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...

Purpose: CBL is a negative regulator of activated receptor tyrosine kinases (RTK). In this study, we determined the frequency of CBL mutations in acute leukemias and evaluated the oncogenic potential of mutant CBL. Experimental Design: The cDNA of 300 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL) patients and 82 human leukemic cell lines was screened for aberrations in the linker and RING finger domain of CBL. The oncogenic potential of identified mutants was evaluated in hematopoietic cells. Results: We identified 3 of 279 AML/MDS patients expressing CBL exon 8/9 deletion mutants. Three of four cases at diagnosis expressed deleted transcripts missing exon 8 or exon 8/9. In remission samples a weak or no expression of mutant CBL was detected. No aberrations were found in normal hematopoietic tissues. One of 116 sequenced AML/MDS cases carried a R420G missense mutation. All AML/MDS patients with identified CBL mutants belonged to the core binding factor and 11q deletion AML subtypes. Functionally, CBL negatively regulated FMSlike tyrosine kinase 3 (FLT3) activity and interacted with human FLT3 via the autophosphorylation sitesY589 and Y599 and colocalized in vivo. Expression of CBLDexon8 and CBLDexon8+9 in FLT3-WT-Ba/F3 cells induced growth factor^independent proliferation associated with autophosphorylation of FLT3 and activated the downstream targets signal transducer and activator of transcription 5 (STAT5) and protein kinase B (AKT). FLT3 ligand^dependent hyperproliferation of CBL mutant cells could be abrogated by treatment with the FLT3 PTK inhibitor PKC412 (midostaurin). Conclusion: CBL exon8/9 mutants occur in genetically defined AML/MDS subtypes and transform hematopoietic cells by constitutively activating the FLT3 pathway. This phenotype resembles the one of mutated RTKs and suggests that CBL mutant AML patients might benefit from treatment with FLT3 PTK inhibitors.CBL, a known negative regulator of activated receptor tyrosine kinases (RTK), is localized on human chromosome 11q23, a region frequently associated with chromosomal aberrations. Translocations t(4;11) and t(11;14), and mixed-lineage leukemia fusion genes involving CBL have been described in patients with leukemia and lymphoma (1 -3). CBL oncogenes were initially identified in the murine system. CBL-70Z, carrying an internal deletion of 17 amino acids, was isolated from the 70Z/ 3 mouse pre-B-cell lymphoma cell line (4). CBL-70Z deregulates the cellular tyrosine kinase machinery, as NIH3T3 serumstarved cells expressing CBL-70Z showed significantly increased endothelial growth factor receptor (EGFR) kinase activity after EGF stimulation (5). p95CBL, expressed in the murine reticulum sarcoma cell line J-774, lacks internal 111 amino acids, comprising whole exons 8 and 9 (6). CBL70Z and p95CBL mutations both target the linker and RING finger domain, which points to a mutation-sensitive region within the CBL protein. Recently the first human CBL mutation has been reported in a patient...

FLT3 (fms-like tyrosine kinase 3) is constitutively activated in about 30% of patients with acute myeloid leukemia (AML) and represents a disease-specific molecular marker. Although FLT3-LM (length mutation) and TKD (tyrosine kinase domain) mutations have been considered to be mutually exclusive, 1% to 2% of patients carry both mutations. However, the functional and clinical significance of this observation is unclear. We demonstrate that FLT3-ITD-TKD dual mutants induce drug resistance toward PTK IntroductionActivating mutations in the FLT3 ( fms-like tyrosine kinase-3) gene play an important role in the pathogenesis of acute myeloid leukemia (AML) and can be found in approximately 25% to 35% of patients with AML. About 30% of AML patients have length mutations in the juxtamembrane domain of FLT3 (FLT3-LM), which consist of internal tandem duplications (ITD) and in some cases also of additional insertions. [1][2][3][4] Another 7% of patients carry point mutations in the tyrosine kinase domain of FLT3 (FLT3-TKD). [5][6][7] Both kinds of mutations confer interleukin-3 (IL-3)-independent growth to IL-3-dependent hematopoietic cell lines 5,[8][9][10][11] and induce a myeloproliferative syndrome in the mouse bone marrow transplantation model. 12 Because of their essential pro-proliferative and antiapoptotic role in AML cells, FLT3 mutants represent a promising molecular target for FLT3 PTK inhibitors. Three compounds with selective FLT3 inhibitory activity are currently evaluated in phase 1/2 clinical trials in patients with AML: PKC412, MLN518, and SU11248. [13][14][15] Initial results have shown that 30% to 50% of patients show a hematological response, but primary and secondary drug resistance represents a major problem. In contrast to the well-characterized mechanisms of drug resistance toward conventional cytotoxic agents, the molecular mechanisms of PTK inhibitor resistance in vivo are poorly understood.We have previously shown that FLT3-ITD-transformed cells can acquire mutations in the TKD domain (FLT3-ITD-TKD dual mutants) during prolonged exposure to FLT3 PTK inhibitors.These mutations cause resistance by reducing inhibitor affinity to the receptor. 16 Similar mechanisms of drug resistance have been described in imatinib-treated patients with chronic myeloid leukemia (CML) that can acquire mutations in the PTK domain of BCR-ABL. [17][18][19][20][21][22] In vitro and in vivo such mutants induce molecular drug resistance by changing the structural conformation and subsequently the access of the inhibitor to the PTK domain. Interestingly, some groups have shown that such mutations in the PTK domain of BCR-ABL can be found in untreated patients with CML and will be selected during inhibitor treatment. 19,23,24 Most patients with AML carry either a FLT3-length mutation (FLT3-LM) or a mutation in the tyrosine kinase domain (FLT3-TKD). However, in a subgroup of patients with AML both mutations can be detected. In a study from Thiede et al, 7 17 (1.7%) AML patients of a total of 979 had both a FLT3-LM and a TKD m...

MEIS1 is a three-amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor that plays key roles in normal hematopoiesis and leukemogenesis. Expression of Meis1 is ratelimiting in MLL-associated leukemias and potently interacts with Hox and NUP98-HOX genes in leukemic transformation to promote self-renewal and proliferation of hematopoietic progenitors. The oncogenicity of MEIS1 has been linked to its transcriptional activation properties. To further reveal the pathways triggered by IntroductionMultiple lines of evidence now point to the key roles of MEIS1, a three-amino acid loop extension class homeodomain-containing homeobox (HOX) cofactor, in both normal hematopoiesis and leukemogenesis. In the context of normal hematopoiesis, Meis1 is preferentially expressed in primitive bone marrow (BM) cells enriched for hematopoietic stem cells (HSCs), 1 and Meis1 knockout mice are embryonic lethal presenting with severely impaired hematopoiesis. 2,3 Deregulated expression of Meis1 accelerates the onset of disease induced by various Hox and NUP98-HOX fusion genes, including genes without leukemogenic potential on their own (reviewed in Argiropoulos and Humphries 4 ). Meis1 expression is also essential and rate limiting for the leukemogenicity of multiple MLL fusions, 5,6 and HOX and MEIS1 gene expression is frequently dysregulated in leukemia patient samples (reviewed in Argiropoulos and Humphries 4 ).Despite the well-documented role of Meis1 in the promotion of acute myelogenous leukemia (AML), the regulatory networks controlled by Meis1 are not fully known. The identification of Flt3, Cd34, Erg1,7,9 and Trib2 10 as Meis1 target genes have provided valuable insight into the function of Meis1. A group of these genes can replace Meis1 and serve as collaborating genes in leukemic transformation, albeit, with decreased potencies compared with Meis1. [10][11][12] However, as in the case with Flt3, the target may be dispensable. 13 Thus, the potency of Meis1 to induce leukemia appears to be related to its ability to modulate multiple pathways. Interesting in this regard is the recently reported link between Meis1 and cell-cycle control. In the context of mixed lineage leukemia (MLL)-fusion leukemias, Meis1 overexpression was observed to correlate with increased cell-cycle entry and modest up-regulation of Bmi1. 5 Moreover, gene expression profiling of murine Mll-AF9 leukemic BM cells transduced with Meis1 short-hairpin RNA showed reduced expression of genes associated with cell-cycle entry, consistent with the impaired cell growth of these cells. 6 The linkage to cell cycle gains currency in light of 2 recent reports that showed a correlation between Meis1 activity, retinal progenitor cell proliferation, and cyclin D1 expression in the developing chick and zebrafish. 14,15 Together, these data provide the basis for further investigation into the role of Meis1 in cell-cycle regulation.Experiments with the Drosophila melanogaster ortholog of Meis1, homothorax (hth), have shown that HTH fused to the potent ...

Purpose: Mutations in the receptor tyrosine kinase FLT3 are found in up to 30% of acute myelogenous leukemia patients and are associated with an inferior prognosis. In this study, we characterized critical tyrosine residues responsible for the transforming potential of active FLT3-receptor mutants and ligand-dependent activation of FLT3-WT. Experimental Design: We performed a detailed structure-function analysis of putative autophosphorylation tyrosine residues in the FLT3-D835Y tyrosine kinase domain (TKD) mutant. All tyrosine residues in the juxtamembrane domain (Y566, Y572, Y589, Y591, Y597, and Y599), interkinase domain (Y726 andY768), and COOH-terminal domain (Y955 andY969) of the FLT3-D835Y construct were successively mutated to phenylalanine and the transforming activity of these mutants was analyzed in interleukin-3-dependent Ba/F3 cells. Tyrosine residues critical for the transforming potential of FLT3-D835Y were also analyzed in FLT3 internal tandem duplication mutants (FLT3-ITD)and the FLT3 wild-type (FLT3-WT) receptor. Result: The substitution of the tyrosine residues by phenylalanine in the juxtamembrane, interkinase, and COOH-terminal domains resulted in a complete loss of the transforming potential of FLT3-D835Y-expressing cells which can be attributed to a significant reduction of signal tranducer and activator of transcription 5 (STAT5) phosphorylation at the molecular level. Reintroduction of single tyrosine residues revealed the critical role of Y589 and Y591in reconstituting interleukin-3-independent growth of FLT3-TKD-expressing cells. Combined mutation of Y589 and Y591 to phenylalanine also abrogated ligand-dependent proliferation of FLT3-WT and the transforming potential of FLT3-ITD-with a subsequent abrogation of STAT5 phosphorylation. Conclusion: We identified two tyrosine residues,Y589 and Y591, in the juxtamembrane domain that are critical for the ligand-dependent activation of FLT3-WTand the transforming potential of oncogenic FLT3 mutants.FLT3 is a member of the class III protein receptor tyrosine kinase family (RTK) that is characterized by five extracellular immunoglobulin-like domains, a juxtamembrane domain (JM), and two protein tyrosine kinase domains (TKD) split by an interkinase domain (IK; ref. 1). The class III receptors also include KIT, FMS, platelet-derived growth factor receptor-a (PDGFRA), and platelet-derived growth factor receptor-h (PDGFRB). Binding of FLT3 ligand (FL) to its receptor induces dimerization, phosphorylation, and subsequent activation of downstream signaling pathways such as signal tranducer and activator of transcription 5 (STAT5), Ras/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase/AKT (2 -6). FLT3 has been shown to play an important role in normal hematopoiesis and is highly expressed in CD34 + hematopoietic progenitor cells (2, 7 -9).Activating mutations of FLT3 are found in 30% of patients with acute myelogenous leukemia (AML) and are associated with an inferior clinical outcome (10 -12). FLT3 internal tandem dup...

FLT3-internal tandem duplications (FLT3-ITDs IntroductionMutations in the FMS-like tyrosine-kinase 3 (FLT3) are one of the most frequently found genetic alterations in patients with acute myeloid leukemia (AML), 1-13 myelodysplastic syndromes (MDSs; 10%-15%), 2,14 and acute lymphoblastic leukemia (ALL; 1%-3%). 9,16,17 FLT3 belongs to the class III of receptor tyrosine kinases, which are characterized by the presence of an extracellular immunoglobulin-like domain, a transmembrane and the cytoplasmic juxtamembrane (JM) domain, and the tyrosine kinase domain (TKD). 18 The class III receptors also include KIT, CSF-1, PDGFRA, and PDGFRB. 19,20 Activation of FLT3 by FLT3 ligand (FL) leads to receptor oligomerization and transphosphorylation of specific tyrosine residues, 21 which activates the downstream signaling pathways including STAT5, Ras/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/AKT. [22][23][24][25] FLT3 is highly expressed in CD34 ϩ hematopoietic progenitor cells and plays an important role in normal hematopoiesis. [26][27][28][29] Three distinct activating mutations of FLT3 in hematologic malignancies have been reported: point mutations (FLT3-JM-PM) 30,31 and internal tandem duplications (FLT3-ITD) in the JM domain and mutations in the tyrosine-kinase domain (FLT3-TKD). 1,8,9,12,16,17,32 The crystal structure of FLT3 has shown that the JM domain acts as an autoinhibitory domain in the inactive state. 33 The JM domain is highly conserved across all members of class III RTKs. Hence many tumors in humans show activating mutations of JM in class III RTKs. 34-37 FLT3-ITDs, found in a majority of acute leukemia patients, are in-frame duplications of a fragment of the JM domain. FLT3-ITDs are highly heterogeneous and vary in length from 2 to 68 AAs. These duplications are thought to disrupt the autoinhibitory mechanism and result in constitutive activation of the catalytic domain of FLT3. Activated FLT3 mutants promote cell proliferation and inhibit apoptosis, leading to factorindependent growth of murine hematopoietic cells in vitro and a myeloproliferative phenotype in vivo. 38 FLT3-ITDs are present in the leukemic blasts of 20% to 30% of all AML patients. Recent studies have also shown that FLT3-ITDs are found in the leukemic stem cells. 39 Furthermore, the presence of a FLT3-ITD has been recognized as an independent poor prognostic factor in AML and is associated with a decreased survival due to an increased relapse rate. 8,9,11,12,[40][41][42][43] Several factors influence the poor prognosis seen in AML patients harboring FLT3-ITDs (eg, a high FLT3-ITD/wildtype ratio). 9,44 A recent study has reported that the detection of FLT3-ITD mutation in less mature progenitor populations, for example, CD34 ϩ /CD33 Ϫ , might be associated with drug resistance. 43 In the present study, we asked whether any common duplicated motif exists in AML patients carrying FLT3-ITDs, which might be responsible for the transforming potential. To address this question, we sequenced and analyzed th...

Our results indicate that olfactory and gustatory functions are significantly decreased in GPA. As the olfactory function of these patients was comparable to patients with RA, chemosensory impairment may not simply be a consequence of the involvement of the upper respiratory tract, but rather a common complication of systemic autoimmune diseases.

In acute myeloid leukemia (AML) FMS-like tyrosine kinase-3 (FLT3) has been shown to be mutated in about one third of patients. Until now, two distinct activating mutations are known: FLT3-length mutations (FLT3-LM) in the juxtamembrane (JM) domain in 20–25% and FLT3-point mutations in the tyrosine-kinase domain (FLT3-TKD) in 7–10% of patients. Here, we have characterized a new class of activating point mutations (PM) that cluster in a 16 amino acid stretch of the juxtamembrane domain of FLT3 (FLT3-JM-PM). Stable expression of four distinct FLT3-JM-PM in IL-3 dependent murine Ba/F3 cells led to factor-independent growth, hyperresponsiveness to FLT3-ligand and resistance to apoptotic cell death compared to FLT3-WT-expressing cells. As a molecular mechanism, we could show activation of STAT5 and upregulation of Bcl-x(L) by all FLT3-JM-PM. A selective FLT3-inhibitor, PKC412, was able to abrogate the factor-independent growth of FLT3-JM-PM. Mapping of the FLT3-JM-PM on the crystal structure of FLT3 showed that these mutations probably reduce the stability of the JM domain in the autoinhibitory conformation, and provide a structural basis for their transforming capacity. Our results show that point mutations in the autoinhibitory JM domain represent a new class of gain-of-function mutations able to activate the transforming potential of FLT3.