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Differential requirement for wild-type Flt3 in leukemia initiation among mouse models of human leukemia.

ABSTRACT: FLT3 is one of the most frequently mutated genes in acute leukemias. However, the role in leukemogenesis of wild-type (wt) FLT3, which is highly expressed in many hematologic malignancies, is unclear. We show here that in mouse models established by retroviral transduction of leukemic fusion proteins, deletion of Flt3 strongly inhibits MLL-ENL and to lesser extent p210(BCR-ABL)-induced leukemogenesis, but has no effect in MLL-AF9 or AML1-ETO9a models. Flt3 acts at the level of leukemic stem cells (LSCs), as a fraction of LSCs in MLL-ENL, but not in MLL-AF9-induced leukemia, expressed Flt3 in vivo, and Flt3 expression on LSCs was associated with leukemia development in this model. Furthermore, efficiency of MLL-ENL, but not of MLL-AF9-induced leukemia induction was significantly enhanced after transduction of Flt3(+) compared to Flt3(-) wt myeloid progenitors. However, Flt3 is not required for immortalization of bone marrow cells in vitro by MLL-ENL and does not affect colony formation by MLL-ENL LSCs in vitro, suggesting that in vitro models do not reflect the in vivo biology of MLL-ENL leukemia with respect to Flt3 requirement. We conclude that wt Flt3 plays a role in leukemia initiation in vivo, which is, however, not universal.

SUBMITTER: Kamezaki K

PROVIDER: S-EPMC3959278 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Differential requirement for wild-type Flt3 in leukemia initiation among mouse models of human leukemia.

Kamezaki Kenjiro K Luchsinger Larry L LL Snoeck Hans-Willem HW

Experimental hematology 20131120 3

FLT3 is one of the most frequently mutated genes in acute leukemias. However, the role in leukemogenesis of wild-type (wt) FLT3, which is highly expressed in many hematologic malignancies, is unclear. We show here that in mouse models established by retroviral transduction of leukemic fusion proteins, deletion of Flt3 strongly inhibits MLL-ENL and to lesser extent p210(BCR-ABL)-induced leukemogenesis, but has no effect in MLL-AF9 or AML1-ETO9a models. Flt3 acts at the level of leukemic stem cell ...[more]

PMID: 24269847

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Project description:BackgroundMidostaurin combined with chemotherapy is currently used to treat newly diagnosed acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 (FLT3)-mutations. However, midostaurin acts as an antagonist to some chemotherapeutic agents in leukemia cell lines without FLT3 mutations. All-trans retinoic acid (ATRA) induces apoptosis when used in combination with midostaurin in FLT3-mutated AML cells. This combination has been shown to be safe in AML patients. However, the effect of this combination has not been investigated in AML without FLT3 mutations.MethodsCell proliferation was assessed by a cell counting assay. Cell death was evaluated by cell viability and Annexin-V assays. Cell differentiation was assessed by CD11b expression profiling and morphological analysis. To explore the underlying mechanisms, we studied the role of caspase3/7, Lyn, Fgr, Hck, RAF, MEK, ERK, AKT, PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε by Western blot analysis and immunoprecipitation assays. Antitumor activity was also confirmed in mouse xenograft models established with AML cells.ResultsIn this study, 0.1 - 0.25 μM midostaurin (mido(L)) combined with ATRA induced differentiation while 0.25 - 0.5 μM midostaurin (mido(H)) combined with ATRA triggered apoptosis in some AML cell lines without FLT3-mutations. Midostaurin combined with ATRA (mido-ATRA) also exhibited antitumor activity in mouse xenograft models established with AML cells. Mechanistically, mido(H)-ATRA-induced apoptosis was dependent on caspase-3/7. Mido(L)-ATRA inhibited Akt activation which was associated with decreased activity of Lyn/Fgr/Hck, resulted in dephosphorylation of RAF S259, activated RAF/MEK/ERK, along with upregulating the protein levels of C/EBPβ, C/EBPε and PU.1. A MEK specific inhibitor was observed to suppress mido(L)-ATRA-induced increases in the protein levels of C/EBPs and PU.1 and mido(L)-ATRA-induced differentiation. Furthermore, inhibition of Akt activity promoted mido(L)-ATRA-induced downregulation of RAF S259 phosphorylation and mido(L)-ATRA-induced differentiation. Therefore, Lyn/Fgr/Hck-associated Akt inhibition activated RAF/MEK/ERK and controlled mido(L)-ATRA-induced differentiation by upregulation of C/EBPs and PU.1. Mido(L)-ATRA also promoted assembly of the signalosome, which may facilitate RAF activation.ConclusionsMidostaurin combined with ATRA exerts antitumor activity against AML with wild-type FLT3 mutations in vitro and in vivo. These findings may provide novel therapeutic strategies for some AML patients without FLT3 mutations and imply a new target of midostaurin.

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Differential requirement for wild-type Flt3 in leukemia initiation among mouse models of human leukemia.

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Differential requirement for wild-type Flt3 in leukemia initiation among mouse models of human leukemia.

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