DOI: 10.5061/dryad.xgxd254rf
title: Prognostic, biological, and structural implications of FLT3-JMD point mutations in acute myeloid leukemia: an analysis of Alliance studies

description: A FLT3 gene frequently undergoes mutations in acute myeloid leukemia
 (AML), with internal tandem duplications (ITD) and tyrosine kinase domain
 (TKD) point mutations (PMs) being most common. Recently, PMs and deletions
 in the FLT3 juxtamembrane domain (JMD) have been identified, but their
 biological and clinical significance remains poorly understood. We
 analyzed 1660 patients with de novo AML and found FLT3-JMD mutations,
 mostly PMs, in 2% of the patients. Patients with FLT3-JMD mutations had a
 higher relapse rate and shorter disease-free survival than those with
 FLT3-TKD, whereas their relapse rate, disease-free and overall survival
 were not significantly different from those of FLT3-ITD-positive patients.
 In vitro experiments showed that FLT3-JMD PMs transformed hematopoietic
 cells and responded well to type I and II FLT3 inhibitors. Molecular
 dynamics simulations were used to explore the conformational changes of
 JMD PMs relative to wild-type FLT3. These mutations exhibited constrained
 domain motions with wider gate openings, potentially enhancing drug
 binding. Altered residue interactions and structural changes shed light on
 their unique functional mechanisms, with increased allosteric pathways
 suggesting reduced interactions with other residues. We conclude that
 patients with FLT3-JMD PMs represent uncommon but important subset with
 distinct molecular and biological features, and may benefit from FLT3
 inhibitors.
description: Viably frozen tumor samples were thawed and stained with Live
 Dead green stain (Thermo Fisher Scientific L23105) and CD45-Amcyan (clone
 2D1). Single live BLASTS were sorted into the wells of a PCR plate
 containing 3uL of DirectPCR Lysis-Reagent for cells (peQlab 31-301-C) with
 0.2mg/mL Proteinase K (Qiagen). To amplify FLT3 exons 13-15 PCR was
 performed with outer primers, left: (CCTGATTGTCTGTGGGGAGT)
 right:(GTTGACACCCCAATCCACTC), using Q5 High-Fidelity 2X Mater Mix (NEB) in
 the same plate as cell lysis. Nested PCR was performed using 2uL of PCR
 product from the outer PCR as template, primers
 left:(AGGAATTCTGTTTCATCGCTGA) right:(TCTTTGTTGCTGTCCTTCCAC) and the same
 PCR master mix as above. Product size of 594bp was confirmed by 1% agarose
 gel. ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher Scientific) was
 used according to the manufacturer’s instructions. Clean PCR product was
 Sanger sequenced in the OSUCCC Genomics Shared Resource on an Applied
 Biosystems 3730xl using both the left and the right the nested primers.
 Sequences were aligned to reference (NCBI: NG_007066.1) using Sequencher
 5.4.6 (Gene Codes Corporation).
description: # Prognostic, biological, and structural implications of FLT3-JMD point
 mutations in acute myeloid leukemia: an analysis of Alliance studies
 [https://doi.org/10.5061/dryad.xgxd254rf](https://doi.org/10.5061/dryad.xgxd254rf) ## Description of the data and file structure These AB1 files were analyzed to produce figure 1C, Single-cell analysis of the *FLT3*-ITD/JMD locus of tumor samples showing the frequency of various clones within *FLT3* mutated primary patient samples by Single cell nested amplification sanger sequencing (scNASS).  This figure appears in the manuscript entitled “Prognostic, biological, and structural implications of FLT3-JMD point mutations in acute myeloid leukemia: an analysis of Alliance studies”.  ### Files and variables Samples included in the scNASS experiment: C-12-5002 C-98-0747 C-12-3095 C-10-4190 C-97-0386 The AB1 file naming scheme is described below. Example file name:  Shelley_5002-1_F_694860_A01.ab1 Each section of the file name is separated by an “_” giving us 5 parts. 1.      Name of laboratory personnel that carried out the experiment. 2.      The last 4 digits of the sample identifier followed by the individual cell number. 3.      “F” is for forward read and “R” is for reverse read. 4.      Order tracking number used by our genomics core. 5.      Well location within the 96-well plate. There are 525 files in this archive. ## Code/software Sequencher 5.4.6 (Gene Codes Corporation) was used to view and align this data for the manuscript.  However, any software that can align AB1 sequence files can be used.  ## Access information Data was derived from the following sources: * All patients investigated had newly diagnosed de novo AML treated similarly on frontline Cancer and Leukemia Group B (CALGB) protocols.