested in human trials.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrroles/pharmacology , Receptors, Platelet-Derived Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor/metabolism , Animals , Cell Division/drug effects , Female , Humans , Inhibitory Concentration 50 , Kinetics , Mice , Mice, Nude , Models, Chemical , Neoplasm Transplantation , Phosphorylation , Sunitinib , Time Factors , Tumor Cells, CulturedABSTRACT
Acute myeloid leukemia (AML) is associated with dysregulated hematopoietic cell proliferation and increased bone marrow angiogenesis, each regulated by signaling through receptor tyrosine kinases (RTKs). SU5416 is a small molecule inhibitor of VEGF receptors, c-kit and FLT3 and therefore provides a novel opportunity to target both angiogenesis and proliferation in AML. SU5416 was assessed in a phase II hematological malignancy trial in the US, where partial responses were observed in two of 33 patients. Since AML provides a unique platform to evaluate mechanism of action of small molecule inhibitors, investigation of the effect of SU5416 on FLT3 expression and phosphorylation in blood and bone marrow was an additional focus of this trial. Phosphorylated FLT3 was detected by immunoprecipitation/Western analysis in peripheral blood samples from 17 of 22 patients, and seven exhibited strong inhibition of phosphorylation immediately following a 1h SU5416 infusion, demonstrating that SU5416 can modulate RTK phosphorylation in humans. Although no clear correlation with clinical response was observed, analysis of patient plasma drug levels suggested that a threshold SU5416 concentration of 15 microM was associated with FLT3 inhibition. This observation was supported by data from an ex vivo model where AML cells were spiked into human blood, established to mimic the clinical setting and enable more rigorous analysis of effect of SU5416. In addition, FLT3 protein levels were downregulated in patient bone marrow samples, analyzed by an RIA assay. To identify putative predictors of response, patient plasma was analyzed for levels of secreted ligands of SU5416 targets; SCF and FLT3 ligand. Baseline levels of SCF in patients with stable or progressive disease were significantly higher than those in normal donors, whereas FLT3 ligand levels in patients who exhibited progressive disease were significantly lower than those in normal donors. The translational and clinical analyses described in this report provide some insights into the mechanism and duration of action of SU5416.
Subject(s)
Indoles/pharmacology , Leukemia, Myeloid/drug therapy , Proto-Oncogene Proteins/drug effects , Pyrroles/pharmacology , Receptor Protein-Tyrosine Kinases/drug effects , Acute Disease , Adult , Aged , Bone Marrow/chemistry , Down-Regulation/drug effects , Female , Humans , Indoles/therapeutic use , Leukemia, Myeloid/blood , Leukemia, Myeloid/pathology , Male , Membrane Proteins/blood , Middle Aged , Mutation , Phosphorylation/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/blood , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Pyrroles/therapeutic use , Receptor Protein-Tyrosine Kinases/blood , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Stem Cell Factor/blood , fms-Like Tyrosine Kinase 3ABSTRACT
PURPOSE: In a recent phase II study of onartuzumab (MetMAb), patients whose non-small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit. EXPERIMENTAL DESIGN: Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA. RESULTS: A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean≥5 copies/cell by FISH); however, benefit was maintained in "MET IHC-positive"/MET FISH-negative patients (HR, 0.37; P=0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P=0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P=0.09) in favor of onartuzumab treatment. CONCLUSIONS: MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Carcinoma, Non-Small-Cell Lung/drug therapy , Proto-Oncogene Proteins c-met/biosynthesis , Quinazolines/administration & dosage , Adolescent , Adult , Aged , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Disease-Free Survival , ErbB Receptors/biosynthesis , Erlotinib Hydrochloride , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , In Situ Hybridization, Fluorescence , Male , Middle Aged , Neoplasm Staging , RNA, Messenger/biosynthesisABSTRACT
Fifteen patients with refractory AML were treated in a phase 1 study with SU11248, an oral kinase inhibitor of fms-like tyrosine kinase 3 (Flt3), Kit, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) receptors. Separate cohorts of patients received SU11248 for 4-week cycles followed by either a 2- or a 1-week rest period. At the starting dose level of 50 mg (n = 13), no dose-limiting toxicities were observed. The most frequent grade 2 toxicities were edema, fatigue, and oral ulcerations. Two fatal bleedings possibly related to the disease, one from a concomitant lung cancer and one cerebral bleeding, were observed. At the 75 mg dose level (n = 2), one case each of grade 4 fatigue, hypertension, and cardiac failure was observed, and this dose level was abandoned. All patients with FLT3 mutations (n = 4) had morphologic or partial responses compared with 2 of 10 evaluable patients with wild-type FLT3. Responses, although longer in patients with mutated FLT3, were of short duration. Reductions of cellularity and numbers of Ki-67(+), phospho-Kit(+), phospho-kinase domain-containing receptor-positive (phospho-KDR(+)), phospho-signal transducer and activator of transcription 5-positive (phospho-STAT5(+)), and phospho-Akt(+) cells were detected in bone marrow histology analysis. In summary, monotherapy with SU11248 induced partial remissions of short duration in acute myeloid leukemia (AML) patients. Further evaluation of this compound, for example in combination with chemotherapy, is warranted.
Subject(s)
Indoles/toxicity , Leukemia, Myeloid, Acute/drug therapy , Pyrroles/toxicity , Aged , Female , Follow-Up Studies , Genotype , Humans , Indoles/pharmacokinetics , Indoles/therapeutic use , Leukemia, Myeloid, Acute/genetics , Male , Metabolic Clearance Rate , Middle Aged , Mutation , Proto-Oncogene Proteins/genetics , Pyrroles/pharmacokinetics , Pyrroles/therapeutic use , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Sunitinib , fms-Like Tyrosine Kinase 3ABSTRACT
FLT3 (fms-related tyrosine kinase/Flk2/Stk-2) is a receptor tyrosine kinase (RTK) primarily expressed on hematopoietic cells. In blasts from acute myelogenous leukemia (AML) patients, 2 classes of FLT3 activating mutations have been identified: internal tandem duplication (ITD) mutations in the juxtamembrane domain (25%-30% of patients) and point mutations in the kinase domain activation loop (7%-8% of patients). FLT3-ITD mutations are the most common molecular defect identified in AML and have been shown to be an independent prognostic factor for decreased survival. FLT3-ITD is therefore an attractive molecular target for therapy. SU11248 is a recently described selective inhibitor with selectivity for split kinase domain RTKs, including platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and KIT. We show that SU11248 also has potent activity against wild-type FLT3 (FLT3-WT), FLT3-ITD, and FLT3 activation loop (FLT3-Asp835) mutants in phosphorylation assays. SU11248 inhibits FLT3-driven phosphorylation and induces apoptosis in vitro. In addition, SU11248 inhibits FLT3-induced VEGF production. The in vivo efficacy of SU11248 was investigated in 2 FLT3-ITD models: a subcutaneous tumor xenograft model and a bone marrow engraftment model. We show that SU11248 (20 mg/kg/d) dramatically regresses FLT3-ITD tumors in the subcutaneous tumor xenograft model and prolongs survival in the bone marrow engraftment model. Pharmacokinetic and pharmacodynamic analysis in subcutaneous tumors showed that a single administration of an efficacious drug dose potently inhibits FLT3-ITD phosphorylation for up to 16 hours following a single dose. These results suggest that further exploration of SU11248 activity in AML patients is warranted.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Pyrroles/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Acute Disease , Amino Acid Substitution , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Bone Marrow Transplantation , Endothelial Growth Factors/biosynthesis , Enzyme Inhibitors/therapeutic use , Female , Humans , Indoles/therapeutic use , Intercellular Signaling Peptides and Proteins/biosynthesis , Leukemia, Myeloid/enzymology , Leukemia, Myeloid/pathology , Lymphokines/biosynthesis , Mice , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Phosphorylation/drug effects , Point Mutation , Protein Processing, Post-Translational/drug effects , Protein Structure, Tertiary , Proto-Oncogene Proteins/genetics , Pyrroles/therapeutic use , Receptor Protein-Tyrosine Kinases/genetics , Recombinant Fusion Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Sunitinib , Tandem Repeat Sequences , Transfection , Tumor Cells, Cultured/enzymology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3ABSTRACT
Increased bone marrow angiogenesis and vascular endothelial growth factor (VEGF) levels are adverse prognostic features in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDSs). VEGF is a soluble circulating angiogenic molecule that stimulates signaling via receptor tyrosine kinases (RTKs), including VEGF receptor 2 (VEGFR-2). AML blasts may express VEGFR-2, c-kit, and FLT3. SU5416 is a small molecule RTK inhibitor (RTKI) of VEGFR-2, c-kit, and both wild-type and mutant FLT3. A multicenter phase 2 study of SU5416 was conducted in patients with refractory AML or MDS. For a median of 9 weeks (range, 1-55 weeks), 55 patients (33 AML: 10 [30%] primary refractory, 23 [70%] relapsed; 22 MDS: 15 [68%] relapsed) received 145 mg/m2 SU5416 twice weekly intravenously. Grade 3 or 4 drug-related toxicities included headaches (14%), infusion-related reactions (11%), dyspnea (14%), fatigue (7%), thrombotic episodes (7%), bone pain (5%), and gastrointestinal disturbance (4%). There were 11 patients (20%) who did not complete 4 weeks of therapy (10 progressive disease, 1 adverse event); 3 patients (5%) who achieved partial responses; and 1 (2%) who achieved hematologic improvement. Single agent SU5416 had biologic and modest clinical activity in refractory AML/MDS. Overall median survival was 12 weeks in AML patients (range, 4-41 weeks) and not reached in MDS patients. Most observed toxicities were attributable to drug formulation (polyoxyl 35 castor oil or hyperosmolarity of the SU5416 preparation). Studies of other RTKI and/or other antiangiogenic approaches, with correlative studies to examine biologic effects, may be warranted in patients with AML/MDS.