Structural and functional characteristics of FLT3_ITD627E and of FLT3_ITD627A. (A) Integration site (top panel) and DNA sequence (bottom panel) of ITD_627E. The crystal structure of the cytoplasmatic part of human FLT3¹⁶  (protein code: 1RJB) was integrated from the Protein Data Bank²¹  (PDB) and analyzed using PyMOL software (DeLano Scientific, Palo Alto, CA). The juxtamembrane (JM) domain (aa 572-609) is in yellow. The β1 sheet (aa 610-615) and β2 sheet (aa 624-630) of the FLT3 tyrosine kinase domain 1 (TKD1) are shown in green with the β sheets connected by the nucleotide binding loop (NBL, aa 616-623) in orange. The integration site of the ITD627E allele in the β2 sheet of TKD1 (aa 627) is shown in red. By PCR amplification of the FLT3 coding sequence and DNA sequencing, ITD_627E (31 amino acids) was found to integrate at nucleotide 1880 of FLT3, thereby generating a single nucleotide change from GCA to GAA. This resulted in an amino acid substitution from alanine to glutamate at amino acid position 627 (ITD627E; bottom panel). An additional mutation in FLT3_ITD627E was excluded by sequencing the entire coding sequence of the FLT3_ITD627E allele. The coding sequence of FLT3 was amplified by PCR with the following primers: FLT3mRNA-f: TGCCGCTGCTCGTTGTTTT; FLT3mRNA-r: AGAAGGCCTTGGATGCAGA. PCR products were subcloned into pCR4-TOPO vectors (Invitrogen) and plasmid DNA from single bacterial clones was isolated and FLT3 inserts were sequenced. (B) In 32D cells stably transfected with FLT3_wt receptor, a standard JM ITD598/599 integrating in the JM domain of FLT3 between codons 598 and 599 (32D_ITD598/599) and in 32D_ITD627E cells, protein expression and phosphorylation of FLT3 (Y591) and of STAT5 (Y694/Y699) was determined by immunoblotting. (C) 32D_FLT3WT, 32D_ITD598/599, 32D_ITD627E, and 32D_ITD627A cells were grown in 10% WEHI-conditioned medium and then subjected to growth factor withdrawal. The ITD mutant ITD627A is identical in length and position of integration to ITD627E (93 bp/31 aa) with the amino acid at codon 627 reverted to wild type (alanine). The percentage of cells featuring a sub-G1 DNA peak as determined by flow cytometry 48 hours and 72 hours after growth factor withdrawal are shown (mean values of 3 independent experiments ± SD). (D) Stably transfected 32D_ITD598/599, 32D_ITD627E and 32D_ITD627A cells were seeded at a fixed density (1000 cells/mL) in 35 mm dishes and grown for 7 days in RPMI 1640/methylcellulose medium (Methocult; StemCell Technologies, Vancouver, BC) supplemented with 10% FCS but in the absence of growth factors. The total number of colonies from 2 dishes of each cell line is shown. As expected, 32D_WT cells did not form colonies under these conditions (data not shown). (E) Kaplan Meier plot of survival of mice injected with 32D_ITD598/599 (n = 5) and with 32D_ITD627E cells (n = 5). The percentage of surviving mice (y-axis) is plotted with respect to time in days (x-axis). It has been previously reported that mice injected with 32D FLT3_wt cells do not develop disease within an observation period up to 3 months.¹²