FLT3/ITD Mutation Signaling Includes Suppression of SHP-1

Peili Chen,Mark Levis,Patrick Brown,Kyu-Tae Kim,Jeffrey Allebach,Donald Small

doi:10.1074/jbc.m411974200

Abstract

Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by approximately 3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations.

Highlights

Deregulation of signaling through protein kinases has been identified as one of the most important mechanisms in human cancers [1, 2]
The fact that this is not seen in TF-1/internal tandem duplication (ITD) cells even in the absence of pretreatment with pervanadate implies that sufficient levels of phosphatases are not induced to counterbalance FLT3/ITD kinase activity
We show that transfection of TF-1 cells with mutated, constitutively activated FLT3 results in increased phosphotyrosine levels and factor-independent growth

Summary

Introduction

Deregulation of signaling through protein kinases has been identified as one of the most important mechanisms in human cancers [1, 2]. The SHP-2 mutations found in JMML, D61Y and E76K, exhibit elevated phosphatase activity compared with wild-type SHP-2 Expression of these mutants in COS-7 cells causes prolonged activation of ERK2 in response to epidermal growth factor stimulation and is associated with increased cell proliferation [42]. More recent studies have shown that Ba/F3 cells expressing those same mutants demonstrate enhanced growth factor-independent survival [43] These experimental data suggested that the SHP-2 mutations found in JMML might contribute to leukemogenesis by activating Ras/ERK signaling pathways and might explain the characteristic GM-CSF hypersensitivity of JMML hematopoietic progenitors. In a mast cell line expressing a c-Kit activation loop mutant, increased degradation of SHP-1 through a ubiquitin-dependent proteolytic pathway has been reported [57] These observations together provide strong support that SHP-1 functions as a tumor suppressor during myelopoiesis. We report for the first time that SHP-1 activity is suppressed by FLT3/ITD mutations and that this suppression is associated with a cell growth and a survival advantage

Methods

Results

Conclusion