Cell Cycle 8:21, 3488-3492; November 1, 2009; 2009 Landes Bioscience

EXTRA VIEW
3488 Cell Cycle Volume 8 Issue 21
Key words: Alox5, BCR-ABL, Ph
+
leuke-
mia, leukemic stem cells, zileuton
Submitted: 06/11/09
Accepted: 08/19/09
Previously published online:
www.landesbioscience.com/journals/cc/
article/9852
*Correspondence to:
Shaoguang Li; Email: shaoguang.li@umassmed.edu
C
ancer stem cells (CSCs) are believed
to be the initiating cells for many
types of blood cancer and some solid
tumors, and curative therapies of these
cancers require eradicating CSCs.
Specic targeting of CSCs but not nor-
mal stem cell counterparts is a correct
strategy for developing new anti-can-
cer therapies, and the success of this
approach relies on identication of spe-
cic target genes in CSCs. Using BCR-
ABL-induced chronic myeloid leukemia
(CML) as a cancer model, we recently
identied arachidonate 5-lipoxygenase
(5-LO) gene (Alox5) as a critical regu-
lator for leukemia stem cells (LSCs) in
CML. Without Alox5, BCR-ABL fails to
induce CML in mice due to the impair-
ments of the functions of LSCs. The lack
of Alox5 does not signicantly affect the
functions of normal hematopoietic stem
cells. In addition, Zileuton, a specic
5-LO inhibitor, also causes the impair-
ments of the functions of LSCs in a
similar manner. Our results prove the
principle that CSC-specic genes that
play key roles in cancer development
can be identied and inhibition of these
genes can lead to eradication of these
cells for cure. Here, we further discuss
the mechanisms of Alox5 in CML, and
the use of Zileuton as a potential and
promising drug in eradicating LSCs
in CML and other myeloproliferative
diseases. We believe that our discovery
of the role of Alox5 in regulating the
function of LSCs in CML reminds us
of viewing CSCs at a different angel.
We predict that CSCs in other types of
cancer also utilize specic regulatory
pathways to control their survival and
self-renewal, and inhibition of these
pathways profoundly suppresses CSCs
but not their normal stem cell counter-
parts. Specic targeting of CSCs with-
out causing signicant harm to normal
stem cells should be a correct direction
to go in developing novel therapeutic
strategies in the future.
Introduction
Human Philadelphia chromosome arises
from a chromosomal translocation
between chromosome 9 and 22[t(9;22)
(q34;q11)], resulting in the formation
of a chimeric gene called BCR-ABL.
1

Philadelphia chromosome-positive (Ph
+
)
leukemias induced by the BCR-ABL
oncogene include chronic myeloid leuke-
mia (CML) and B cell acute lymphoblas-
tic leukemia (B-ALL). CML is a clonal
expansion of BCR-ABL-expressing
hematopoietic stem cells, and often ini-
tiates in a chronic phase and eventually
progresses to a terminal blastic phase.
The BCR-ABL tyrosine kinase inhibi-
tor imatinib mesylate is the standard
of care for Ph
+
leukemia, and induces
a complete hematologic and cytoge-
netic response in most CML patients.
2

However, imatinib is ineffective in sup-
pressing BCR-ABL mutants that have
mutations in the kinase domains of
BCR-ABL, and among these, the BCR-
ABL-T315I mutant, which is present in
1520% of imatinib-resistant patients, is
not inhibited by either imatinib or dasa-
tinib and other clinically available BCR-
ABL kinase inhibitors.
3
Imatinib will
The Alox5 gene is a novel therapeutic target in cancer stem cells of
chronic myeloid leukemia
Yaoyu Chen,
1
Dongguang Li
2
and Shaoguang Li
1,
*
1
Division of Hematology/Oncology; Department of Medicine; University of Massachusetts Medical School; Worcester, MA USA;
2
School of Computer and
Security Science; Edith Cowan University; Mount Lawley, WA Australia
www.landesbioscience.com Cell Cycle 3489
EXTRA VIEW EXTRA VIEW
Alox5, it was striking that recipients of
BCR-ABL transduced bone marrow cells
from Alox5
-/-
donor mice failed to develop
CML, albeit leukemia cells transiently
existed. Specically, myeloid leukemia
cells grew initially, reached a peak after
2 weeks, then started to decline, and even-
tually disappeared after 7 weeks. In con-
trast, recipients of BCR-ABL transduced
bone marrow cells from wild type donor
mice developed and died of CML within
4 weeks. In the same CML mice receiv-
ing BCR-ABL transduced Alox5
-/-
bone
marrow cells, non-BCR-ABL-expressing
myeloid cells were found to continue to
grow while BCR-ABL-expressing myeloid
cells gradually declined and disappeared
in the absence of Alox5. Because CML
develops from LSCs, together these results
suggest that loss of Alox5 leads to a func-
tional defect in LSCs rather than in nor-
mal HSCs.
To show the effect of Alox5 on LSCs,
FACS-sorted wild type and Alox5
-/-
LSCs
were mixed in a 1:1 ratio and then trans-
planted into the same recipient mice. At
day 25 after transplantation, more than
90% of myeloid leukemia cells in periph-
eral blood of the mice were wild-type
(CD45.1
+
) in origin, and all these mice
developed CML and died. These results
show that the ability of Alox5
-/-
LSCs to
induce CML is signicantly lower than
that of wild type LSCs, demonstrating
that the Alox5 deciency severely impairs
the function of LSCs. This idea was more
rmly demonstrated by a failure of Alox5
-/-

LSCs to induce CML when sorted Alox5
-/-

LSCs were transplanted into lethally
irradiated recipient mice. In these mice,
myeloid leukemia cells could be detected
initially in peripheral blood of recipi-
ent mice receiving BCR-ABL transduced
Alox5
-/-
bone marrow cells; however, these
leukemia cells soon disappeared, and the
mice became leukemia-free and survived.
Importantly, the Alox5 deciency did not
cause a functional defect in normal HSCs,
because wild-type or Alox5
-/-
bone mar-
row cells re-populated lethally irradiated
recipient mice in a similar degree. These
results indicate that loss of Alox5 speci-
cally causes the impairment in LSCs but
not normal HSCs.
Because LSCs (GFP
+
Lin
-
c-Kit
+
Sca-1
+
)
contain BCR-ABL-expressing long-term
Our Strategy for Targeting LSCs
A straightforward strategy for targeting
LSCs is through inhibiting development-
related genes that play roles in regulation
of both normal HSCs and LSCs, as many
of these genes have been identied and
initially found to play critical roles in the
development of normal HSCs. For exam-
ple, Wnt/-catenin, hedgehog and Bim-1
have been known to play important roles
in normal embryonic or HSCs, and later
reported to functionally regulate CML
stem cells.
14-17
As expected, these genes
essential for normal development of HSCs
should serve as effective targets for inhib-
iting LSCs.
17,18
Specically, cyclopamine,
a hedgehog inhibitor, reduces CML stem
cell population and prolongs survive of
CML mice.
18
However, a potential prob-
lem of this strategy is that normal stem
cells could be simultaneously inhibited
during a long-term cancer treatment, lead-
ing to severe side effects potentially. One
strategy for inhibiting LSCs is to target
genes that play crucial roles in functional
regulation of LSCs but not normal HSCs,
and obviously, the challenge for taking
this approach is to rst identify these key
stem cell-specic genes, as expression of
many genes can be altered by an oncogene
including BCR-ABL in CML and not all
these genes are essential for survival and
self-renewal of LSCs. The Alox5 gene is
probably the rst LSC-specic gene iden-
tied and shown to play a critical role in
LSCs but not normal HSCs,
13
and our
identication of this gene in LSCs shows a
good example for how we design a strategy
for targeting LSCs. In our study, the Alox5
gene was picked for further functional
study based on our comparison of gene
expression proles between normal HSCs
and LSCs by DNA microarray analysis, in
which Alox5 is shown to be upregulated by
BCR-ABL, which was conrmed by real-
time PCR analysis. Alox5 has been shown
to be associated with many important sig-
naling pathways including p53 and PI3K,
and is thought to be involved in many dif-
ferent types of diseases.
19,20
For the above-
mentioned reasons, we decided to test the
role of Alox5 in the development of CML
induced by BCR-ABL in mice. Although
we anticipated to observe an attenuation
of CML development in the absence of
unlikely provide a cure, as CML stem
cells exist.
4
CD34
+
Lin
-
cells from bone
marrow of CML patients contain CML
stem cells and are thought to be respon-
sible for disease progression and resistance
to imatinib.
4
These CML stem cells are
insensitive to imatinib both in vitro and
in vivo.
5,6
Specically, CML stem cells
were found in the undivided CD34
+
cell
population that was insensitive to ima-
tinib inhibition after being cultured with
imatinib, although imatinib killed almost
all dividing cells.
5
Furthermore, the BCR-
ABL transcript could still be detected in
CD34
+
cells in the bone marrow of CML
patients after a long-term treatment with
imatinib.
6
Leukemia Stem Cells in CML
Leukemia stem cells (LSCs) in many
types of hematologic malignancies are
believed to be a cell population required
for initiating and sustaining growth of
leukemia cells.
7-11
In our study of LSCs
in CML induced by BCR-ABL in mice,
we show that LSCs are similar pheno-
typically to normal hematopoietic stem
cells (HSCs).
12
In these CML mice,
BCR-ABL-expressing HSCs (GFP
+
Lin
-
c-
Kit
+
Sca-1
+
) are able to induce secondary
CML in recipient mice,
12
indicating that
this cell population functions as LSCs.
Mechanistically, it is reasonable to think
that BCR-ABL causes aberrant expres-
sion of genes responsible for consequently
turning a normal stem cell into a cancer
stem cell. To support this idea, we show
that the Alox5 gene is overexpressed in
LSCs in CML mice, and these BCR-ABL-
expressing cells also express the same cell
surface markers that are expressed on
normal HSCs.
13
Although the underly-
ing molecular mechanisms for the dif-
ferent properties between normal HSCs
and LSCs are still unclear, our identi-
cation of Alox5 as a critical regulator of
LSCs provides us with the rst clue for
further investigation. As described above,
bone marrow CD34
+
Lin
-
CML stem cells
in human patients are insensitive to the
BCR-ABL kinase inhibitor imatinib, and
are difcult to be eradicated.
5,6
This cell
population isolated from CML patients
will provide a more physiological system
for studying CML stem cells.
3490 Cell Cycle Volume 8 Issue 21
myeloproliferative disease (MPD). We
ask this question because we postulate
that some MPDs may have a common
cellular origin same as LSCs in CML,
and if so, the development of these MPDs
should be affected by Alox5. To test this
idea, we chose to determine whether
Alox5 plays a role in MPD induced by
the TEL-PDGFR oncogene.
24,25
In
mice, recipients of TEL-PDGFR trans-
duced bone marrow cells rapidly develops
MPD that recapitulates some aspects of
human CMML, including leukocyto-
sis, splenomegaly, and extramedullary
hematopoiesis.
24,25
To examine the role
of Alox5 in myeloid leukemia induced by
TEL-PDGFR, we transduced wild type
or Alox5
-/-
donor bone marrow cells in
B6 background with TEL-PDGFR to
induce leukemia. We found that recipi-
ents of TEL-PDGFR transduced bone
marrow cells from 5-FU-treated wild-type
donor mice developed and died of myeloid
leukemia within 40 days, whereas recipi-
ents of TEL-PDGFR transduced bone
marrow cells from Alox5
-/-
donor mice
were largely resistant to the induction of
myeloid leukemia (Fig. 1A). This defec-
tive disease phenotype correlated with
less spleen weight (Fig. 1B). In addition,
FACS analysis of myeloid leukemia cells
in peripheral blood showed that Gr-1
+

myeloid leukemia cells grew initially,
reached a peak level quickly, and then
decline to a low level at 60 days after the
induction of leukemia (Fig. 1C). These
results indicate that myeloid leukemia
induced by TEL-PDGFR cannot fully
develop in the absence of Alox5. It will be
interesting to further study what cell lin-
eage is affected by the Alox5 deciency
in TEL-PDGFR induced leukemia. It
is possible that the development of other
types of MPDs also requires Alox5, and
that the Alox5 pathway may represent a
major regulatory network in MPDs. On
the other hand, Alox5 is dispensable in
regulation of self-renewal and differen-
tiation of normal HSCs. This different
requirement of Alox5 in LSCs and nor-
mal HSCs provides a unique opportu-
nity to develop specic anti-LSC therapy
without causing harm to normal HSCs.
Importantly, Alox5 may serve as an effec-
tive target in preventing the development
of CML and perhaps other MPDs.
then analyzed LSCs in bone marrow of
the treated mice by FACS at days 20 and
90 after the induction of CML by BCR-
ABL. We nd that the ratio between
the percentage of LT-LSCs and that of
ST-LSCs/MPP cells is similarly increased
as shown in recipients of BCR-ABL trans-
duced Alox5
-/-
bone marrow cells, suggest-
ing that inhibition of 5-LO by Zileuton
also causes the blockade of differentiation
of LT-LSCs. These results indicate that
targeting of LSCs with Zileuton could
be benecial to improving treatment of
CML, and we tested this idea in mice.
We treated CML mice with a placebo,
Zileuton or imatinib alone, or two agents
in combination. As expected, all placebo-
treated mice developed and died of CML
within 4 weeks after the induction of
CML by BCR-ABL, and Zileuton alone
was even more effective than imatinib in
prolonging survival of CML mice. The
therapeutic effect of Zilueton is likely
through inhibiting LSCs, because at the
early stage of the disease Zileuton treat-
ment caused a less marked reduction of
white blood cell counts than did imatinib
treatment, presumably because Zileuton
targeted LSCs and imatinib inhibited
more differentiated leukemia cells. About
7 weeks after the treatment with Zileuton,
GFP
+
Gr-1
+
leukemia cells in peripheral
blood of the mice gradually declined and
dropped from over 50% to less than 2%,
indicating that myeloid leukemia is even-
tually eliminated. Treatment of CML
mice with both Zileuton and imatinib had
a better therapeutic effect than with either
Zileuton or imatinib alone in prolonging
survival of the mice. These ndings are
consistent with those from our genetic
studies using Alox5
-/-
mice, demonstrat-
ing that targeting of the Alox5 pathway is
potentially curative for CML.
Alox5 is Also Required for
Myeloproliferative Disease
Induced by TEL-PDGFR
The specic role of the Alox5 gene in reg-
ulating the function of LSCs but not nor-
mal HSCs suggests different mechanisms
of self-renewal and differentiate between
LSCs in CML and normal HSCs. We
wonder whether Alox5 is also required
for the development of other types of
(LT), short-term (ST) HSCs and multipo-
tent progenitor (MPP) cells, in our study
we further investigated which of these
cell populations is affected by the Alox5
deciency. At an early time point after
the induction of CML by BCR-ABL, the
numbers of LSCs were similar between
recipients of BCR-ABL transduced wild-
type and Alox5
-/-
bone marrow cells.
Among these LSCs, the percentage or
total number of LT-LSCs was about half
of those of ST-LSCs/MPP cells. After 90
days, few LSCs could be found in bone
marrow of mice receiving BCR-ABL trans-
duced Alox5
-/-
bone marrow cells, and the
percentage or total number of LT-LSCs
was about eight-fold higher than those of
ST-LSCs/MPP cells. This reversal of the
LT-LSCs to ST-LSCs/MPPs ratio suggests
that the Alox5 deciency causes a blockade
of differentiation of LT-LSCs. Although
more experiments are needed to conrm
this check point between LT-LSCs and
ST-LSCs, if true, this could be the rst
evidence showing how differentiation of
LT-LSCs is mechanistically regulated.
Expression of Alox5 is also differentially
expressed in human CML.
21,22
We believe
that the Alox5 pathway represents a major
molecular network that regulates the func-
tion of LSCs, and much more work needs
to be done to fully dissect this pathway.
Zileuton is a Promising Drug for
Eradicating LSCs in Human CML
Several drugs, such as cyclopamine
(inhibiting the function of hedgehog)
and rapamycin (restoring the function
of Pten), have been reported to have
an inhibitory effect on leukemia stem
cells,
18,23
and their effectiveness in treat-
ing human leukemias remains to be seen.
Our identication of the role of the Alox5
gene in regulating LSCs in CML pro-
vides a unique opportunity to develop a
novel anti-stem cell therapy for CML, as
Zileuton, a FDA-approved drug for treat-
ing human asthma,
22
specically inhibits
the enzymatic activity of arachidonate
5-lipoxygenase (5-LO),
23
the product of
the Alox5 gene. In our Alox5 study, we
further tested whether Alox5 is a poten-
tial therapeutic target in LSCs.
13
With
Zileuton, we treated recipients of BCR-
ABL transduced bone marrow cells, and
www.landesbioscience.com Cell Cycle 3491
ABL kinase activity alone is insufcient
to completely shut down BCR-ABL, and
that other strategies need to be developed.
Our demonstration of the role of Alox5 in
regulating the function of LSCs but not
normal HSCs identies the rst LSC-
specic gene in CML, and provides us
with an exciting opportunity to explore
the regulatory molecular pathways in
LSCs. In our Alox5 study, we have pri-
marily linked the Alox5 pathway to acti-
vation of -catenin, as we showed that
the loss of Alox5 caused downregulation
of -catenin expression in LSCs, but not
in normal HSCs. Several important ques-
tions remain to be answered: (1) How
does BCR-ABL activate Alox5 expression
in LSCs? (2) How do Alox5 and -catenin
interact to regulate the function of LSCs?
(3) What are other signaling molecules
in the Alox5 pathway? (4) Is the 5-LO
inhibitor Zileution effective in eradicating
human cancer stem cells in CML patients?
(5) Does LTB4 produced by 5-LO play a
in LSCs, and the key is to select candidate
genes for further functional tests. Prior to
the functional tests, we always conrm
expression change of a gene of interest by
real-time PCR. The function of a candi-
date gene can be primarily determined
based on the literature or database search.
The last step is to functionally test a can-
didate gene using knockout or transgenic
strains.
Future Directions
Imatinib effectively inhibits BCR-ABL
kinase activity, but does not remove BCR-
ABL protein, which may partially explain
why imatinib does not eradicate LSCs in
CML. In fact, we have shown that total
number and percentage of LSCs in bone
marrow of CML mice gradually increase
with time during imatinib treatment,
although the BCR-ABL kinase activity
is greatly inhibited by imatinib.
26
These
results indicate that inhibition of BCR-
Our Strategies for Identifying New
Therapeutic Targets in Cancer
Stem Cells
Targeting of cancer stem cells (CSCs) has
become an important issue in develop-
ing new therapies of cancer. As described
above, we believe that the key to eradicat-
ing CSCs is to identify critical pathways
responsible for regulating self-renewal and
differentiation of CSCs but not normal
stem cells. Our Alox5 study has shown
that this strategy is feasible.
13
Using CML
induced by BCR-ABL as a model disease,
here we propose a strategy for identifying
new therapeutic targets in CSCs (Fig. 2).
Isolation of LSCs and normal HSCs is a
key initial step to obtaining high-quality
RNA for microarray analysis. In our study,
BCR-ABL-expressing HSCs (GFP
+
Lin
-
c-
Kit
+
Sca-1
+
) represent LSCs in CML mice.
It is important to realize that the microar-
ray analysis will show many genes that
were up and downregulated by BCR-ABL
Figure 1. Alox5 is essential for the induction of MPD by TEL-PDGFR. Kaplan-Meier survival curves for recipients of TEL-PDGFR-transduced bone
marrow cells from wild type or Alox5
-/-
donor mice. All recipients of BCR-ABL-transduced bone marrow cells from wild type donor mice developed
MPD and died within 40 days after bone marrow transplantation (days post BMT), whereas the majority of recipients of TEL-PDGFR-transduced
bone marrow cells from Alox5
-/-
donor mice survived.
3492 Cell Cycle Volume 8 Issue 21
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role in regulating the function of LSCs?
We believe that our Alox5 story has shown
a new strategy for how to effectively target
cancer stem cells for developing curative
therapies.
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Figure 2. Our strategy for identifcation of genes that play key roles in regulating the functions of LSCs. One of the most critical steps in this ap-
proach is to analyze DNA microarray data to provide a short list of candidate genes.