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Microbiology and Molecular Genetics
B.S., 1997 Banaras Hindu University, India
M.S., 1999 Banaras Hindu University, India
Thesis Advisor: Ian Whitehead, Ph.D
Department of Microbiology and Molecular Genetics
Thursday, January 8, 2009
Chronic Myelogenous Leukemia (CML) is a malignant disorder of the hematopoietic stem cells characterized by the expansion of myeloid elements. Approximately 95% of CML patients carry the hallmark Philadelphia chromosome that results from a reciprocal translocation between chromosomes 9 and 22. This chromosomal rearrangement produces p210 Bcr-Abl, an in-frame 210 kD chimeric protein. The Abl-derived tyrosine kinase is constitutively activated in CML and is necessary for p210 Bcr-Abl-mediated transformation. Bcr contains a centrally located Rho-specific guanine nucleotide exchange factor (RhoGEF) domain which is retained in p210 Bcr-Abl. A variant of Bcr-Abl (designated p185 Bcr-Abl) which lacks the RhoGEF domain is associated with acute lymphoblastic leukemia, but not CML. RhoGEF domains have been described in over 70 mammalian proteins and are known to harbor potent oncogenic activity. Although this domain has been shown to exhibit independent signaling activity in vivo, its contribution to p210 Bcr-Abl transformation has not yet been determined. In a previous study we have reported that the RhoGEF domain is subject to autoinhibition in the context of Bcr. In this current study we report that it is constitutively activated in the p210 Bcr-Abl fusion protein. p210 Bcr-Abl is capable of activating RhoA independent of its tyrosine kinase activity, and mutations within the RhoGEF domain predicted to eliminate the catalytic activity inhibit RhoA activation. The RhoGEF defective p210 Bcr-Abl mutant retains similar levels of auto- and trans-kinase activity as its wild type counterpart. The transforming potential of the RhoGEF mutant is impaired in an anchorage-independent growth assay in Rat1 fibroblast cells. However, the mutant is able to confer the phenotype of growth factor independence in myeloid cells, suggesting that some, but not all parameters of p210 Bcr-Abl-mediated transformation are dependent upon a catalytically active RhoGEF domain. This represents the first tyrosine kinase independent gain of function associated with p210 Bcr-Abl that contributes towards the transformed phenotype. We evaluated this RhoGEF defective mutant in a murine bone marrow transduction-transplantation model for CML. Although, both Bcr-Abl and mutant transplanted mice succumbed to leukemia, the mutant transplanted mice displayed a significant survival advantage and survived longer than the wild type counterparts. Consistent with previous reports p210 Bcr-Abl-transplanted mice primarily succumb to myeloid-specific leukemias. In contrast, mice transplanted with the mutant predominantly develop lymphoid-based leukemias. These observations suggest an important role for RhoGEF signaling in disease progression, and may account for the differences in clinical outcome associated with p210 Bcr-Abl and p185 Bcr-Abl expression.