Modulation of FLT3 Inhibitor-induced Cytotoxicity in Acute Myeloid Leukemia by FLT3 Ligand
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Date
2005-06
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The Ohio State University
Abstract
Acute myeloid leukemia (AML) is a cancer of hematopoietic cells that develops rapidly in the bone marrow and quickly spreads to the blood. According to the American Cancer Society, there will be 11,960 new cases of AML in 2005 and about 9,000 patient deaths as a result. An internal tandem duplication (ITD) in the juxtamembrane domain of the FMS-like tyrosine kinase 3 receptor (FLT3) is present in approximately 25% of AML patients and predicts for poor prognosis. This mutation causes constitutive activation of the FLT3 receptor in the absence of the natural FLT3 ligand (FL). Unlike the wild-type (WT) counterpart, FLT3 ITD confers numerous oncogenic properties, such as uninterrupted proliferation, resistance to apoptosis and a block in differentiation. Selective FLT3 inhibitors have been developed, but clinical trials using these inhibitors as single agents have not been overwhelmingly successful. We hypothesize that one or more factors normally present within the bone marrow may be impacting negatively FLT3 inhibitor efficacy in these early trials. In particular, FL has been shown to promote the expansion of hematopoietic precursors, and is constitutively expressed by most bone marrow stromal tissues. Moreover, overexpression of FL by leukemic blasts has also been reported in AML.
To test our hypothesis, AML cells were treated with the novel FLT3 inhibitor, THRX-165724, in the presence or absence of exogenous FL. After 48 hours, cell viability was determined by MTS cell proliferation assay, and apoptosis was assessed after staining with propidium iodide and analysis of DNA content. Three cell lines were tested, representing the common FLT3 genotypes seen in AML (FLT3WT/WT, FLT3ITD/WT, and FLT3ITD/-). Molm-13 and MV4-11 cells (both FLT3 ITD+) underwent apoptosis with a 50% inhibitory concentration (IC50) of approximately 100-120 nM. However, exogenous FL decreased Molm-13 (FLT3ITD/WT) sensitivity to FLT3 inhibition 9-fold (p=0.0135), while FL modulation of THRX-165724-induced cytotoxicity of MV4-11 cells (FLT3ITD/-) was found not to be significant. No inhibitor-induced cytotoxicity or apoptosis in THP-1 cells (FLT3WT/WT) was observed by treatment of up to 5 µM inhibitor, irrespective of presence or absence of FL.
We next examined the downstream pathways (MAPK, STAT5, and AKT) implicated in FLT3 signaling. Cells were treated with THRX-165724, and protein lysates prepared. Using standard Western blotting techniques, we observed that FLT3 signaling along the MAPK pathway was rescued from inhibition by the FLT3 ligand. With combined U0126 (a MAPK inhibitor) and THRX-165724 treatment, reversal of these FL-induced changes in cytotoxicity and apoptosis was seen. These data, if confirmed in a larger study with primary AML samples, may help to provide an explanation for some of the suboptimal clinical results—the majority of FLT3 ITD+ patients identified to date have the FLT3ITD/WT genotype, analogous to the Molm-13 cell line.
Description
This project was developed in coordination with the larger study “FL Attenuates FLT3 inhibitor effects in AML” being conducted by the following researchers affiliated with the Ohio State University Medical Center: Rebecca B. Klisovic, Susan P. Whitman, Jacob J. Bryan, Himani Pandya, Martin Guimond, Brad Blaser, Kati Maharry, Roger Briesewitz, Clara D. Bloomfield, and Michael A. Caligiuri.
Keywords
THRX-165724, internal tandem duplication, FLT3 ITD, MAP kinase, apoptosis, AML, U0126, FLT3 Inhibition, FL, ERK