Overview
When chromosome 22 exchanges, or translocates, genetic material with chromosome 9, it creates a mutation known as the Philadelphia chromosome. The translocation combines two genes, BCR and ABL, that function normally apart but are oncogenic (cancer causing) when brought together. The Human Philadelphia chromosome-positive leukemias such as chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) are among the most common blood malignancies. Our laboratory has developed an efficient and accurate mouse model of human Philadelphia chromosome-positive leukemia induced by the BCR/ABL oncogene, with a goal of understanding the molecular basis of the disease. We focus on the genetic dissection of the signaling pathways utilized by BCR/ABL to induce leukemias and on the identification of potential molecular and cellular targets for leukemia treatment. We are also studying the molecular mechanisms for survival and self-renewal of leukemic stem cells.
Research details
Molecular basis of human Ph+ leukemias
The human Philadelphia chromosome arises from a translocation between Chromosomes 9 and 22, and results in formation of the chimeric and constitutively activated BCR-ABL tyrosine kinase. Philadelphia chromosome-positive (Ph+) leukemias induced by the BCR-ABL oncogene include chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). CML often initiates in a chronic phase and eventually progresses to a terminal blastic phase, in which either acute myeloid or acute B-lymphoid leukemia develops. Some Ph+ leukemia patients, however, have B-ALL as their first clinical appearance. It is generally believed that shutting down the kinase activity of BCR-ABL will completely inhibit its functions, leading to inactivation of its downstream signaling pathways. Therefore, current therapeutic efforts have focused on targeting BCR-ABL kinase activity using kinase inhibitors.
The BCR-ABL tyrosine kinase inhibitor imatinib mesylate (Gleevec) is the standard of care for Ph+ leukemia. Imatinib induces a complete hematologic response in chronic phase CML patients. However, imatinib does not completely eliminate BCR-ABL-expressing leukemic cells, and patients frequently present with drug resistance. Imatinib prolongs survival of mice with BCR-ABL-induced CML, but does not cure the disease. Recently, three BCR-ABL kinase inhibitors, dasatinib, AP23464, and AMN107, have been shown to inhibit almost all imatinib-resistant BCR-ABL mutants; the exception is the T315I mutant, which is present in 15-20 percent of imatinib-resistant patients. Dasatinib is also a potent inhibitor of SRC family kinases, but the role of the anti-SRC activity of this compound in Ph+ leukemia therapy is unclear. For unknown reasons, imatinib is much less effective in treating CML blastic phase patients and patients with Ph+ B-ALL, which has not been shown to be related to the BCR-ABL kinase domain mutations, the most common type of imatinib resistance. Because imatinib is a strong inhibitor of BCR-ABL kinase activity, the inability of imatinib to cure CML and B-ALL in mice suggests that inactivation of BCR-ABL kinase activity alone is insufficient to control the disease.
We have previously shown that the three SRC family kinases LYN, HCK, and FGR are activated by BCR-ABL in lymphoid leukemic cells and are required for the development of B-ALL. We reasoned that inhibition of BCR-ABL kinase activity by imatinib might not inactivate SRC kinases activated by BCR-ABL in lymphoid leukemic cells, and this may explain the relatively poor activity of imatinib against Ph+ B-ALL and lymphoid blast crisis. During the past year, we further investigated the relationship between SRC kinase activation and BCR-ABL kinase activity, and we have begun to study molecular mechanisms for survival and self-renewal of leukemic stem cells.
Activation of SRC kinases by BCR-ABL does not depend on its kinase activity
We tested the hypothesis that imatinib may not inactivate SRC kinases activated by BCR-ABL using a BCR-ABL-expressing pre-B cell line. The cells were treated with or without imatinib. Compared to cells bearing the empty vector, Western blot analysis showed that SRC kinases were activated in cells expressing one of two forms of BCR-ABL (P190 and P210), and imatinib treatment markedly inhibited BCR-ABL kinase activity but did not result in a decrease in SRC activation. These results indicate that while imatinib was very effective in inhibiting BCR-ABL phosphorylation, it was unable to affect BCR-ABL-stimulated phosphorylation of SRC kinases. To further demonstrate this finding, we used the P190 or P210 form of BCR-ABL to transform mouse bone marrow (BM) cells. These cells were then treated with imatinib. Imatinib inhibited BCR-ABL phosphorylation, resulting in decreased phosphorylation of the downstream signaling molecule CRKL, but did not affect BCR-ABL-stimulated phosphorylation of SRC kinases. These observations indicate that, in imatinib-treated BCR-ABL-expressing cells, SRC kinases are still active, and that activation of SRC kinases by BCR-ABL is independent of its kinase activity.
Progression to lymphoid blast crisis CML requires activation of SRC kinases
Chronic phase CML advances to blastic phase. We tested genetically whether SRC kinases play a role in CML transition to lymphoid blast crisis using a serial transplantation assay. Mice were transplanted with BCR-ABL transduced BM cells from either wild type or Lyn-/-Hck-/-Fgr-/- mice to induce CML, and BM cells from the CML mice were subsequently transferred into recipient mice. Mice receiving wild type CML BM cells developed B-ALL, shown by GFP+/B220+ leukemic cells in peripheral blood, whereas none of the mice receiving Lyn-/-Hck-/-Fgr-/- CML BM cells developed this disease. These results indicate that CML transition to lymphoid blast crisis requires SRC kinases.
Identification of leukemic stem cells in mice
To identify CML stem cells, we tested whether BCR-ABL-expressing HSCs function as the stem cells. We first sorted HSCs (Lin-KIT+Sca-1+) from C57BL/6J BM cells and then transduced with BCR-ABL retrovirus, followed by transferring into recipient mice. The mice developed and died of CML. To definitively confirm that BCR-ABL-expressing HSCs are CML stem cells, we isolated BM cells from primary CML mice, and sorted out the BCR-ABL-expressing HSCs (GFP+Lin-KIT+Sca-1+) by FACS. The sorted cells were transferred into recipient mice, and the mice developed and died of CML, indicating that BCR-ABL-expressing HSCs function as CML stem cells. We identified the cell types of the residual GFP+ cells in dasatinib-treated B-ALL mice as pro-/pre-B cells, and these progenitor leukemic cells may have acquired self-renewal capacity and function as B-ALL stem cells. To test this hypothesis, we sorted by FACS the B220+/CD19+/GFP+ cells from BM of B-ALL mice followed by transplantation of the cells into recipient mice. These mice developed B-ALL, and leukemic cells in peripheral blood were CD19+/CD43+ pro-B cells. We believe that CD19+/B220+/CD43+ pro-B cells expressing BCR-ABL can function as B-ALL stem cells. To support this idea, we transferred purified BCR-ABL-expressing CD19+/B220+/CD43+ pro-B cells into recipient mice; these cells induced leukemia and also had potential to differentiate.
Lab staff
Principal Investigator: Shaoguang Li, M.D., Ph.D.
Postdoctoral Fellow: Con Sullivan, Ph.D. , Yiguo Hu, M.D., Ph.D.
Predoctoral Associates: Yaoyu Chen, M.D., M.S., Cong Peng, B.S., M.S., Christopher McCarty, B.S., M.S., Haojian Zhang, B.S., M.S.
Research Assistant II: Lori Douglas, B.S.
Research Administrative Assistant: Patricia Cherry
Publication listings
(Selected from 2001 to present)
Li D, Li S. 2008. Models of signal transduction in cancer. Drug Discovery Today 4(2):61-66.
Li S, Li D. 2008. Stem cell and kinase activity-independent pathway in resistance of leukaemia to BCR-ABL kinase inhibitors. J Cell Mol Med 11(6):1251-1262.
Li S. 2008. Src-family kinases in the development and therapy of Philadelphia chromosome-positive chronic myeloid leukemia and acute lymphoblastic leukemia. Leuk Lymphoma 49(1):19-26.
Li S. 2007. Src kinase signaling in leukeamia. Int J Biochem Cell Biol 39:1483-1488.
Peng C, Brain J, Hu Y, Kong L, Goodrich A, Grayzel D, Read M, Pak R, Li S. 2007. Inhibition of heat shock protein 90 prolongs survival of mice with BCR-ABL-T315I-induced leukemia and suppresses leukemic stem cells. Blood 110(2):678-685.
Peng C, Li D, Li S. 2007. Heat shock protein 90: a potential therapeutic target in leukemic progenitor and stem cells harboring mutant BCR-ABL resistant to kinase inhibitors. Cell Cycle 6(18):2227-2231.
Hu Y, Swerdlow S, Duffy TM, Weinmann R, Lee FY, Li S. 2006. Targeting multiple kinase pathways in leukemic progenitors and stem cells is essential for improverd treatment of Ph+ leukemia in mice. Proc Natl Acad Sci USA 103(45):16870-16875.
Zaleskas VM, Krause DS, Lazarides K, Patel N, Hu Y, Li S, Van Etten R. 2006. Molecular pathogenesis and therapy of polycythemia induced in mice by JAK2 V617F. PLoS One 1:218.
Li S. 2005. Src kinases as targets for B cell acute lymphoblastic leukaemia therapy. Expert Opin Ther Targets 9(2):329-341.
Hu Y, Liu Y, Pelletier S, Buchdunger E, Warmuth M, Fabbro D, Van Etten RA, Li S. 2004. Requirement for Src kinases for BCR-ABL-induced B-lymphoblastic leukaemia but not chronic myeloid leukaemia. Nat Genet 36(5):453-461.
Pelletier S, Hong D, Hu Y, Liu Y, Li S. 2004. Lack of the adhesion molecules P-selectin and intercellular adhesion molecule-1 accelerates the development of BCR/ABL-induced chronic myeloid leukemia-like myeloproliferative disease in mice. Blood 104(7):2163-217.
Dash AB, Williams IR, Kutok JL, Tomasson MH, Anastasiadou E, Kindahl K, Li S, Van Etten RA, Borrow J, Housman D, Druker BJ, Gilliland DG. 2002. A murine model of CML blast crisis induced by cooperation between BCR/ABL and NUP98/HOXA9. Proc Natl Acad Sci U S A 99(11):7622-7627.
Sattler M, Mohi MG, Pride YB, Quinnan LR, Malouf NA, Podar K, Gesbert F, IwasakiH, Li S, Van Etten RA, Neel BG, Gu H, Griffin JD. 2002. Critical role for Gab2 in transformation by BCR/ABL. Cancer Cell 1:479-492.
Li S, Couvillon A, Brasher B, Van Etten RA. 2001. Tyrosine phosphorylation of Grb2 by Bcr/Abl oncoprotein and epidermal growth factor-receptor: a novel regulatory mechanism for tyrosine kinase signaling . EMBO J 20(23):6793-6804.
Li S, Gillessen S, Tomasson MH, Dranoff G, Gilliland DG, Van Etten RA. 2001. Interleukin-3 and granulocyte-macophage colony-stimulating factor are not required for induction of chronic myeloid leukemia-like disease in mice by the P210 BCR/ABL oncogene. Blood 97:1442-1450.
Tomasson MH, Williams IR, Li S, Kutok J, Gillesen S, Dranoff G, Van Etten RA, Gilliland DG. 2001. Induction of myeloproliferative disease in mice by tyrosine kinase fusion oncogenes does not require GM-CSF or IL-3. Blood 97:1435-1441.
