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

Abstract

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...

Divergent cytotoxic effects of PKC412 in combination with conventional antileukemic agents in FLT3 mutation-positive versus -negative leukemia cell lines

“…The Tyr599Phe point mutation is a novel one, which has not been previously reported among cell lines nor patients with FLT3 mutations. 34,35 Interestingly, the Tyr599Phe and seven-codon duplication were found in the same allele. An ITD of 30 bp found in MV4-11 cells resulted in an additional histidine in the middle of the duplication.…”

The major lung cancer-derived mutants of ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/ERBB2 inhibitor HKI-272

“…Ba/F3 cells rely on continuous supplementation of recombinant interleukin-3 (IL-3) for their growth; introduction of dominant oncogenes renders these cells IL-3 independent (Azam et al, 2003;Jiang et al, 2004). Ba/F3 cells therefore represent an ideal model to assay for oncogenicity and to analyse different oncogene mutants for their sensitivity to targeted drugs.…”

Optimization of allele specific PCR (AS-PCR) for the early detection of FLT3 (D835Y) mutation in Acute Myeloid Leukemia patients at Tabuk, Saudi Arabia.

“…PCR-RFLP (restriction fragment length polymorphism) using EcoRV is one of the methods of detection for FLT3-TKD. It is important to note that digestion by EcoRV could not detect all FLT3-TKD mutations (13), however, sequence analysis showed that 50 -68% of FLT3-TKD mutations were substitutions of the first nucleotide of codon D835, most commonly from G to T (14). Traditionally, FLT3-ITD, FLT3-TKD, and NPM1 mutations are detected by PCR followed by electrophoresis or Taqman RQ-PCR (15).…”