Imatinib restores normal maturation and trafficking of KIT juxtamembrane domain mutants Free

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Mutations of the KIT tyrosine kinase receptor are critical event in gastrointestinal stromal tumors (GISTs) oncogenesis. The most frequent KIT mutations in GISTs are reported in the juxtamembrane domain coded by the exon 11, but the prognosis and the response to imatinib therapy are uncertain. KIT deletions of codons 562-579 have been reported to be more strongly associated with metastases than deletions of codons 550-561 and deletions in the distal part of exon 11 (565-579) translates to the worse response to imatinib therapy.

To gain more understanding about these differential biological effects of KIT exon 11 deletions, NIH3T3 cells were stably transfected with the human wild type KIT gene or two mutated KIT (KITdel557-558 or KITdel564-581). The differential effects of the mutation type, on KIT expression, activation and response to imatinib were analyzed.

Transfected cell lines expressed a functional KIT, and mutants are constitutively activated and have transforming activity. Wild type KIT was mainly present at the plasma membrane, in a mature unphosphorylated form, and its activation by SCF, induced phosphorylation and raft-dependent internalization. By constrast, cells transfected with mutant had a weak expression at the cell surface of the KIT mature form but expressed mainly the intracellular immature form, which was constitutively phosphorylated. Constitutive activation of mutants did not lead to higher internalization. Moreover, immunocytochemistry analysis revealed that, mutant KIT was mainly localized in perinuclear areas, in transfected cells and in GISTs patients with homozygous deletions, while it was cytoplasmic in KIT WT. Interestingly imatinib not only dramatically decreased KIT tyrosine kinase activity but also enhanced KIT maturation and expression at cell surface.

In conclusion, we showed, in NIH3T3 cells transfected with the most frequent of KIT human mutations reported in GISTs, that constitutively activated mutants bypassed the normal post-translational maturation and trafficking. Activation of the immature intracellular form of KIT may be a general mechanism of gain-of-function mutations of TKR in tumors. Moreover we highlighted a new mechanism of imatinib action where the blockade of the immature form of KIT resulting in the restoration of a normal KIT trafficking. These results suggest that both normal and bypass pathways should be targeted and might change common use of tyrosine kinase receptor inhibitors in next years.