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(Druker et al ., 2001b). However, imatinib was unable to abrogate BCR-
ABL-expressing leukemic cells (Marley et al ., 2000) and induced cellular
and clinical drug resistance (Branford et al ., 2002; Gorre et al ., 2001;
le Coutre et al ., 2000; Mahon et al ., 2000; Shah et al ., 2002; von Bubnoff
et al ., 2002; Weisberg and Griffin, 2000), suggesting that the use of imatinib
as a single agent may not prevent eventual disease progression to terminal
blast crisis or cure CML. Moreover, imatinib is much less effective in treat-
ing CML blast crisis patients (Druker et al ., 2001a; Talpaz et al ., 2000).
Recently, a newly developed Abl kinase inhibitor (termed BMS-354825
and produced by Bristol-Myers Squibb) has been shown to have an
inhibitory effect on almost all imatinib-resistant BCR-ABL mutants (Shah et
al ., 2004), offering some hope for overcoming imatinib resistance. However,
the BCR-ABL-T315I mutant that frequently appears in patients resistant to
imatinib therapy is still resistant to BMS-354825 (Shah et al ., 2004). Another
novel Abl kinase inhibitor, named AP23464, has also been effective against
several frequently observed imatinib-resistant BCR-ABL mutants, but inef-
fective against the BCR-ABL-T315I mutant (O'Hare et al ., 2004). While
clinical trials are ongoing to determine the effectiveness of these drugs in
treating human Ph + leukemia patients who are resistant to imatinib, our
preliminary results have shown that imatinib does not eradicate leukemic
cells in mice. Therefore, it is critical to develop new therapies that are syn-
ergistic with available treatment strategies and capable of killing BCR-ABL-
expressing leukemic stem cells (LSCs). The success of this approach requires
an understanding of the signaling pathways utilized by BCR-ABL in LSCs
to induce Ph + leukemias. Molecular pathways involved in regulation of the
self-renewal and survival of Ph + LSCs are largely unknown. We have iden-
tified BCR-ABL-expressing hematopoietic stem cells (HSCs) (GFP + Lin c-
Kit + Sca-1 + ) as CML stem cells (Hu et al ., 2006), providing a valuable assay
system for studying the biology of LSCs. Using this LSC assay system, we
will be able to identify and test molecular targets in LSCs.
2.3.1.2. Experimental design
Bone marrow cells are isolated from the long bones of CML mice that
are untreated or treated with imatinib. BCR-ABL-expressing or non-
BCR-ABL-expressing (transduced with the empty GFP vector) HSCs
(GFP + Lin c-Kit + Sca-1 + ) are stored by fluorescence-activated cell sorting
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