Identification of a novel type of ITD mutations located in nonjuxtamembrane domains of the FLT3 tyrosine kinase receptor

Abstract

IntroductionFms-like tyrosine kinase 3 (FLT3) belongs to the class III receptor tyrosine kinase (RTK) family that includes FMS, platelet-derived growth factor receptor (PDGFR), and c-KIT. 1 FLT3 plays an important physiologic role in self-renewal and differentiation of hematopoietic stem and progenitor cells. [2][3][4] In acute myeloid leukemia (AML), activating mutations in the FLT3 gene occur in 30% to 40% of adult patients and have been demonstrated to play a crucial role in driving proliferation and survival of the leukemic clone. Twenty percent to 30% of AML patients harbour an internal tandem duplication (ITD) in the juxtamembrane (JM) region of FLT3. 5,6 ITDs found in FLT3 are always in frame and range from 3 base pairs (bp) to more than 400 bp. 7 Ten percent of AML patients have mutations within the activation loop of the second kinase domain, predominantly substitutions of aspartate at residue 835 (D835). However, additional activating mutations in this region have also been described. [8][9][10] Expression of FLT3_ITD receptors results in autophosphorylation of FLT3 and subsequent activation of downstream signaling. 11-13 Consequently, FLT3_ITD mutations render hematopoietic cells growth factor-independent by promoting cell proliferation and inhibition of apoptosis and lead to myeloproliferative disease in a murine transplantation model. 11,[13][14][15] Generally, it is believed that the ITD insertions within FLT3 occur in the zipper or linker peptide segment of JM (JM-Z) near the JM hinge region, thereby disrupting the autoinhibitory function of the JM-domain. 16 However, a detailed analysis of FLT3_ITD insertion sites has not been performed so far. Therefore, we have addressed this issue and have determined ITD insertion sites of 753 unselected FLT3_ITD-positive AML cases by direct cDNA sequencing. Methods Sequence analysis of FLT3_ITD from 753 unselected FLT3_ITD-positive AML casesMononucleated bone marrow cells from patients participating in standard diagnostic procedures and treated according to protocols of the AML Cooperative Group (AMLCG) study group or according to other intensive AML therapy protocols were obtained by Ficoll density gradient centrifugation. Some of the patients have already been described previously. 7 mRNA was extracted with the MagnaPureLC mRNA Kit I (Roche Diagnostics, Mannheim, Germany). cDNA synthesis of mRNA of an equivalent of 5 to 10 ϫ 10 6 cells was performed using SuperscriptII (Gibco BRL/Invitrogen, Karlsruhe, Germany) and random hexamer primers (Pharmacia, Freiburg, Germany). PCR and evaluation was performed as described previously. 7 Mutation detection was done by standard agarose gel electrophoresis and in parallel by fragment analysis on a capillary sequencer as described previously. 17 Direct sequencing of PCR products was performed as described 7 and primers were the same as used for PCR or RT-PCR amplification, respectively.Blood samples from AML patients were collected after informed consent was obtained in accordance with the Declaration of Helsinki. Labor...

Prognostic impact of white blood cell count in intermediate risk acute myeloid leukemia: relevance of mutated NPM1 and FLT3-ITD

“…[14][15][16][17][18] However, the number of mutated alleles, rather than its presence or the insertion site of the ITD, has been shown to affect outcome. [17][18][19]34,35 Likewise, in our study we also found that patients with the genotypic combination 'NPMc + with FLT3-ITD' and high levels of the mutant allele (i.e. an FLT3-ITD/FLT3 ratio >1) had significantly worse long-term outcome.…”

Repression of Gadd45α by activated FLT3 and GM-CSF receptor mutants contributes to growth, survival and blocked differentiation

“…(b) Expression of GADD45A mRNA in AML. We determined the average levels of GADD45A mRNA in each AML cluster identified by Valk et al 38 Plot shows normalized expression of GADD45A in each AML cluster (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) and in the normal control (CD34 þ and normal bone marrow (NBM)) (*Po0.05, # Po0.001, t-test). (c) Cluster details including number of patients, most common abnormality and fold expression change.…”

“…1 Recent screens have also identified a range of other point mutations in FLT3 in AML. [2][3][4] FLT3-ITD and FLT3-TKD mutants induce distinct disease states in mouse models 5 and although FLT3-ITD mutations are associated with a poor prognosis, [6][7][8][9] the prognostic implications of FLT3-TKD mutations are less clear. 9,10 The signaling events induced by the activated FLT3 mutants presumably converge to modulate gene expression changes contributing to survival, proliferation and maintenance of differentiation arrest.…”

Up-regulation of homeodomain genes, DLX1 and DLX2, by FLT3 signaling

“…40 PKC412 is also not specific for FLT3 signaling and potentially could affect other pathways that interfere with PAI-1 activation. In addition, a novel type of ITD mutation not localized in the juxtamembrane region of the FLT3 receptor was recently discovered by Breitenbuecher et al 41 This mutation was detected in about 29% of FLT3/ITD-positive patients and was associated with persistence of phosphorylated-ERK1/2 despite PKC412 treatment.…”

“…In addition, it has been reported that ERK-mediated phosphorylation of Smad1-3 impairs the nuclear transport and signaling activity of these proteins. [40][41][42] Thus, inhibition of FLT3, which is known to signal in part by phospho-ERK1/2, would be predicted to enhance nuclear transport of phosphorylated Smad2. However, further studies are required to reveal this mechanism.…”