Prognostic, biological, and structural implications of FLT3-JMD point mutations in acute myeloid leukemia: an analysis of Alliance studies

Abstract

The 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.

Fig. 1. FLT3-JMD point mutations prevalence and association with prognosis in AML.

A Frequencies of molecular alterations detected in the FLT3-JMD region of AML patients in the Alliance dataset. lg-like, ligand binding domain; TMD, transmembrane domain; JMD, juxtamembrane domain; TKD1, tyrosine kinase domain-1; KID, kinase insert domain; TKD2, tyrosine kinase domain-2. B Associated co-mutations in FLT3-JMD mutated patients. C Single-cell analysis of the FLT3-ITD/JMD locus of tumor samples showing the frequency of various clones within FLT3 mutated primary patient samples. D Kaplan–Meier plot showing overall survival of patients <60 years old with different FLT3 mutations.

Fig. 2. Biological characteristics of FLT3-JMD point mutations and their sensitivity to FLT3 inhibitors.

A Growth Curves of Ba/F3 cells expressing FLT3 Y572C, V579A, V592A, ITD-V592A or ITD in addition to the parental cell line in the presence or absence of IL3 for 7 days. B Effect of gilteritinib on cell viability of Ba/F3 cells expressing FLT3 Y572C, V579A, V592A, ITD-V592A or ITD in addition to parental cell line (MTT assay, 72 h, n = 18). C Response of primary samples with FLT3-JMD point mutations to gilteritinib treatment (CellTiter-Glo Luminescent Cell Viability Assay, 72 h). D Western blot analysis of phosphorylated FLT3 in BaF3 cells expressing FLT3 Y572C, V579A, V592A, ITD-V592A or ITD. E Western blot analysis of cell signaling in BaF3 cells expressing FLT3 Y572C, V579A, V592A, ITD-V592A or ITD with or without gilteritinib (Gilt) treatment.

Fig. 3. Effect of FLT3-JMD point mutations on FLT3 domain motions and drug dissociation.

A Structure of FLT3 with the C-lobe and N-lobe domains shown in green; probabilities of domain distance distributions for the three mutants in comparison to the wild-type FLT3. B Schematic showing a gate controlling the drug binding site accessibility; probabilities of gate opening distance distributions for the three mutants in comparison to the wild-type FLT3. C Top binding poses of gilteritinib showing a similar binding pose in the three mutants as compared to the wild-type.

Fig. 4. Structural mechanisms in mutant FLT3 kinases.

A Altered secondary structures surrounding the mutated residues in the three mutants as compared to the wild-type FLT3. B Residue interaction network maps for the three mutants as compared to the wild-type FLT3.