Point mutations in the juxtamembrane domain of FLT3 define a new class of activating mutations in AML

Abstract

IntroductionFMS-like tyrosine kinase-3 (FLT3) has been shown to be mutated in about one-third of patients with acute myeloid leukemia (AML), representing one of the most frequently occurring mutations in this disease. 1,2 Until now, two distinct clusters of activating mutations are known: FLT3-internal tandem duplications (FLT3-ITDs) in the juxtamembrane (JM) domain in 20% to 25% of patients, and point mutations (PMs) in the tyrosine-kinase domain (FLT3-TKD) in 7% to 10% of patients. [3][4][5][6][7][8][9] Recently, the crystal structure of the autoinhibited form of FLT3 was resolved. 10 The structure conforms to the prototypical conformation common to other inactive kinases that have a "closed" activation loop, but the remarkable feature is the complete JM domain serving as a critical autoinhibitory loop and interacting with all key features of FLT3. This domain can be divided into three distinct parts: the JM binding motif (JM-B), JM switch motif (JM-S), and the zipper or linker peptide segment (JM-Z). According to that model, the JM-B region is nearly buried in the FLT3 structure. It serves as an autoinhibitory domain, which in an inactive state prevents the N lobe from rotating toward the C lobe of the tyrosine kinase domain (TKD) to generate the activated kinase fold.The cytoplasmatic juxtamembrane domain is highly conserved between different members of the class III receptor tyrosine kinase (RTK) family. A variety of tumors in animals and humans have been described that harbor activating mutations in the JM domain. [11][12][13][14] The most frequently occurring activating mutations in AML, FLT3-ITDs, occur primarily in the JM-Z domain. They represent a heterogenous group of mutations, where a fragment of the JM domain, varying in length from 2 to 204 nucleotides (nt), is duplicated and inserted in a direct head-to-tail orientation always maintaining the reading frame.Recently, we discovered a novel missense point mutation in the JM domain of FLT3 in the AML cell lines Mono-Mac (MM)-1 and MM-6, changing valine with alanine at position 592. 15 By performing a LightCycler (Roche, Mannheim, Germany) mutational screening of FLT3 in 785 AML patient samples, we were able to identify two other point mutations: F594L in two AML patients and Y591C in 1 AML patient. In addition, Stirewalt et al 16 found additional point mutations in the JM domain of FLT3 (V579A and F590GY591D) in AML patients by using single-stranded conformational polymorphism analyses (polymerase chain reaction [PCR]/SSCPs).Here, we have studied the functional significance of this new class of activating mutations in patients with AML: PMs that cluster in a 16-aa stretch of the JM domain (FLT3-JM-PMs).We could clearly demonstrate that FLT3 receptors harboring one of these JM point mutations, when expressed in Ba/F3 cells, Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Sp556/3-1) and the Deutsche Krebshilfe (10-1997-Sp2).Reprints: Karsten Spiekermann, Department of Medicine III, University Hospital Grosshadern, CCG "Leukemia," GSF...

Cooperating gene mutations in acute myeloid leukemia: a review of the literature

“…Their prevalence seems to be lower than 1% in all AML subtypes and has not yet been established in cytogenetically normal AML (CN-AML). 19,20 Lastly, a novel gain-of-function mutation (K663Q), located not in the AL but in the N-terminal part of TKD, has been recently identified in AML. 21 Interestingly, a recent study stressed the importance of functional studies, since among the different FLT3 mutations reported outside the JMD and the TKD, some were not associated with kinase activation, and did not contribute to leukemogenesis.…”

“…20,23,24 However, there is increasing evidence suggesting that the downstream cellular responses to FLT3-ITD and FLT3-TKD mutation are not equivalent. In murine bone marrow transplantation models as well as in transgenic mice, FLT3-ITD induces a myeloproliferative disease that closely resembles human chronic myelomonocytic leukemia, and FLT3-TKD an oligoclonal lymphoid disorder with longest latency, but neither FLT3-ITD nor FLT3-TDK mutations induce AML.…”

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.…”

Drug resistance in mutant FLT3-positive AML

“…Occurring less frequently are additional point mutations in the kinase domain, including N841I (Jiang et al, 2004) and Y842C (Kindler et al, 2005). In addition, novel activating point mutations have been identified in a 16 amino-acid stretch of the FLT3 juxtamembrane, which generally have a weaker transforming potential than FLT3-ITD and tyrosine kinase domain mutants (Reindl et al, 2006). A diagram of FLT3 mutations is shown in Figure 1.…”