title: Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518.
abstract: FLT3 is constitutively activated by internal tandem duplications (ITDs) in the juxtamembrane domain or by activation loop mutations in acute myeloid leukemia (AML). We tested the sensitivity of 8 activation loop mutations to the small molecule FLT3 inhibitor, MLN518. Each FLT3 activation loop mutant, including D835Y, D835A, D835E, D835H, D835N, D835V, D835del, and I836del, transformed Ba/F3 cells to factor-independent proliferation and had constitutive tyrosine kinase activation, as assessed by FLT3 autophosphorylation and activation of downstream effectors, including STAT5 and ERK. MLN518 inhibited FLT3 autophosphorylation and phosphorylation of STAT5 and ERK in FLT3-ITD-transformed Ba/F3 cells with an IC(50) (50% inhibition of cell viability) of approximately 500 nM. However, there was a broad spectrum of sensitivity among the 8 activation loop mutants, with IC(50) ranging from approximately 500 nM to more than 10 microM for the inhibition of phosphorylation of FLT3, STAT5, and ERK. The relative sensitivity of the mutants to MLN518 in biochemical assays correlated with the cellular IC(50) for cytokine-independent proliferation of FLT3-transformed Ba/F3 cells in the presence of MLN518. Thus, certain activation loop mutations in FLT3 simultaneously confer resistance to small molecule inhibitors. These findings have implications for the evaluation of responses in clinical trials with FLT3 inhibitors and provide a strategy to screen for compounds that can overcome resistance.
authors.affiliation: Division of Hematology/Oncology, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115, USA. Jennifer_Clark@dfci.harvard.edu
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Clark
authors.forename: Jan
authors.initials: J
authors.lastname: Cools
authors.forename: David P
authors.initials: DP
authors.lastname: Curley
authors.forename: Jin-Chen
authors.initials: JC
authors.lastname: Yu
authors.forename: Nathalie A
authors.initials: NA
authors.lastname: Lokker
authors.forename: Neill A
authors.initials: NA
authors.lastname: Giese
authors.forename: D Gary
authors.initials: DG
authors.lastname: Gilliland
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D009154:Mutation; D020125:Mutation, Missense; D010597:Pharmacogenetics; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D011799:Quinazolines / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D017384:Sequence Deletion; D014161:Transduction, Genetic; D051941:fms-Like Tyrosine Kinase 3
chemical_list: D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011799:Quinazolines; C464670:tandutinib; C497970:FLT3 protein, human; C497971:Flt3 protein, mouse; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3