Abstract
High-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the activation of the anti-apoptotic transcription factor NFκB, via the IKK complex. Here, we show that constitutive activation of the receptor tyrosine kinase Flt3 is responsible for IKK activation. Chemical inhibition or knockdown of Flt3 with small interfering RNAs reduced NFκB activation in MDS and AML cell lines, as well as in primary CD34+ bone marrow cells from patients, causing apoptosis. Epistatic analysis involving the simultaneous inhibition of Flt3 and IKK suggested that both kinases act in the same anti-apoptotic pathway. An IKK2 mutant with a constitutive kinase activity and a plasma membrane-tethered mutant of NEMO that activates IKK1/2 prevented the cytocidal action of Flt3 inhibition. Flt3 phosphorylates IKK2 in vitro, and Flt3 inhibition reduced the phosphorylation of IKK2 in MDS or AML cell lines. IKK2 and Flt3 physically associated in MDS and AML cells, and Flt3 inhibition disrupted this interaction. Flt3 inhibition only killed CD34+ bone marrow cells from high-risk MDS and AML patients, in correlation with blast numbers and NFκB activity, yet had no lethal effect on healthy CD34+ cells or cells from low-risk MDS. These results suggest that Flt3 inhibitors might exert an anti-neoplastic effect in high-risk MDS and AML through inhibition of NFκB.
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Abbreviations
- Flt3:
-
Fms-like tyrosine kinase-3
- Flt3I:
-
Flt3 inhibitor
- AML:
-
Acute myeloid leukemia
- BMMNC:
-
Bone marrow mononuclear cells
- NFκB:
-
Nuclear factor-κB
- DAPI:
-
4′,6-diaminidino-2-phenylindole
- DiOC6(3):
-
3,3′ dihexyloxacarbocyanine iodide
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- IκB:
-
Inhibitor of NFκB
- IKK:
-
IκB kinase
- MDS:
-
Myelodysplastic syndrome
- PI:
-
Propidium iodide
- Z-VAD.fmk:
-
N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
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Acknowledgements
We are indebted to Jalil Abdelali (Institut Gustave Roussy, Villejuif, France) for support in confocal microscopy. Guido Kroemer is supported by Agence Nationale de Recherche, Fondation de France, Cent pour Sang la Vie, Cancéropôle Ile-de-France, Institut National du Cancer, Ligue Nationale contre le Cancer, and European Community (Active p53, Apo-Sys, TransDeath, RIGHT, ChemoRes, ApopTrain). Jennifer Grosjean received a post-doctoral fellowship by Cancéropôle Ile-de-France. Lionel Ades receives a scholarship from Assistance Publique-Hopitaux de Paris and Caisse Nationale d’Assurance Maladie des Professions Indépendantes. M.T. receives a Ph.D. fellowship from Université Paris Sud, Paris 11.
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Author contributions: J.G-R. performed the experiments and analyzed the data. L.A., S.B., C.F., T.B. and S.d.B. provided bone marrow samples and essential clinical information on patients. A.I and P.F. participated in the conception of the study. G.K. conceived and directed the study. J.G-R and G.K. wrote the paper.
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10495_2008_243_MOESM1_ESM.pdf
Effects of Flt3 ligation and inhibition on apoptosis and NFκB activation of KG1 cells. KG1 cells were cultured overnight in the absence or presence of Flt3I (2 µM) and/or Flt3L (10 µM) and the frequency of apoptotic events was estimated by determining the number of ΔΨ lowm (DiOC6(3)low) cells. In addition, the IκBα degradation was assessed by immunoblot (B) and the nuclear localization of NFκB was determined by EMSA (C) or immunofluorescence detection of p65 (D). (PDF 34 kb)
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Grosjean-Raillard, J., Adès, L., Boehrer, S. et al. Flt3 receptor inhibition reduces constitutive NFκB activation in high-risk myelodysplastic syndrome and acute myeloid leukemia. Apoptosis 13, 1148–1161 (2008). https://doi.org/10.1007/s10495-008-0243-4
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DOI: https://doi.org/10.1007/s10495-008-0243-4