The two major imatinib resistance mutations E255K and T315I enhance the activity of BCR/ABL fusion kinase

doi:10.1016/j.bbrc.2004.05.113

Biochemical and Biophysical Research Communications

Volume 319, Issue 4, 9 July 2004, Pages 1272-1275

https://doi.org/10.1016/j.bbrc.2004.05.113 Get rights and content

Abstract

The resistance to the tyrosine kinase inhibitor imatinib in BCR/ABL-positive leukemias is mostly associated with mutations in the kinase domain of BCR/ABL, which include the most prevalent mutations E255K and T315I. Intriguingly, these mutations have also been identified in some patients before imatinib treatment. Here we examined the effects of these mutations on the kinase activity of a BCR/ABL kinase domain construct that also contained the SH3 and SH2 domains. When expressed in COS7 cells, the BCR/ABL construct with either E255K or T315I exhibited not only the resistance to imatinib but also the increase in activity to induce autophosphorylation as well as tyrosine phosphorylation of various cellular proteins, which included STAT5. The mutant kinases also showed increased activities in in vitro kinase assays. These results raise a possibility that the major imatinib resistance mutations E255K and T315I may confer the growth advantage on leukemic cells to expand in the absence of selective pressure from imatinib treatment.

Section snippets

Materials and methods

Expression plasmids. Expression plasmids for the BCR/ABL tyrosine kinase domain were prepared by subcloning 1470-bp BCR/ABL reverse transcription-polymerase chain reaction (RT-PCR) products, obtained from imatinib-resistant CML patients after written informed consent, into the pCMV-tag1 vector (Stratagene, La Jolla, CA). In brief, a 1470-bp fragment of BCR/ABL cDNA was amplified from CML cells by using a BCR-specific upstream primer (5^′-CGGGATCCACTCAGCCACTGGATTTAAG-3^′) and an ABL-specific

Results

First, we expressed the BCR/ABL kinase domain constructs (Fig. 1) in COS7 cells and examined its ability to induce tyrosine phosphorylation of cellular proteins. As shown in Fig. 2A, anti-phosphotyrosine blotting of cellular substrates demonstrated that the BCR/ABL construct with either E255K or T315I induced significantly higher levels of tyrosine phosphorylation of various cellular proteins as compared with those induced by the construct without mutation. Whereas imatinib abolished the

Discussion

The present study has revealed that the E255K or T315I mutation confers not only the resistance to imatinib but also the increase in kinase activity upon BCL/ABL, which may play an important role in expansion of the mutant clones before imatinib therapy. This is in accordance with a recent study on the mechanisms of imatinib resistance, which revealed that mutations associated with imatinib resistance destabilize an autoinhibited, inactive conformation of BCR/ABL to which imatinib

Acknowledgements

This study was supported in part by grants from Ministry of Education, Culture, Sports, Science and Technology of Japan.

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The two major imatinib resistance mutations E255K and T315I enhance the activity of BCR/ABL fusion kinase

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

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Cancer Cell

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J. Biol. Chem.

Cell