The NH₂-Terminal Coiled-Coil Domain and Tyrosine 177 Play Important Roles in Induction of a Myeloproliferative Disease in Mice by Bcr-Abl

Abstract

Bcr-Abl, a fusion protein generated by t(9;22)(q34;q11) translocation, plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). It has been shown that Bcr-Abl contains multiple functional domains and motifs and can disrupt regulation of many signaling pathways and cellular functions. However, the role of specific domains and motifs of Bcr-Abl or of specific signaling pathways in the complex in vivo pathogenesis of CML is not completely known. We have previously shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human CML. We have also shown that the Abl kinase activity within Bcr-Abl is essential for Bcr-Abl leukemogenesis, yet activation of the Abl kinase without Bcr sequences is not sufficient to induce MPD in mice. In this study we investigated the role of Bcr sequences within Bcr-Abl in inducing MPD using this murine model for CML. We found that the NH₂-terminal coiled-coil (CC) domain was both essential and sufficient, even though not efficient, to activate Abl to induce an MPD in mice. Interestingly, deletion of the Src homology 3 domain complemented the deficiencies of the CC-deleted Bcr-Abl in inducing MPD in mice. We further demonstrated that the Grb2 binding site at Y177 played an important role in efficient induction of MPD. These studies directly demonstrated the important roles of Bcr sequences in induction of MPD by Bcr-Abl.

ACKNOWLEDGMENTS

We thank Ben Hentel and Jonathan Schatz for their help in flow cytometry analyses.

This work was supported by National Cancer Institute grant CA68008 (to R.R.) and by ACS grant RPG-97-131-01-LBC (to R.R.). R.R. is a recipient of The Leukemia and Lymphoma Society Scholar Award.

ADDENDUM

It was reported, after submission of our results, that Y177 was required for the induction of MPD in mice by Bcr-Abl (34). Similar to our results, Y177F was shown to induce a massive abdominal T-cell malignancy. However, in that report only a few Y177F mice had moderately increased neutrophils in peripheral blood, bone marrow, and spleen, and some Y177F mice developed B-lymphoid leukemia (34). The differences between our results and those reported (34) may be due to different retroviral titers and/or different retroviral transduction methods used. Bcr-Abl has been shown to induce a variety of hematological neoplasms, including acute B-lymphocytic leukemia; pre-B-cell lymphoma; T-cell leukemia and/or lymphoma; macrophage, erythroid, and mast cell tumors; myelomonocytic leukemia; and myeloproliferative disease (6, 8, 9, 16-18, 20, 22, 24, 37, 47, 50). The nature of the hematological neoplasms induced by Bcr-Abl has been shown to be influenced by the genetic background of mouse strains, by treatment of bone marrow cells (5-FU treated versus non-5-FU treated), as well as by infection conditions or the promoters used in transgenic animals (8, 9, 16-18, 20, 24, 47), which may ultimately affect what cells Bcr-Abl expression is targeted into and, therefore, what disease Bcr-Abl induces.