Mutations in the juxtamembrane and kinase domains of FLT3 are common in AML, but it is not known whether alterations outside these regions contribute to leukemogenesis. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in AML patients without known FLT3 mutations and experimentally tested the consequences of each candidate leukemogenic allele. This approach identified gain-of-function mutations that activated downstream signaling and conferred sensitivity to FLT3 inhibition and alleles that were not associated with kinase activation, including mutations in the catalytic domain. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation and underscore the importance of functional studies to distinguish "driver" mutations underlying tumorigenesis from biologically neutral "passenger" alterations.
Epidermal growth factor receptor (EGFR) plays critical roles in many biological processes and in tumorigenesis. Here, we show that two mutated EGFRs found in lung and other malignancies, EGFR-G719S and EGFR-L858R, could transform Ba/F3 cells to interleukin-3 (IL-3)-independent growth, in a ligand-independent manner, an activity associated with the transforming function of other mutated tyrosine kinases. The mutated receptors are autophosphorylated in the absence of IL-3 without EGF stimulation, and their expression led to the constitutive activation of signal transducers and activators of transcription 5, extracellular signal-regulated kinase 1/2 (ERK1/2), ERK5, and AKT. In wild-type EGFRexpressing Ba/F3 cells, the major EGF-mediated signaling pathways were still intact. Gefitinib inhibited the growth of mutant EGFR-transformed Ba/F3 cells. Strikingly, the gefitinib sensitivity of cells expressing the L858R mutant was significantly greater than that of cells expressing the G719S mutant form, suggesting that distinct EGFR mutations may be differentially sensitive to small-molecule inhibitors. Furthermore, our data showed an antiproliferative effect of gefitinib on the EGFR-transformed Ba/F3 cells. Our results provide a model system to study the function of mutated EGFR and the differential effects of pharmacologic EGFR inhibition on the distinct mutant forms of this tyrosine kinase. (Cancer Res 2005; 65(19): 8968-74)
The FLT3 receptor tyrosine kinase plays an important role in normal hematopoietic development and leukemogenesis. Point mutations within the activation loop and inframe tandem duplications of the juxtamembrane domain represent the most frequent molecular abnormalities observed in acute myeloid leukemia. Interestingly these gain-of-function mutations correlate with different clinical outcomes, suggesting that signals from constitutive FLT3 mutants activate different downstream targets. In principle, mass spectrometry offers a powerful means to quantify protein phosphorylation and identify signaling events associated with constitutively active kinases or other oncogenic events. However, regulation of individual phosphorylation sites presents a challenging case for proteomics studies whereby quantification is based on individual peptides rather than an average across different peptides derived from the same protein. Here we describe a robust experimental framework and associated error model for iTRAQ-based quantification on an Orbitrap mass spectrometer that relates variance of peptide ratios to mass spectral peak height and provides for assignment of p value, q value, and confidence interval to every peptide identification, all based on routine measurements, obviating the need for detailed characterization of individual ion peaks. Moreover, we demonstrate that our model is stable over time and can be applied in a manner directly analogous to ubiquitously used external mass calibration routines. Application of our error model to quantitative proteomics data for FLT3 signaling provides evidence that phosphorylation of tyrosine phosphatase SHP1 abrogates the transformative potential, but not overall kinase activity, of FLT3-D835Y in acute myeloid leukemia. Molecular & Cellular Proteomics 9:780 -790, 2010.Deregulated tyrosine phosphorylation is a well established molecular hallmark in the development of both solid tumors and hematopoietic malignancies (1, 2). In acute myeloid leukemia (AML), 1 mutations of the Fms-like tyrosine kinase 3 (Flt3) gene or its overexpression represent the most frequent molecular abnormalities, observed in ϳ35 and ϳ90% of patients, respectively (3, 4). Internal tandem duplication (ITD) in the juxtamembrane region occurs in ϳ25% of patients, whereas another 7% of cases manifest as point mutations in the activation loop at Asp-835 (typically D835Y). Both mutation classes induce constitutive tyrosine kinase activity and confer IL-3-independent growth of the factor IL-3-dependent BaF3 and 32D cells. Interestingly, the ITD and D835Y mutants respond differently to the current suite of targeted therapeutics (5), and in vitro studies suggest that they can activate different downstream targets, including STAT5a (6 -8). Collectively, these observations motivate continued efforts to delineate oncogenic pathways in further detail to expand our understanding of the molecular basis for disease progression and to identify additional therapeutic entry points.Over the past decade, mass spectrometry-based pr...
Members of the inhibitor of apoptosis protein (IAP) family play a role in mediating apoptosis. Studies suggest that these proteins may be a viable target in leukemia because they have been found to be variably expressed in acute leukemias and are associated with chemosensitivity, chemoresistance, disease progression, remission, and patient survival. Another promising therapeutic target, FLT3, is mutated in about one third of acute myelogenous leukemia (AML) patients; promising results have recently been achieved in clinical trials investigating the effects of the protein tyrosine kinase inhibitor PKC412 on AML patients harboring mutations in the FLT3 protein. Of growing concern, however, is the development of drug resistance resulting from the emergence of point mutations in targeted tyrosine kinases used for treatment of acute leukemia patients. One approach to overriding resistance is to combine structurally unrelated inhibitors and/or inhibitors of different signaling pathways. The proapoptotic IAP inhibitor, LBW242, was shown in proliferation studies done in vitro to enhance the killing of PKC412-sensitive and PKC412-resistant cell lines expressing mutant FLT3 when combined with either PKC412 or standard cytotoxic agents (doxorubicin and Ara-c). In addition, in an in vivo imaging assay using bioluminescence as a measure of tumor burden, a total of 12 male NCr-nude mice were treated for 10 days with p.o. administration of vehicle, LBW242 (50 mg/kg/day), PKC412 (40 mg/kg/day), or a combination of LBW242 and PKC412; the lowest tumor burden was observed in the drug combination group. Finally, the combination of LBW242 and PKC412 was sufficient to override stromal-mediated viability signaling conferring resistance to PKC412. [Mol Cancer Ther 2007;6(7):1951–61]
Drug resistance resulting from emergence of imatinib-resistant BCR-ABL
Targeted cancer therapies impede cancer cell growth by inhibiting the function of activated oncogene products. Patients with non-small cell lung cancer and somatic mutations of EGFR can have a dramatic response to treatment with erlotinib and gefitinib; different somatic mutations are associated with different times to progression and survival. In this study, the relative and absolute potencies of two distinct EGFR tyrosine kinase inhibitors, erlotinib and an investigational irreversible inhibitor, HKI-272, were found to vary significantly in a panel of Ba/F3 cells transformed by representative EGFR somatic mutations. HKI-272 more potently inhibited the primary exon 20 insertion mutants, the secondary erlotinib-resistance mutants including T790M and many erlotinib-sensitive mutants including L858R. In contrast, erlotinib is a more potent inhibitor of the major exon 19 deletion mutants than is HKI-272. Analyses of EGFR autophosphorylation patterns confirmed the mutation-specific variation in relative potency of these tyrosine kinase inhibitors. Our finding that distinct EGFR inhibitors are more effective in vitro for different mutant forms of the protein suggests that tyrosine kinase inhibitor treatment could be tailored to specific EGFR mutations. More broadly, these results imply that the development and deployment of targeted therapies should focus on inhibition of specific cancer-causing mutations, not only on the mutated target.
IntroductionPhosphatidylinositol 3-kinase (PI3K) is an integral component of signaling pathways involved in the development and progression of myeloid leukemias, such as chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). CML is caused by BCR-ABL, which is the product of a reciprocal t(9;22) chromosome translocation in a hematopoietic stem cell. 1 The 210 kDa BCR-ABL protein is expressed in CML patients, and the p190 kDa BCR-ABL protein occurs in Philadelphia chromosome positive (Ph ϩ ) acute lymphoblastic leukemia (ALL) patients and stems from a different breakpoint in the BCR gene. 2,3 Constitutive activation of the PI3K/Akt (protein kinase B) kinases occurs in human leukemias. 4,5 AML is characterized by a complete or partial block in cellular differentiation and aberrant proliferation of myeloid progenitor cells. Approximately 30% of AML patients and a portion of ALL patients express a mutated form of the class III receptor tyrosine kinase, FLT3 (Fms-Like Tyrosine kinase-3; STK-1, human Stem Cell Tyrosine Kinase-1; or FLK-2, Fet al Liver . 6 Constitutively activated FLT3 occurs most often as internal tandem duplications (ITDs) within the juxtamembrane domain, 7 and is observed in approximately 20% to 25% of AML patients, but in less than 5% of patients with myelodysplastic syndrome. [7][8][9][10][11][12][13][14][15] We have previously demonstrated that FLT3-ITD induces AKT activation in Ba/F3 cells. 16 Moreover, FLT3-ITD leukemia cell lines and primary AML blasts display constitutive AKT phosphorylation, which is inhibited by the mutant FLT3 inhibitor, PKC412.The downstream effector of PI3K, AKT, is frequently hyperactivated in human cancers. Recent studies have shown that AKT is activated through phosphorylation in the majority of cases of AML. [17][18][19][20] Because FLT3-ITD mutations have been associated with AKT activation, 21 it has been suggested that the PI3K/AKT signaling pathway represents a critical, and shared, downstream target of these oncogenes. The signaling pathways used by FLT3 to transform cells are only partially known. In response to ligand, wild-type FLT3 activates pathways typical for type III tyrosine kinase receptors, including the PI3K/AKT, RAS/mitogen-activated protein kinase (MAPK), and STAT pathways. Of these pathways, studies demonstrated that STAT5 is activated by signaling from FLT3-ITD (Y589 and Y591) and that this is required for transformation in vivo. 22 There is a PI3K binding site outside of the kinase insert in the carboxy tail of the wt FLT3 receptor, 23 Mutations in N-RAS are common in AML, with a smaller number of mutations in K-RAS. In Ba/F3 cells, expression of most RAS point mutations, such as NRAS G12D (a common point mutation in AML), induces at least transient factor-independent proliferation, and studies with RAS mutants, which selectively activate downstream pathway (including Raf (Ras*T35S), phosphatidylinositol 3-kinase (Ras*Y40C) or RalGEFs (Ras*E37G) have suggested the activation of PI3K is essential.An important downstream effector of th...
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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