FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells

Abstract

Abstract

FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30~40% of acute myelogenous leukemia (AML) patients. Thus, the drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from AML patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. A tyrosine kinase inhibitor midostaurin (PKC412) markedly decreases in FLT3-ITD retention and increases in the PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

* Grant Support

Japan Society for the Promotion of Science (18K07208 to YO, 19H03722 to TN and 20K08719 to RA)

Friends of Leukemia Research Fund (to YO)

Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics (to YO)

Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care (To YO)

* E-mail contacts, Kouhei Yamawaki koyamawa{at}ncc.go.jp, Isamu Shiina shiina{at}rs.kagu.tus.ac.jp, Takatsugu Murata t_murata{at}rs.tus.ac.jp, Satoru Tateyama tateyama.satoru.ma{at}m-chemical.co.jp, Yutarou Maekawa maekawa.yt{at}om.asahi-kasei.co.jp, Mariko Niwa 1320586{at}ed.tus.ac.jp, Motoyuki Shimonaka simonaka{at}rs.kagu.tus.ac.jp, Koji Okamoto kojokamo{at}ncc.go.jp, Toshihiro Suzuki toshsuzu{at}med.teikyo-u.ac.jp, Toshirou Nishida tnishida{at}ncc.go.jp

*

Abbreviations

AML

acute myeloid leukemia

BFA

brefeldin A

EGFR

epidermal growth factor receptor

ER

endoplasmic reticulum

ERK

extracellular signal-regulated kinase

FGFR

fibroblast growth factor receptor

FLT3

fms-like tyrosine kinase 3

GADS

GRB2-related adaptor downstream of Shc

GIST

gastrointestinal stromal tumor

ITD

internal tandem duplication

JM

juxta-membrane

MCL

mast cell leukemia

M-COPA

2-methylcoprophilinamide

PDGFR

platelet-derived growth factor receptor

pFLT3

phospho-FLT3

PM

plasma membrane

RET

rearranged during transfection

RTK

receptor tyrosine kinase

STAT

signal transducers and activators of transcription

TGN

trans-Golgi network

TKI

tyrosine kinase inhibitor

wt

wild-type.