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Flumatinib, a selective inhibitor of BCR-ABL/PDGFR/KIT, effectively overcomes drug resistance of certain KIT mutants.

Zhao J, Quan H, Xu Y, Kong X, Jin L, Lou L - Cancer Sci. (2014)

Bottom Line: Interestingly, our in vitro study revealed that flumatinib effectively overcame the drug resistance of certain KIT mutants with activation loop mutations (i.e., D820G, N822K, Y823D, and A829P).Our in vivo study consistently suggested that flumatinib had superior efficacy compared with imatinib or sunitinib against 32D cells with the secondary mutation Y823D.Molecular modeling of flumatinib docked to the KIT kinase domain suggested a special mechanism underlying the capability of flumatinib to overcome the drug-resistance conferred by activation loop mutations.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

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KIT mutants, downstream signaling effectors ERK1/2, and signal transducer and activator of transcription-3 (STAT3), are constitutively phosphorylated in transformed 32D cell lines. Total cell lysates were analyzed by Western blotting, and the levels of phosphorylated (p-) and total proteins were determined using specific antibodies. rmSCF, recombinant mouse stem cell factor; WT, wild-type.
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fig01: KIT mutants, downstream signaling effectors ERK1/2, and signal transducer and activator of transcription-3 (STAT3), are constitutively phosphorylated in transformed 32D cell lines. Total cell lysates were analyzed by Western blotting, and the levels of phosphorylated (p-) and total proteins were determined using specific antibodies. rmSCF, recombinant mouse stem cell factor; WT, wild-type.

Mentions: The IL-3-dependent murine cell line 32D was transfected by retroviral vectors expressing WT KIT or 1 of 17 KIT mutants and selected for IL-3-independent growth. These transforming primary mutations mapped to the extracellular domain (Del [T417Y418D419] ins Ile, and Y503-F504 ins AY),6,18 the juxtamembrane region (encoded by exon 11) (V559D, Del[V559V560], D579-H580 ins IDPTQLPYD),2 or activation loop of the kinase domain (D816H/V/Y, and N822K).5,7 Considering that GISTs with KIT exon 11 mutants commonly become imatinib-resistant due to acquisition of secondary mutations in the kinase domain (i.e., V654A, T670I, D816H, D820G, N822K, Y823D, and A829P),13,18 we constructed imatinib-resistant double mutants by introducing each of these secondary mutations into the imatinib-sensitive mutant V559D. All of these mutants transformed 32D cells to IL-3-independent growth in the absence of rmSCF, and WT KIT transformed 32D cells to rmSCF-dependent growth. As expected, all transformed cells were GFP positive (data not shown). The 32D cells transformed by any of the KIT mutants showed constitutive phosphorylation of KIT and downstream signaling effectors ERK1/2 and STAT3 (Fig. 1). Consistent with a previous study,19 we observed differential phosphorylation of two KIT bands of approximately 160 and 145 kDa, representing the fully glycosylated cell surface receptor, and incompletely processed internalized forms of KIT, respectively.


Flumatinib, a selective inhibitor of BCR-ABL/PDGFR/KIT, effectively overcomes drug resistance of certain KIT mutants.

Zhao J, Quan H, Xu Y, Kong X, Jin L, Lou L - Cancer Sci. (2014)

KIT mutants, downstream signaling effectors ERK1/2, and signal transducer and activator of transcription-3 (STAT3), are constitutively phosphorylated in transformed 32D cell lines. Total cell lysates were analyzed by Western blotting, and the levels of phosphorylated (p-) and total proteins were determined using specific antibodies. rmSCF, recombinant mouse stem cell factor; WT, wild-type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317885&req=5

fig01: KIT mutants, downstream signaling effectors ERK1/2, and signal transducer and activator of transcription-3 (STAT3), are constitutively phosphorylated in transformed 32D cell lines. Total cell lysates were analyzed by Western blotting, and the levels of phosphorylated (p-) and total proteins were determined using specific antibodies. rmSCF, recombinant mouse stem cell factor; WT, wild-type.
Mentions: The IL-3-dependent murine cell line 32D was transfected by retroviral vectors expressing WT KIT or 1 of 17 KIT mutants and selected for IL-3-independent growth. These transforming primary mutations mapped to the extracellular domain (Del [T417Y418D419] ins Ile, and Y503-F504 ins AY),6,18 the juxtamembrane region (encoded by exon 11) (V559D, Del[V559V560], D579-H580 ins IDPTQLPYD),2 or activation loop of the kinase domain (D816H/V/Y, and N822K).5,7 Considering that GISTs with KIT exon 11 mutants commonly become imatinib-resistant due to acquisition of secondary mutations in the kinase domain (i.e., V654A, T670I, D816H, D820G, N822K, Y823D, and A829P),13,18 we constructed imatinib-resistant double mutants by introducing each of these secondary mutations into the imatinib-sensitive mutant V559D. All of these mutants transformed 32D cells to IL-3-independent growth in the absence of rmSCF, and WT KIT transformed 32D cells to rmSCF-dependent growth. As expected, all transformed cells were GFP positive (data not shown). The 32D cells transformed by any of the KIT mutants showed constitutive phosphorylation of KIT and downstream signaling effectors ERK1/2 and STAT3 (Fig. 1). Consistent with a previous study,19 we observed differential phosphorylation of two KIT bands of approximately 160 and 145 kDa, representing the fully glycosylated cell surface receptor, and incompletely processed internalized forms of KIT, respectively.

Bottom Line: Interestingly, our in vitro study revealed that flumatinib effectively overcame the drug resistance of certain KIT mutants with activation loop mutations (i.e., D820G, N822K, Y823D, and A829P).Our in vivo study consistently suggested that flumatinib had superior efficacy compared with imatinib or sunitinib against 32D cells with the secondary mutation Y823D.Molecular modeling of flumatinib docked to the KIT kinase domain suggested a special mechanism underlying the capability of flumatinib to overcome the drug-resistance conferred by activation loop mutations.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

Show MeSH
Related in: MedlinePlus