Inhibition Of N822k T > A Mutation-Induced Constitutive C-Kit Activation In Aml Cells Triggers Apoptotic And Autophagic Pathways Leading To Death

INTERNATIONAL JOURNAL OF MEDICAL SCIENCES(2019)

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Abstract
Background: The D816V mutation of c-KIT can constitutively activate tyrosine kinase, thereby promote core binding factor acute myeloid leukemia (CBF-AML) cell proliferation and inhibit apoptosis. Previous studies have indicated similar proliferation and apoptosis between N822K and D816V mutations.The current study aims to determine the occurrence and potential functions of N822K mutation-induced c-KIT activation in AML cells, and explore possible mechanisms of poor prognosis of CBF-AML.Methods: c-KIT N822K mutation status in AML cells was determined by exon 17 sequencing. The level of c-KIT expression was detected by flow cytometry (FCM) and colony formation was assessed after hu-SCF stimulation. After exposure to sunitinib (a kind of tyrosine kinase inhibitor, TKI), cell proliferation inhibition was tested by MTT, cell cycle and apoptosis were measured by FCM, autophagy was assessed by fluorescence microscopy and immunoblotting.Results: Kasumi-1 cell line was detected to bear c-KIT N822K (T>A) mutation. After hu-SCF stimulation, CD117 expression was decreased and the colony formation efficiency was not altered in Kasumi-1 cells. After sunitinib inhibited the c-KIT activity, the colony formation efficiency was reduced, and the half-maximal inhibitory concentration (IC50) of sunitinib was low (0.44 +/- 0.17 mu M) at 48 hours. Moreover, cells were arrested in G0/G1 phase, corresponding to an increase of apoptosis ratio. Acidic vesicular organelles (AVO) were observed along with an altered expression of autophagy-related proteins in Kasumi-1 cells.Conclusions: Our data indicated that inhibition of N822K T>A mutation-induced constitutive c-KIT activation in AML cells triggered apoptotic and autophagic pathways leading to death, and c-KIT N822K mutation may have clinical application as a CBF-AML treatment target.
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Key words
c-KIT, function mutation, biological behavior, CBF-AML, treatment target
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