A BCR-ABL Mutant Lacking Direct Binding Sites for the GRB2, CBL and CRKL Adapter Proteins Fails to Induce Leukemia in Mice

Kara J Johnson,Thomas O'Hare,Amie S Corbin,Ian J Griswold,Marc Loriaux,Brian J Druker,Michael W Deininger,Christophe Nicot

doi:10.1371/journal.pone.0007439

Abstract

The BCR-ABL tyrosine kinase is the defining feature of chronic myeloid leukemia (CML) and its kinase activity is required for induction of this disease. Current thinking holds that BCR-ABL forms a multi-protein complex that incorporates several substrates and adaptor proteins and is stabilized by multiple direct and indirect interactions. Signaling output from this highly redundant network leads to cellular transformation. Proteins known to be associated with BCR-ABL in this complex include: GRB2, c-CBL, p62DOK, and CRKL. These proteins in turn, link BCR-ABL to various signaling pathways indicated in cellular transformation. In this study we show that a triple mutant of BCR-ABL with mutations of the direct binding sites for GRB2, CBL, p62DOK and CRKL, is defective for transformation of primary hematopoietic cells in vitro and in a murine CML model, while it retains the capacity to induce IL-3 independence in 32D cells. Compared to BCR-ABL, the triple mutant's ability to activate the MAP kinase and PI3-kinase pathways is severely compromised, while STAT5 phosphorylation is maintained, suggesting that the former are crucial for the transformation of primary cells, but dispensable for transformation of factor dependent cell lines. Our data suggest that inhibition of BCR-ABL-induced leukemia by disrupting protein interactions could be possible, but would require blocking of multiple sites.

Highlights

The BCR-ABL tyrosine kinase is the molecular hallmark of chronic myeloid leukemia (CML) and its kinase activity is required for disease induction [1,2]
All clones were maintained in the presence of IL-3 prior to assessment of factor independent growth
The parental 32D cells were unable to proliferate in the absence of IL3, whereas wild type BCR-ABL, the Y177F, DSH2, deletion of the C-terminal prolinerich region (DPro) and the triple mutant BCR-ABL grew at comparable rates, indicating the triple mutant is capable of inducing factor independent growth

Summary

Introduction

The BCR-ABL tyrosine kinase is the molecular hallmark of chronic myeloid leukemia (CML) and its kinase activity is required for disease induction [1,2]. Since the tyrosine kinase activity of BCR-ABL is essential for its oncogenic activity in vitro and in vivo [1,2], much effort has been directed at determining which of its substrates are required for leukemogenesis. A number of BCR-ABL substrates have been identified, including BCR-ABL itself, CBL, CRKL, the p85 kDa regulatory subunit of phosphoinositide (PI) 3-kinase, p62DOK, RAS-GAP, paxillin, and SHC [9]. Co-immunoprecipitation experiments have shown that BCR-ABL forms stable complexes with several of these substrates including CRKL, SHC, CBL, p62DOK, and PI3-kinase [9,10,11,12]. In the complicated network of interactions that results, the role and relative importance of individual components has been difficult to establish

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