Author

Yiguo Hu

Date of Award

8-2007

Level of Access

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Shaoguang Li

Second Committee Member

Carol J. Bult

Third Committee Member

Thomas Gridley

Abstract

Over 95% of CML and 20% of ALL are caused by Philadelphia chromosome. Ph chromosome is generated by a reciprocal translocation between chromosomes 9 and 22. This translocation generates a fusion gene, BCR-ABL. BCR-ABL oncoprotein activates multiple signal transduction pathways in cells responsible for the protection from apoptosis, stimulation of growth factor-independent proliferation, adhesion/invasion ability and induction of resistance to genotoxic drugs and radiation. It is generally believed that shutting down the kinase activity of BCR-ABL by imatinib will completely inhibits all its functions, leading to inactivation of its downstream signal pathways and cure of the disease. Imatinib mesylate is the preferred treatment for Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) in chronic phase, but is much less effective in CML blast crisis or Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Here, we show that BCR-ABL activates the Src kinases Hck, Lyn and Fgr in B-lymphoid cells. Upon retroviral transduction of BCRABL, marrow from mice lacking all the three Src kinases efficiently induces CML in recipient mice but is defective for induction of B-ALL. We also find that Src kinases activiated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells, suggesting that imatinib does not inactivate all BCR-ABL-activated signal pathways. Dasatinib, a dual inhibitor, which is more potent at inhibiting BCR-ABL and Src kinases activity, and affords complete remission of B-ALL but not CML. These results implicate Src family kinases as therapeutic targets in Ph+ B-ALL and suggest that inhibition of Src and BCR-ABL kinases benefits patients with Ph+ acute leukemia. Although Src kinases are required for BCR-ABL induced B-ALL in mouse model, inhibition of both Src and BCR-ABL kinase activities by dasatinib affords complete controlling B-ALL, maintained as long as treatment is continued, dasatinib cannot complete eradication of leukemic stem cells of B-ALL, as disease would relapse within one month after stopping drug. It is because that dasatinib cannot eradicate B-ALL stem cells, BCR-ABL+ pro-B cells. Leukemia is generated from leukemia stem cells. We found that BCR-ABL expressing LSK population can function as CML stem cells. Inhibition of BCR-ABL kinase activity by imatinib (Gleevec) cannot eradicate BCR-ABL+ leukemic stem cells. We showed that (3-catenin is acquired for leukemic stem cell renewal to maintain the stem cell pool. BCR-ABL disturbs BCL2 family protein functions. BCR-ABL increased Bcl-2 expression and enhanced Bcl-2/Bax heterodimer formation. Bax mutations are commonly detected in human hematopoietic malignancies, especially in acute lymphoblastic leukemia. In BCR-ABL-induced leukemia, Bax deficiency promotes leukemia process. In addition, BCR-ABL-transformed bone marrow cells were less sensitive to induction of apoptosis by the kinase inhibitor imatinib. These results demonstrate the pro-apoptotic role of Bax in BCR-ABL signaling and leukemogenesis.

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