A Bcr-Abl Mutant Lacking Direct Binding Sites for the Grb2, Cbl and CrkL Adapter Proteins Fails to Induce Leukemia in Mice.
Authors: Johnson Kara J. , Griswold Ian J. , Corbin Amie , Deininger Michael W.N. , Druker Brian J. 
Published: October 2019

The Bcr-Abl tyrosine kinase is detectable in greater than 95% of cases of chronic myelogenous leukemia (CML) and its kinase activity is required for induction of this disease. A number of signaling proteins are associated with and phosphorylated by Bcr-Abl. Proteins known to associate directly with Bcr-Abl include Grb2, c-Cbl, CrkL and p62Dok. Mutations of the direct binding sites for these proteins in Bcr-Abl abolish the direct interactions, but do not completely eliminate interactions, presumably due to the ability of many of these proteins to interact both directly and indirectly with Bcr-Abl. Individual mutations of the Grb2 and c-Cbl binding domains change the phenotype of disease induced in murine bone marrow transplantation assays from a myeloproliferative disorder to a B or T-lymphoid leukemia with prolonged latency. Thus, due to the complexity of Bcr-Abl signaling and a lack of a one to one correlation between direct binding sites, specific signaling proteins, and specific phenotypes, we created a triple mutant lacking the direct binding sites for Grb2, c-Cbl and CrkL. Stable myeloid cell lines were generated in the myeloid progenitor cell line, 32D, expressing the wild type and triple mutant forms of Bcr-Abl. Cell proliferation assays were performed in the presence and absence of WEHI (an IL-3 source) to assess growth factor requirements. Expression of the triple mutant in cell lines was able to confer growth factor dependence when expressed at levels comparable to wild type. Lysates from cell lines were analyzed by immunoprecipitation and immunoblotting and demonstrated that nearly all associations between Bcr-Abl and Grb2, c-Cbl, CrkL and p62Dok were eliminated in the triple mutant. Despite the lack of interaction, these proteins remained tyrosine phosphorylated at levels which correlate with Bcr-Abl expression. Phosphorylation was inhibited by treatment of cell lines with imatininb, indicating that the activity of Bcr-Abl is required for their phosphorylation, either directly or indirectly. Analysis of the activation of various signaling pathways (Akt, MAPK, MEK, Stat5), shows that only Stat5 remains phosphorylated in triple mutant cell lines. Despite inducing factor independent growth of 32D cells, the triple mutant was unable to induce the outgrowth of hematopoetic progenitors in B-cell lymphoid outgrowth assays. To test leukemogencity in vivo, murine bone marrow transplantation/transduction assays were also carried out using MSCV-MIGR1 vector constructs. The triple mutant failed to induce leukemia in the mice. In summary, a triple mutant of Bcr-Abl lacking the binding sites for Grb2, c-Cbl and CrkL is able to confer growth factor independence in cell lines. Although the associations of these proteins with Bcr-Abl are nearly eliminated, they are still tyrosine phosphorylated and this is dependent on the activity of Bcr-Abl. Despite its ability to transform cell lines, the triple mutant was not able to induce the outgrowth of hematopoetic progenitors in B-cell outgrowth assays, nor induce leukemia in mice. Although the phosphorylation of Stat5 correlates with factor independent growth, this is not sufficient to induce transformation in vivo suggesting that interactions with other signaling pathways disrupted in this triple mutant of Bcr-Abl are necessary. To our knowledge this is the only kinase active variant of Bcr-Abl that has failed to induce leukemia in vivo.