Positions

• 2013-Present: Professor, University of Massachusetts Medical School, Division of Hematology/Oncology, Department of Medicine, Worcester, MA
• 2008-2013: Associate Professor, University of Massachusetts Medical School, Division of Hematology/Oncology, Department of Medicine, Worcester, MA
• 2007-2008: Associate Professor, The Jackson Laboratory, Bar Harbor, ME
• 2001-2007: Assistant Professor, The Jackson Laboratory, Bar Harbor, ME

Societies

• 2007-present American Association for the Advancement of Science (AAAS)
• 2006-present American Association for Cancer Research (AACR)
• 2002-present American Society of Hematology (ASH)

Current Research

Molecular Basis of Human Philadelphia Chromosome-positive 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, other BCR-ABL kinase inhibitors, such as dasatinib, have been shown to inhibit almost all imatinib-resistant BCR-ABL mutants; the exception is the T315I mutant, which is present in 15-20% 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 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 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.

Identifying and targeting leukemic stem cells

To identify CML stem cells, we tested whether BCR-ABL-expressing HSCs function as the stem cells. We first sorted HSCs (Lin-c-kit+Sca-1+) were sorted out from C57BL/6 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 bone marrow cells from primary CML mice, and sorted out the BCR-ABL-expressing HSCs (GFP+Lin-c-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.

One of major focuses in the lab is to understand the biology of leukemia stem cells (LSCs), and to identify selective and effective target genes that play key roles in survival and self-renewal of these LSCs. Based on our DNA microarray data and genetic validation of candidate genes in our leukemia mouse models, we have identified a group of genes that are essential for the functions of LSCs, shedding light on developing an anti-stem cell therapy for CML.