The Structural Basis for Autoinhibition of FLT3 by the Juxtamembrane Domain

Griffith, Jeffrey K.; Black, James; Faerman, C. H.; Swenson, Lora; Wynn, Michael; Fan, Li; Lippke, Judith A.; Saxena, Kumkum

doi:10.1016/s1097-2765(03)00505-7

Cited by 436 publications

(395 citation statements)

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“…The structure of the FLT3 kinase in its autoinhibited state was recently elucidated 24 and allowed us to ask whether this might reveal a mechanism by which mutation of N841 could lead to the 24 Residues highlighted by this study are indicated and are shown in "stick" representation. Y572 is at the N-terminal residue of the JM domain visible in the crystal structure.…”

Section: Resultsmentioning

confidence: 97%

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML

Jiang

Páez

Lee

et al. 2004

Blood

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The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)-independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC 50 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to smallmolecule FLT3 inhibitors such as PKC412. IntroductionFLT3, a class 3 receptor tyrosine kinase, is targeted and activated by somatic mutation in acute myeloid leukemia (AML). Internal tandem duplication (ITD) mutations of the FLT3 juxtamembrane (JM) occur in approximately 24% of patients with AML and in 15% of patients with secondary AML 1,2 and are associated with shortened disease-free survival. 3 Mutations in the activation loop (AL), typically D835Y, occur in approximately 7% of patients with AML and 3% of patients with myelodysplastic syndromes (MDS) 4,5 and in patients with T-cell ALL (T-ALL). 6 An increased frequency of FLT3 mutations has also been associated with mutations involving the mixed-lineage leukemia (MLL) gene. 7 ITD and AL mutations result in constitutive FLT3 kinase activity. When FLT3 receptors harboring such mutations are introduced into mammalian cells, downstream signaling pathways are activated that lead to factor-independent growth in vitro and to leukemogenesis in vivo. 8,9 For example, the production of FLT3-ITD mutant proteins in primary murine bone marrow cells results in a lethal myeloproliferative phenotype. 10 Thus, FLT3 is a leukemia oncogene, and activating FLT3 mutations likely contribute significantly to the development of leukemia in humans.Several small-molecule inhibitors block the kinase activity of FLT3 with high potency 10-13 (eg, PKC412, MLN518, SU11248) and can prolong the lifespans of mice harboring leukemias expressing mutant FLT3 receptors. 10,14 In phase-1 and -2 clinical trials, FLT3 inhibitors have reduced FLT3 phosphorylation 15-17 and leukemia...

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Section: Resultsmentioning

confidence: 97%

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML

Jiang

Páez

Lee

et al. 2004

Blood

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“…The two activating mutations act through two distinctively different mechanisms. The FLT3-ITD mutation activates the kinase activity through releasing the negative constraint that the juxtamembrane region poses on the kinase domain of FLT3 [15]. Thus, the mutation does not involve any structural changes in the kinase domain.…”

Section: Discussionmentioning

confidence: 99%

“…The ITD occurs as an in-frame duplication of a sequence of varying length, leading to release of the negative regulatory constraint that the juxtamembrane domain poses on the kinase domain of FLT3 [15,16]. This is found in about 17-26% of patients with AML, and is the most common mutation in FLT3 [1,2].…”

Section: Introductionmentioning

confidence: 99%

Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation

Razumovskaya¹,

Masson²,

Khan

et al. 2009

Experimental Hematology

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Objective. Fms-like tyrosine kinase-3 (FLT3), a growth factor receptor normally expressed in hematopoietic progenitor cells, has been shown to have an important role in the development of acute myeloid leukemia c due to activating mutations. FLT3 mutations are found in approximately one third of AML patients and correlate with a poor prognosis, thus making the FLT3 receptor a potential therapeutic target. The aim of the investigation is to analyze the kinetics and specificity of FLT3 autophosphorylation in wild-type FLT3 as well as in the oncogenic FLT3 mutants.Methods. We have used Ba/F3 cells stably expressing either wild-type, ITD or D835Y mutants of FLT3 in order to compare the site selectivity of tyrosine phosphorylation sites. By the use of a panel of phosphospecific antibodies directed against potential tyrosine phosphorylation sites in FLT3, we identified several novel phosphorylation sites in FLT3 and studied the kinetics and specificity of ligand-induced phosphorylation in living cells.Results. Eight phosphorylated tyrosines (pY589, pY591, pY599, pY726, pY768, pY793, pY842 and pY955) were investigated and shown to be differentially phosphorylated in the wild-type versus the mutated receptors. Furthermore, we show that tyrosines 726, 793 and 842 are novel phosphorylation sites of FLT3 in intact cells. Conclusion.In this study, we have looked at the site-specific phosphorylation in the wild-type FLT3 in comparison to the mutants found in AML. We observed not only quantitative changes but more importantly, qualitative differences in the phosphorylation patterns of the wild-type and the mutated FLT3 receptors, which might contribute to the understanding of the mechanisms by which FLT3 contributes to AML in patients with mutations in FLT3.

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“…24 The ITD disrupts the autoinhibitory function of the juxtamembrane domain, resulting in constitutive autophosphorylation of FLT3 and activation of its downstream effectors. 25,26 The FLT3-ITD mutation produces growth factor-independent proliferation in leukemia cell lines and a fatal myeloproliferative syndrome in murine models. 27 Interestingly, however, when FLT3-ITD was expressed in cells from knockout mice lacking FLT3 ligand, the receptor was only weakly activated.…”

Section: Flt3 Mutationsmentioning

confidence: 99%

Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia

2012

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Acute myeloid leukemia (AML) is a highly heterogenous disease with multiple signaling pathways contributing to its pathogenesis. A key driver of AML is the FMS-like tyrosine kinase receptor-3 (FLT3). Activating mutations in FLT3, primarily the FLT3-internal tandem duplication (FLT3-ITD), are associated with decreased progression-free and overall survival. Identification of the importance of FLT3-ITD and the FLT3 pathway in the prognosis of patients with AML has stimulated efforts to develop therapeutic inhibitors of FLT3. Although these inhibitors have shown promising antileukemic activity, they have had limited efficacy to date as single agents and may require use in combination with cytotoxic chemotherapies. Here, we review clinical and preclinical results for the clinically mature FLT3 inhibitors currently in development. We conclude that multitargeted FLT3 inhibitors may have more utility earlier in the course of disease, when in vitro evidence suggests that AML cells are less dependent on FLT3 signaling, perhaps because of upregulation of multiple other signaling pathways. More potent agents may have greater utility in relapsed and heavily pretreated patients, in whom high levels of circulating FLT3 ligand may necessitate use of an agent with a very favorable pharmacokinetic/ pharmacodynamic profile. Novel combination regimens are also discussed.

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The Structural Basis for Autoinhibition of FLT3 by the Juxtamembrane Domain

Cited by 436 publications

References 50 publications

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML

Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation

Targeting the FMS-like tyrosine kinase 3 in acute myeloid leukemia

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