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

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, 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 leukemia 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. Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase 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.

Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD

“…While PTPRJ is presumed to be primarily localized at the plasma membrane ( 37 , 46 , 83 ), FLT3 ITD is retained in its immature HM form in the ER/Golgi system ( 17 , 66 ). The maturation efficiency of FLT3 ITD has been demonstrated to be inversely correlated to its tyrosine phosphorylation ( 17 , 66 ). PTP such as SHP1 and PTP1B have been found to dephosphorylate FLT3 ITD ( 66 ).…”

“…As a consequence, FLT3 ITD mutations presumably disrupt the auto-inhibitory activity of the JM domain ( 4 , 5 ), leading to ligand-independent dimerization, auto-phosphorylation, and constitutive activation of the receptor ( 10 , 11 , 14 ). In contrast to the wt receptor and due to its constitutive kinase activity, ITD-mutated FLT3 is predominantly retained in the endoplasmic reticulum (ER) and Golgi compartments ( 15 – 17 ), from where it strongly promotes activation of signal transducer and activator of transcription 5 (STAT5) ( 15 – 17 ). Due to a residual pool of FLT3 ITD reaching the plasma membrane, constitutive activation of AKT and extracellular signal-regulated kinases 1/2 (ERK1/2) signalling can also be found ( 16 , 18 , 19 ).…”

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIβ activation in GIST cells

“…We recently reported that the FLT3-ITD accumulates in the Golgi area in AML cells, similarly to KIT in GIST cells 19 . However, PKD2 knockdown in the AML cell line, MOLM-14, did not affect FLT3 levels, signals, or mutant localization (Figures S8A and S8B).…”

“…In sharp contrast, during biosynthetic trafficking in GIST cells, the KIT Ex17 mutant and KIT Ex11 accumulate in the Golgi/trans-Golgi network (TGN) area, where they initiate oncogenic signaling [16][17][18] . We further showed that FLT3-internal tandem duplication (FLT3-ITD) in AML triggers growth signals in the Golgi area and the endoplasmic reticulum (ER) 19 . Although the organelles where RTK accumulates differ among cancer types, compartment-dependent signaling is a characteristic feature of mutant RTKs.…”

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 20, 2022. ; https://doi.org/10.1101/2022.12. 19.520889 doi: bioRxiv preprint…”