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Colloquium
Normal and leukemic hematopoiesis: Are leukemias
a stem cell disorder or a reacquisition of stem
cell characteristics?
Emmanuelle Passegue, Catriona H. M. Jamieson, Laurie E. Ailles, and Irving L. Weissman*
Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
. .
Leukemia can be viewed as a newly formed, abnormal hemato-
poietic tissue initiated by a few leukemic stem cells (LSCs) that
undergo an aberrant and poorly regulated process of organogen-
esis analogous to that of normal hematopoietic stem cells. A
hallmark of all cancers is the capacity for unlimited self-renewal,
which is also a defining characteristic of normal stem cells. Given
this shared attribute, it has been proposed that leukemias may be
initiated by transforming events that take place in hematopoietic
stem cells. Alternatively, leukemias may also arise from more
committed progenitors caused by mutations and/or selective ex-
pression of genes that enhance their otherwise limited self-re-
newal capabilities. Identifying the LSCs for each type of leukemia
is a current challenge and a critical step in understanding their
respective biologies and may provide key insights into more
effective treatments. Moreover, LSC identification and purification
will provide a powerful diagnostic, prognostic, and therapeutic
tool in the clinic.
n many respects a leukemic cell resembles a stem cell. Stem
~ cells are defined as clonogenic cells capable of both self-
renewal and multilineage differentiation. The cells from the
hematopoietic system are continually generated from self-
renewing progenitors in the bone marrow called hematopoietic
stem cells (HSCs), or blood-forming stem cells, which have been
isolated in both humans and mice (1-44. HSCs can be divided
into a long-term subset (LT-HSC), capable of indefinite self-
renewal, and a short-term subset (ST-HSC) that self-renew for
a defined interval. HSCs give rise to nonself-renewing oligolin-
eage progenitors, which in turn give rise to progeny that are more
restricted in their differentiation potential, and finally to func-
tionally mature cells. The phenotypic and functional properties
of normal HSCs have been extensively reviewed (5-7~. Recent
studies have demonstrated that normal stem cells and cancer
cells share the ability to self-renew and that many pathways
classically associated with cancer also regulate normal stem cell
development. For most cancers, the target cell of the transfor-
mation events is unknown, but evidence indicates that certain
types of leukemias arise from mutations that accumulate in
HSCs. Conversely, analyses performed on mouse models of
certain types of human leukemias have demonstrated that
restricted progenitors or even differentiated cells may also
become transformed. In this review, we will discuss the notion
of the leukemic stem cell (LSC), with emphasis on myeloid
leukemias, and the potential origin of these cells: HSCs or
committed progenitor cells that have reacquired stem cell char-
acteristics, mainly the ability to self-renew. Finally, we will
describe the research under way to elucidate the molecular
pathways leading to leukemia.
HSCs
The first experimental evidence to indicate the existence of
HSCs was the discovery in 1961 by Till and McCulloch (8) of a
11842-11849 1 PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1
population of clonogenic bone marrow cells capable of gener-
ating myelo-erythroid colonies in the spleen of lethally irradiated
hosts. Occasionally these colonies contained clonogenic cells
that could be further retransplanted into secondary lethally
irradiated hosts and reconstitute the immune system. These were
proposed to be HSCs, i.e., progenitor cells with the essential
characteristic of self-renewal and differentiation potential for all
types of blood cells (1-4~. The development of clonal assays for
all major hematopoietic lineages together with the availability of
multiparameter fluorescence-activated cell sorting (FACS) has
enabled the prospective purification of HSCs from mice and to
highly enrich for HSCs from humans according to the cell-
surface expression of specific molecules and their functional
read-out in vivo and in vitro in stromal long-term colony-
initiating assays (54. After the identification and prospective
isolation of murine HSCs, considerable progress has been made
toward the characterization of the mechanisms controlling their
fates. During or after cell division, the two daughter cells of a
stem cell each have to decide their fate. They can either choose
to remain as HSCs, commit to differentiation, or die by apoptosis
and also to stay in the bone marrow or migrate to the periphery.
These processes of cell-fate decisions must be finely tuned to
maintain a steady-state level of functional HSCs in the bone
marrow and to constantly provide progenitors for the various
hematopoietic lineages.
Surface Markers. Several marker combinations, such as ~Linneg/~°W,
Thyl.1~°W, c-Kithigh, Sca-1+], ~Lin-, Thyl.1~°W, Sca-1+, rhodamine
123~°W] (9) or [Lin-, CD34-/int, c-Kit+, Sca-1+] (4), have been
used to isolate nearly pure mouse HSC populations. Similar
marker combinations [Lin-, Thyl+, CD34+, CD38neg/~°W] (9) are
used to highly enrich human HSC populations. Although there
are some variations in the exact frequencies found, the different
methods of isolation all indicate that HSCs are rare cells. Using
the ~Linneg/~ow Thy1 l~0w, c Kithigh, Sca-1+] markers, ~1:5,000
mouse bone marrow cells have long-term, multilineage, repop-
ulating capability, i.e., is a LT-HSC, whereas ~1:1,000 has a
more limited, short-term, multilineage repopulating capability,
i.e., is a ST-HSC or a multipotent progenitor (MPP). Recently,
the expression of the receptor tyrosine kinase Flk-2 has been
identified as a reliable marker to discriminate between LT-HSC
This paper results from the Arthur M. Sackler Coiloquium of the Nationai Acaclemy of
Sciences, "Regenerative Meclicine," heic] October 18-22, 2002, at the Arnoic] ancl Mabei
Beckman Center of the Nationai Acaclemies of Science and Engineering in Irvine, CA.
Abbreviations: HSC, hematopoietic stem ceit; ET-HSC, iong-term HSC; ST-HSC, short-term
HSC; MPP, muitipotent progenitor; CEP, common iymphoic] progenitor; CMP, common
myeloic] progenitor; GMP, myelomonocytic progenitor; ESC, leukemic stem ceil; CML
chronic myeloic' leukemia; AME, acute myeloic' leukemia; PME, promyelocytic leukemia;
APME, acute PME; Shh, Sonic hec~gehog; RARcY, retinoic acic] receptor a.
*To whom corresponclence shouic] be aciciressecl. E-maii: irv@?stanford.eclu.
@) 2003 by The Nationai Acaclemy of Sciences of the USA
www.pnas.org/cgi/cloi/10. ~ 073/pnas.2034201 ~ 00
OCR for page 27
Stem cells
Oligolineage
progenitors
-
· _
MEP —
, ~
,
~ ~ Lined c-kithi Sca-1hi Thy1a1'° Flk2~(mouse)
LT-HSC - ) J
~ Lint CD34. CD38- CD90+ (human)
ST HSC ~ ~ 31 Line c-kithi Sca-1 hi Thy] .1'° Flk2+ (mouse) |
1
Multipotent _ ~
progenitors MPP ~ | Lin-~°c-kithiSca-1hiThy1.1-Flk2+(mouse) |
CMP 1~) ~ CLP
/ ~ Pro-DC ~ ~c/ ~ ~`
~ ~ Pro-T ~Pro-NK. Pro-E}
Mature cells .. ~ ~
GMP ~
Erythrocytes Platelets Granulocytes Macrophages Dendritic-cells
~ ~ —
T-cells NK-cells B-cells
Fig. 1. Hematopoietic and progenitor cell lineages. HSCs can be divided into LT-HSCs, highly self-renewing cells that reconstitute an animal for its entire life
span, or ST-HSCs, which reconstitute the animal for a limited period. ST-HSCs differentiate into MPPs, which do not or briefly self-renew, and have the ability
to differentiate into oligolineage-restricted progenitors that ultimately give rise to differentiated progeny through functionally irreversible maturation steps.
The CLPs give rise to T lymphocytes, B lymphocytes, and natural killer (NK) cells. The CMPs give rise to GMPs, which then differentiate into monocytes/
macrophages and granulocytes, and to megakaryotic/erythroid progenitors (MEP), which produce megakaryocytes/platelets and erythrocytes. Both CMPs and
CLPs can give rise to dendritic cells. All of these stem and progenitor populations are separable as pure populations by using cell surface markers.
[Thyl.1l°w, Flk-2neg], ST-HSC [Thyl.1~°W, Flk-2+], and MPP
[Thyl.1-, Flk-2+] in combination with the [Lint, c-Kithigh,
and Sca-1+] markers (10~. Morphologically, HSCs and MPPs
resemble lymphocytes.
Differentiation Potential. Whereas LT-HSCs self-renew for the life
of the host, the derivative ST-HSCs retain self-renewal capacity
for ~8 weeks (2) and give rise to the briefly self-renewing MPPs
(11), which then differentiate into oligolineage-restricted pro-
genitors through functionally irreversible maturation steps (see
Fig. 1~. Two kinds of oligolineage-restricted progenitors have
been identified so far in the mouse: the common lymphoid
progenitors (CLPs), which at a clonal level are restricted to give
rise to T lymphocytes, B lymphocytes, and natural killer cells
(12), and the common myeloid progenitors (CMPs), which are
progenitors for the myelo-erythroid lineages (13~. CMPs give
rise to myelomonocytic progenitors (GMPs), which in turn
produce monocytes/macrophages and granulocytes, and to
megakaryotic/erythroid progenitors, which differentiate into
megakaryocytes/platelets and erythrocyte, but still maintain the
potential for B cell lineage differentiation at an extremely low
frequency (134. Interestingly, both CMPs and CLPs can give rise
to dendritic cells (14, 15), suggesting the existence of alternative
commitment pathways to the mutually exclusive developmental
pathways for myeloid and lymphoid lineages. All of these
progenitor populations are separable as pure populations by
using cell surface markers and have been shown to be devoid of
detectable self-renewal activity after transplantation (16~.
Passegue et a/.
In parallel to the clarification of the developmental hierarchy
between HSCs and committed progenitors, considerable
progress has been made toward the identification of molecular
mechanisms regulating lineage commitment within the hema-
topoietic system. Although it is largely beyond the scope of this
review to describe these mechanisms in detail, they appear to
represent a stepwise process characterized by the alternate
expression of specific transcriptional regulators, growth factors,
and growth factor receptors, whose combination determines
lineage commitment and maturation (17, 18~. With the recent
use of DNA microarrays to investigate the gene expression
profile of HSCs, progress has also been made toward the
identification of the downstream effecters genes of the tran-
scription factors (19, 20~. Future gene expression profiling of
defined HSCs and progenitor populations should rapidly ad-
vance our understanding of the molecular regulatory networks
that control the development of all blood cells.
Proliferation, Apoptosis, and Self-Renewal. As HSCs mature from
the long-term self-renewing pool to MPPs, they progressively
lose their potential to self-renew but become more mitotically
active. In young mice, the frequency of HSCs in hematopoietic
tissues is relatively constant (21-23) and HSCs have long been
considered to be a resting cell population, with only a few stem
cells contributing to steady-state hematopoiesis. In fact, recent
studies have shown that in young adult mice ~8-10% of
LT-HSCs randomly enter the cell cycle per day, with all HSCs
entering the cell cycle in 1-3 months (24, 25). Although the rate
PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11843
OCR for page 28
at which human HSCs enter the cell cycle is currently unknown,
assuming a comparable rate as in the mouse would result in a
very large number of cell divisions over a lifetime and agree with
the largest estimates of the number of cell divisions that an HSC
can undergo (26~.
A requirement for continuously dividing and self-renewing
cells, such as HSCs and tumor cells, is the ability to avoid fatal
telomere shortening through the action of the telomerase com-
plex (27, 284. In mice, LT-HSCs contain as much telomerase
activity as cancer cells do, whereas ST-HSCs and MPPs have
significantly less (294. Enriched human stem/progenitor cell
populations show telomere shortening with age (27), as do
mouse LT-HSCs that undergo many divisions during serial
transplantation (30~. These results are consistent with the hy-
pothesis that telomere shortening may limit the replicative
capacity of HSCs. Serial transplantation has often been taken as
a measure of the replicative life span of hematopoietic cells.
However, resting bone marrow HSCs that are in the S/G2/M
phases of the cell cycle transplant less well than HSCs with 2 M
DNA content (31), although they are fully active as LT-HSCs.
Furthermore, after transplantation, a much higher frequency of
HSCs remain in the cell cycle for several months (23, 31), and
whether HSCs have long or short telomeres, they are lost by the
fifth serial transplantation generation (324. Therefore, serial
transplantation of HSCs may create artefacts that renders it as
an inappropriate measure of HSC intrinsic life span.
The number of HSCs, as with many other cell types, is also
regulated by programmed cell death or apoptosis (33~. Prevent-
ing apoptosis by transgenic expression of the antiapoptotic
protein Bc1-2 in LT-HSCs has been shown to cause a gradual
increase in LT-HSC frequency, which takes place without ma-
lignant transformation and despite Bcl-2-mediated decrease of
LT-HSC entry into the cell cycle. Different mouse strains also
show differences in the number and cell cycle status of HSCs,
perhaps indicating differences in susceptibility to apoptosis.
Several chromosomal regions have been shown to contribute to
these polymorphisms (34, 35), but to date few of these regulator
genes have been isolated (36~.
HSCs reside predominantly in the bone marrow but low
numbers of HSCs are also found in peripheral blood (37~. Recent
work has shown that HSCs rapidly and constitutively migrate
through the blood and play a physiological role in functional
reengraftment of unconditioned bone marrow (38~. In viva,
LT-HSCs respond to a variety of conditions by entering the cell
cycle, expanding their numbers by self-renewing proliferation,
and mobilizing into the bloodstream (394. These conditions
include myelosuppresion with chemotherapeutic agents such as
cyclophosphamide and treatment with the cytokines granulocyte
colony-stimulating factor and granulocyte-macrophage colony-
stimulating factor (40, 41~. In vitro, several growth factors have
been identified to induce a potent proliferation of HSCs but have
been unsuccessful in preventing their diffentiation or apoptosis
in long-term cultures. Although progress has been made in
identifying culture conditions that maintain some of the HSC
functions in vitro (42), it has not been possible to identify a
combination of growth factors able to expand the number of
HSCs in culture with retention of their transplantable activity.
Mouse embryonic stem cells can continuously proliferate, with-
out undergoing terminal differentiation, in the presence of
leukemia inhibitory factor, which acts, at least in part, via
activation of the transcription factor STAT3, an essential mech-
anism for self-renewal activity in embryonic stem cells (43~. It
therefore seems likely that cytokines and/or cell-surface mole-
cules expressed by bone marrow stromal cells (44) are necessary
for maintaining the self-renewal activity of HSCs.
At the molecular level, the mechanisms controlling HSC
self-renewal activity are still poorly understood. Interestingly,
the signaling pathways that have been shown to date to be
1 1 844 1 www. pnas.org/cgi/doi/ 1 0.1 073/pnas.20342011 00
involved in the regulation of HSC self-renewal, i.e., Hox genes
and Notch, Sonic hedgehog (Shh), and Wnt signaling pathways,
are also hypothesized to be associated with oncogenesis (45--484.
Expression of some of the Hox genes, such as HoxB4 and HoxA9,
has been intimately tied to HSC self-renewal, because their
overexpression in hematopoietic cells causes the selective ex-
pansion of the HSC population both in vitro and in vivo (49, 50~.
Activation of Notch by its ligand Jagged-1 in cultured HSCs also
results in an increased amount of primitive progenitor activity,
both in vitro and after transplantation in vivo, indicating that
Notch activation promotes HSC self-renewal (51, 52~. Similarly,
HSC-enriched ~Lin-, CD34+, CD38-] human cells exhibit in-
creased self-renewal capability in response to Shh stimulation in
vitro, indicating that the Shh pathway also plays a role in
regulating self-renewal of HSCs as of other types of tissue stem
cells (53~. Recently, the different components of the Wnt
signaling pathway have been elucidated (54~. The Wnt proteins
usually act by binding to cell-surface Frizzled receptors and
activating the dishevelled (Dsh) gene product. Active Dsh
inhibits the association of glycogene synthase kinase 3~3 and axin
with p-catenin, leading to reduced phosphorylation and nuclear
translocation of 13-catenin. Nuclear [3-catenin associates with the
LEF/TCF family of transcription factors to stimulate transcrip-
tion of downstream target genes. The expression of Wnt proteins
in the bone marrow (55) suggests that they may play a role in
hematopoiesis. Furthermore, retroviral transduction of consti-
tutively activated I3-catenin into HSCs leads to their expansion
in vitro, and retroviral transduction of the Wnt pathway inhibitor
axin leads to inhibition of HSC proliferation, increased HSC
death in vitro, and reduced reconstitution in vivo (56~. Soluble
partially purified Wnt proteins obtained from conditioned su-
pernatants have also been shown to influence the proliferation
of CD34+ hematopoietic progenitors isolated from mouse fetal
livers and human bone marrow (57, 58~. Recently, purified
Wnt3A has been shown to act on highly purified LT-HSCs to
cause up to a 300-fold expansion of LT-HSCs identified both
phenotypically and functionally (56, 59~. The molecular mech-
anism by which Wnt signaling influences HSCs remains to be
elucidated, as does the question of whether the Wnt, Notch, and
Shh pathways interact to regulate stem and progenitor cell
self-renewal.
Emerg~ng evidence from stem cell biology has recently provided
new insights into cancer biology by emphasizing the relationship
between stem cells and tumor cells and by formalizing the notion
that tumors might contain some cancer stem cells, which are rare
cells with indefinite proliferation potential that drive the for-
mation and growth of tumors (474. Leukemias are blood cancers,
and the hematopoietic system is one of the best tissues to study
the notion of the cancer or LSCs, because normal HSCs are a
well-characterized cell population and LSCs have recently been
revealed (see below). In the present review we focus on myeloid
leukemias (60-62~. The current paradigm of myeloid leukemias
states that the process giving rise to the aggressive and often fatal
acute myeloid leukemias (AMLs) takes place through an or-
dered progression from the indolent myelodysplastic syndromes
or myeloproliferative disorders to the more aggressive chronic
myeloid leukemias (CMLs) that, if not inherently fatal, ulti-
mately progress to the blast crisis of AML. Many different
translocations and genetic aberrations are found within the
various forms of myeloid leukemia and specific translocations
are often associated with disease subtypes that manifest them-
selves through the accumulation of immature myeloid cells at
varying stages of differentiation (60, 61~.
The Concept of LSCs. Since the early 1970s, the notion of tumor-
igenic LSCs has emerged based on several studies showing that
Passegue et a/.
OCR for page 29
only a small subset of leukemic cells was capable of extensive
proliferation in vitro and in vivo. Using ascites-derived mouse
myeloma cells, separated first from normal hematopoietic cells,
Park and coworkers (63) showed that only 1 in 10,000 to 1 in 100
leukemic cells were able to form colonies in vitro in clonal
colony-forming assays. In addition, only 1-4% of the total
number of leukemic cells transplanted in vivo could form spleen
colonies, even using different types of leukemic cells (64, 654. Progenitors
Because the clonogenic read-out of the leukemic cells perfectly
mirrored the difference in clonogenicity among the normal
hematopoietic cells, the clonogenic leukemic cells were de-
scribed as LSCs. However, not until 1997, in studies published by
Blair and colleagues (66) and Bonnet and Dick (67), was there
a clear demonstration that most of the leukemic cells were
unable to proliferate extensively and only a small, defined subset Mature cells
of cells was consistently clonogenic. In these studies, LSCs for
human AML were identified prospectively-and purified as
tThyl-, CD34+, CD38-] cells from various patient samples.
Although these cells represent a small and variable proportion
of the totality of the AML cells (0.2-1% depending on the
patient), they were the only cells capable of transferring AML
from human patient to nonobese diabetic/severe combined
immunodeficient (NOD/SCID) mice and were referred as SCID
leukemia-initiating cells or SL-IC. The general concept of LSCs
is now well established and serves as a reference for the
identification of other cancer stem cells in solid tumors (47, 68~.
A given leukemia can be viewed as a newly formed aberrant
hematopoietic tissue initiated by tumorigenic leukemic cells that
have kept or reacquired the capacity for indefinite proliferation
through accumulated mutations. This concept suggests that
leukemias are produced by a few LSCs that undergo an aberrant
and poorly regulated process of organogenesis analogous to that
of normal HSCs. Both cell types have extensive proliferative
potential and the ability to give rise to new hematopoietic tissues,
normal in the first case and abnormal in the second. Both normal
and leukemic tissues are composed of a combination of heter-
ogeneous cells, with different phenotypic characteristics and
proliferative potentials. Because most leukemias, similar to most
solid tumors, have a clonal origin, tumorigenic leukemic cells
must give rise to phenotypically diverse progeny including a few
LSCs with indefinite proliferation potential, as well as cells
within the leukemia that may have limited or no proliferative
potential. Although some of the heterogeneity in the tumor
arises as a result of continuing mutagenesis or epigenetic
changes, it is likely that it also arises from the aberrant differ-
entiation of the cancer cells. The variable expression of myeloid
markers by blast cells in CML further supports the notion that
some of the heterogene~ty within the leukemic cells results from
the abnormal differentiation of malignant myeloid cells.
The Controversy About the Origin of LSCs. For most leukemia, as for
most cancers, the target cell of transforming mutations is still
unknown. Because normal stem cells and LSCs share the ability
to self-renew, as well as various developmental pathways, it has
been postulated that LSCs are HSCs that have become leukemic
as the result of accumulated mutations. Because normal stem
cells and LSCs share the ability to self-renew, as well as various
developmental pathways, it is possible that LSCs are HSCs that
have become leukemic as the result of accumulated mutations.
HSCs have the machinery for self-renewal already activated and
therefore may require fewer mutations to maintain it than more
differentiated cells would require to activate it ectopically. HSCs
also persist throughout life and therefore have much greater
opportunities to accumulate mutations than more mature cells,
which persist only for a short period. Conversely, LSCs could also
be a more restricted progenitor or even a differentiated mature
cell, which would have first to reacquire the stem cell capability
for self-renewal before becoming tumorigenic to accumulate
Passegue et al.
HSC LSC Cq! ~
r
) LSC
Fig. 2. Origin of the LSC. A given leukemia can be viewed as a newly formed
abnormal hematopoietic tissue initiated by a few LSCs that undergo an
aberrant and poorly regulated process of organogenesis analogous to that of
normal HSCs. LSCs can either be HSCs, which have become leukemic as the
result of accumulated mutations, or more restricted progenitors, which have
reacquired the stem cell capability of self-renewal. Regardless of their origin,
both types of LSCs give rise to similar end-stage leukemias.
additional mutations. Restricted progenitors could therefore
potentially be transformed either by acquiring mutations that
cause them to self-renew like stem cells, or HSCs themselves
could be accumulating genetic and epigenetic changes, such as
loss of programmed cell death, ultimately leading to the gain of
self-renewal activity in a restricted progenitor population (see
Fig. 2~.
HSCs as LSCs. There is now evidence that certain subtypes of
human AML and CML arise from mutations that accumulate in
HSCs. For most AML subtypes, except for the M3 acute
promyelocytic leukemia (APML) subtype, the only cells capable
of transplanting AML in nonobese diabetic/severe combined
immunodeficient (NOD/SCID) mice have a tCD34+, CD38-]
phenotype, similar to that of normal HSC, whereas the iCD34+,
CD38+] leukemic blast cells cannot transfer the disease to mice
(66, 674. These observations suggest that for most AML subtypes
HSCs rather than committed progenitors are the target for
leukemic transformation, regardless of the extent and the lineage
of differentiation of the leukemic cells present in the peripheral
circulation. The t(8,21) translocation is one of the most frequent
chromosomal abnormalities associated with AML (~15%) (69~.
This translocation involves the AML-1 gene (AML1 also known
as CBFA2, PEBP2aB, and RUNX1) on chromosome 21 and the
ETO (MTG8) gene on chromosome 8 and generates an AML1-
ETO fusion transcription factor (70~. Expression of the AML1-
ETO fusion transcript can be detected in leukemic blast cells but
also in normal bone marrow cells, including HSCs, obtained
from AML patients in remission (71, 72~. However, these
prospectively isolated AML1-ETO-expressing stem cells and
their progeny are not leukemic and could differentiate into
normal myelo-erythroid cells in vitro (714. This observation
suggests that the translocation occurred originally in normal
HSCs and that additional mutation in a subset of these HSCs or
their progeny subsequently led to leukemia. Because the original
AML1-ETO-expressing normal HSC were ~Lin-, CD34+,
CD38-, Thy-1~] and the LSC were ~Lin-, CD34+, CD38-,
Thy-1-i, the subsequent transforming mutation might have
occurred either in downstream Thy-1 negative progenitors or in
HSCs that have lost Thy-1 expression as one of the first
consequences of the neoplastic transformation (71, 724. This
observation is further supported by the finding of Blair and
PNAS | September 30, 2003 1 vo~. 100 | suppl. ~ | 11845
OCR for page 30
colleagues (66) that the severe combined immunodeficient
leukemia-initiating cells from human AML samples were also
Thy-1 (CD90) negative.
Approximately 95% of CML patients possess the Philadelphia
(Ph) chromosome, a shortened chromosome 22 arising from a
reciprocal translocation t(9q34;22qll), which serves as a cyto-
genetic hallmark of the disease, and contains the BCR-ABL
fusion gene, which produces the 210-kDa BCR-ABL chimeric
protein (73~. This fusion protein can be found in myeloid,
erythroid, B lymphoid, and occasionally T lymphoid cells in the
majority of CML patients, suggesting that the original translo-
cation takes place in LT-HSCs. This finding was consistent with
the early demonstration, by analysis of allelic X chromosome
enzyme isoforms, that CML cells were part of a larger clone that
included red blood cells and B cells (74~. More recently, the
BCR-ABL gene could be detected in endothelial cells produced
from cells of a CML patient (754. If confirmed as not the result
of cell fusion, these data might place the level of the BCR-ABL
mutation in a cell type even earlier than the LT-HSCs, such as
the putative hemangioblast cells, a very primitive cell population
with both hematopoietic and endothelial differentiation poten-
tial that remains to be identified. In chronic phase, the leukemic
clone appears to be maintained by a small number of BCR-
ABL-positive tCD34+, CD38-] cells, a population enriched for
HSCs, which differentiate normally and amplify slowly. In
contrast, as these cells enter the intermediate stages of lineage
restriction, their progeny are selectively expanded and generate
an enlarged pool of overproduced tCD34-] progeny. Recent
analyses of purified subsets of primitive CML cells have provided
a coherent explanation for this dichotomous behavior of BCR-
ABL-positive stem and progenitor cells based on the discovery
of an unusual autocrine IL-3/granulocyte colony-stimulating
factor mechanism (624. This mechanism only partially counter-
acts in viva signals that maintain normal HSCs in a quiescent
state but, when active in BCR-ABL-positive HSCs, promotes
their differentiation in favor of their self-renewal. In more
differentiated CML progenitors, the same mechanism has a
more potent mitogenic effect that is then extinguished when the
cells enter the terminal stages of differentiation. Thus, further
expansion of the leukemic clone is limited until additional
mutations are acquired that will further alter the regulatory
mechanisms still operative in the chronic phase cells. These
observations strongly argue for the necessity of additional so-
matic mutations or epigenetic events in HSCs or myeloid
progenitors for the development of fully malignant diseases.
Committed Progenitors as LSCs. Although HSCs are often the
target of genetic events leading to malignant transformations,
committed progenitors or even differentiated cells may also
become transformed. In APML patient samples, the M3 subtype
of AML, it has been shown that the APML-associated fusion
gene PML/retinoic acid receptor or (RARo~), which results from
the t(15,17) balanced reciprocal translocation, was present in
tCD34-CD38+] cell populations but not in [CD34+CD38-]
HSC-enriched cell populations (76~. This observation suggests
that in APML the transformation process may involve a more
differentiated cell type than HSCs and/or pluripotent progen-
itors that have been implicated in the other AML subtypes
(66, 67~.
Further evidence indicating that cells devoid of self-renewing
activity, such as committed progenitors and mature cells, can
also be the target cells for leukemic events came from analyses
of leukemia-associated genes in the mouse. The development of
transgenic and knockout technologies has led to the creation of
a wide variety of physiological disease models of human leuke-
mias, and the promoter elements of several myeloid-specific
human genes have been used to target transgene expression
specifically to committed myeloid cells. The MRP8 gene encodes
; ..,
1 1 846 1 www.pnas.org/cgi/doi/ 1 0.1 073/pnas.20342011 00
a small calcium-binding protein of the S100 family, which is only
expressed in neutrophils, monocytes, and their immediate pro-
genitors, CMPs and GMPs, but not in HSCs (77~. The use of-the
human MRP8 (hMRP8) promoter has allowed the generation of
several accurate mouse models of human leukemia. Transgenic
mice expressing BCR-ABL from this promoter develop a CML-
like disease (78), and mice similarly expressing the fusion gene
PML/RARa exhibit a preleukemic state, which eventually
progresses to APML, similar to human patients with the PML/
RARor translocation (794. Transgenic mice expressing the fusion
protein AML1-ETO from the MRP8 promoter also develop
AML with high frequency, but only after mutagenesis treatment,
indicating the strong requirement of AML1-ETO for additional
mutations (80~. The promoter regions of other myeloid-specific
genes have also been used to drive transgene expression in the
myeloid lineage, including the CDllb promoter (81>, which is
exclusively expressed in differentiating and mature monocytes
and granulocytes, and the human cathepsin G (hCG) promoter
(82), which is expressed in myeloid progenitors but not in mature
myeloid cells. The failure of PML/RARor to cause leukemia
when expressed from the CDllb promoter during late myeloid
differentiation, and the differences in the phenotypes resulting
from hCG- and hMRP8-directed PML/RARor expression most
likely reflect the difference in the expression profile given by
these two promoters and reveal the importance of the develop-
mental stage of the cells targeted by the transgene promoter for
the leukemogenic effect of the leukemic-associated fusion gene
(83~. A recent study using retroviral transduction of highly
purified HSCs, CMPs, and GMPs also has shown that the potent
leukemic-fusion gene MLL-ENL, which results from the t(11,19)
translocation and has been involved in a large subset of AML in
childhood (84), can induce the exact same AML not only from
totipotent HSCs, but also from restricted CMPs and GMPs
(unpublished results). Altogether, these different models dem-
onstrate that myeloid leukemias can also arise from committed
progenitors, because leukemic fusion proteins such as BCR-
ABL, PML/RARor, and MLL-ENL, which were previously
thought to act only in totipotent HSCs, can be transforming at
the level of myeloid progenitors and are able to give rise to
leukemia without HSC involvement. However, in the case of
spontaneously arising human leukemia, it seems likely that HSCs
accumulate the mutations that are necessary for neoplastic
transformation, but that these genetic mutations exert their
effects in committed progenitors, leading to the generation of
LSCs downstream of the HSCs.
Deconstructing the Molecular Pathways Leading to Leukemia
Transgenic mouse models have been used to deconstruct in vivo
the molecular pathways leading to leukemic transformation,
similar to what has been done in normal human epithelial and
fibroblast cells (854. The development of cancer is a stepwise
process in which increasing numbers of somatic mutations give
rise to an increasingly transformed clonal population of cells
(86~. The multistep model of carcinogenesis was originally
postulated to require a clonal event causing increased prolifer-
ation, which, together with mutations blocking cellular differ-
entiation, synergizes to cause transformation. More recently,
protooncogenes that either suppress or promote programmed
cell death, or apoptosis, have been shown to play critical roles in
oncogenesis, as well as in regulating hematopoiesis (87, 884.
Although genetic rearrangements and leukemic-associated fu-
sion genes have a critical function by interfering with the
hematopoietic differentiation programs and thereby dictating
the nature of the leukemia, they require additional cooperative
mutations to induce fully malignant diseases (see Fig. 34.
Differentiation and Leukemia. Leukemia-associated fusion pro-
teins generally function as aberrantly activated signaling mech-
Passegue et a/.
OCR for page 31
Impaired differentiation
BCR-ABL
AML-associated fusion proteins
(AML1-ETO, PML-RARa, MLL-ENL)
Increased cell survival
Bcl2
Bclx'
Fas-receptor / signaling pathway
Increased proliferation ~ ~ ~ Genomic instability
C-myc
Cyclin D1
BCR-ABL ~ ,
Increased self-renewal
Hox genes
Wnt pathway (~-catenin)
Notch pathway
Shh pathway
Fig. 3. Deregulated pathways leading to leukemia. Although leukemias are
heterogeneous in terms of phenotypes, there are general mechanisms under-
lying leukemic transformation such as increased cell survival, increased pro-
liferation capacity, increased self-renewal capacity, genomic instability, and
prevention of differentiation. Examples of such deregulated mechanisms
and/or signaling pathways that have been found in various types of leukemias
are indicated.
anisms or transcriptional regulators that directly interfere with
the hematopoietic differentiation program (18~. The 210-kDa
BCR-ABL fusion protein differs from the normal 145-kDa
c-ABL in its preferential location in the cytoplasm and its
constitutively elevated tyrosine kinase activity. Structure -
function analyses have shown that both of these properties are
critical for the transforming activity of BCR-ABL (73~. Con-
cerning the vast collection of AML-associated fusion proteins,
one of the two components of each fusion protein is generally a
transcription factor (AML1, CBFI3, or RARE whereas the
other partner is more variable in function, but is often involved
in the control of cell survival and apoptosis such as the nuclear
structure protein PML (89~. Moreover, AML-associated fusion
proteins have been shown to affect hematopoietic differentiation
in a variety of experimental models, and the specific stage of
myeloid maturation arrest appears to direct dependent on the
nature of the fusion protein expressed. The abnormal network of
transcriptional regulation induced by these leukemia-associated
fusion genes seems to occur through common mechanisms,
including recruitment of aberrant corepressor complexes, alter-
ation in chromatin remodeling, and disruption of specific sub-
nuclear compartments (89, 904.
Programmed Cell Death and Leukemia. The Bc1-2 family members,
such as Bc1-2, Bcl-xl, Mcl-1, and A1, function as cell death
antagonists against a wide array of apoptotic stimuli, whereas
their binding partners, like Bax, Bad, and Bak, promote apo-
ptosis (873. Thus, gain-of-function mutations in Bc1-2 family
members or loss-of-function mutations in Bax family members
would be expected to predispose toward cancer. Bc1-2 was in fact
discovered upon characterization of a t(14,18) chromosomal
translocation found in follicular lymphoma. Although the role of
Bc1-2 in leukemogenesis has been extensively confirmed in
lymphoid leukemia, deregulation of Bc1-2 family members also
appears to play an important role for the transformation of
myeloid cells. The fusion protein AML1-ETO was shown to
directly up-regulate expression of Bc1-2 by binding to its pro-
moter elements (914. In fact, cells from most human AML have
been found to express Bc1-2 at much higher levels than their
normal counterparts (92~. Furthermore, activating mutations by
retroviral integration in the murine Bcl-xl gene have been
reported for both myeloid and T cell leukemias (93~. Together,
Passegue et a/.
these results suggest that activating mutations in Bc1-2 gene
family members may be critical events in the multistep myeloid
transformation process. Hence, the increased survival provided
by enforced Bc1-2 family member expression may allow sufficient
time for the acquisition of additional oncogenic mutations, a
mechanism thought to underlie the transition from chronic to
acute leukemia (94~. Whereas the deregulation of Bc1-2 expres-
sion is found in many human cancers, overexpression of Bc1-2 in
a transgenic mouse model has been found to be relatively benign
in terms of cellular transformation (954. Hence, enforced ex-
pression of Bc1-2 in the myeloid lineage with the hMRP8
promoter leads to a disease that is similar to human chronic
myelomonocytic leukemia, including monocytosis, splenomegaly
and neutropenia as the mice age (964. However, despite de-
creased survival compared with littermates, these mice rarely
progress to acute leukemia and they require additional muta-
tions to promote AML, as has been shown for lymphoid leuke-
mias (97~.
Although loss-of-function mutations in the Fas receptor
(CD95) or Fas signaling pathway have historically been associ-
ated with lymphoid hyperplasia and autoimmunity (98, 99),
several clinical reports have recently implicated similar muta-
tions in various human leukemias (100, 101~. Granulocytes, and
their myeloblastic progenitors, are known to express high levels
of Fas (102), and several patients with granulocytic leukemias
have been shown to have blasts with functional deficiencies in the
Fas signaling pathway (103~. Interestingly, intercrosses between
hMRP8BC~-2 and Fas-deficient FasiPr/iPr mice lead to the devel-
opment of AML in 15% of the Fas~Pr/~PrhMRP8BC~~2 mice, with an
expansion of myeloblasts in all hematopoietic tissues and sub-
stantially lower numbers of granulocytes in the bone marrow and
blood (96~. These results indicate that Bc1-2 and the Fas receptor
regulate two distinct apoptosis pathways in the myeloid lineage
and that alteration of both of them seems to be required for
transformation of myeloblasts in both mouse and human. Fur-
thermore, increased survival provided by enforced Bc1-2 expres-
sion greatly increases the incidence of CML-like disease in
hMRP8BCR-AB~hMRP8BC~-2 double transgenic mice (78), as well
as the incidence of APML development in hMRP
hMRP8BC~-2 double transgenic mice (104~. Altogether, these
observations demonstrate that prevention of cell death is one of
the crucial events in myeloid leukemogenesis and may even be
the first step that sets the stage for additional mutations.
Self-Renewal and Leukemia. Leukemic cells absolutely require
self-renewal capability to propagate the disease. Self-renewal is
the default pathway in virtually all single-celled organisms, and
regulation of self-renewal versus differentiation or death is a
strict requirement for organisms that have tissue specialization.
Perhaps the reason not all of the Fas~Pr/~PrhMRP8BC~~2 mice
progress to AML is that the leukemic cells acquire an additional
mutation that causes deregulated self-renewal. Current research
focuses on gain-of-function mutations that promote constitutive
self-renewal, such as stabilization of 'S-catenin. Stabilized ,3-cate-
nin has been shown to promote the self-renewal of stem cells and
other types of progenitor cells (47, 105), and activation of
13-catenin and deregulation of the Wnt signaling pathway is a
common phenomenon in cancer (106~. Mutations in other
signaling pathways that promote progenitor self-renewal, such as
Notch and Shh, are also likely to contribute to unregulated
self-renewal of leukemic cells and should be further studied.
Although much work is being done to identify the transcrip-
tion factors and the signal transduction pathways involved in
HSC cell-fate decisions and self-renewal regulation, an alterna-
tive option is to envisage that self-renewal is a default pathway
that can occur only to the extent that death and differentiation
are prevented. In that view, it is interesting that the main effect
of leukemia-inducing genes is to prevent apoptosis and/or to
PNAS | September 30, 2003 I vol. ,00 I suppl. ~ | 11847
OCR for page 32
~ . ~
block cell differentiation. One simplistic view is therefore to
assume that whether the target cell of the transformation event
is an HSC or a restricted progenitor, the impairment of these
cellular pathways will produce self-renewing LSCs. This question
has recently been addressed in the study performed by Cozzio
and colleagues (unpublished results), which showed that retro-
viral transduction of HSCs, CMPs, or GMPs by MLL-ENL
induced the exact same AML. This result raises several inter-
esting questions: either MLL-ENL interferes at a crucial point
in the regulation of the normal self-renewal program, which is
currently unknown, leading to leukemic expansion of any of the
transduced cells, or MLL-ENL, through oncogenic mechanisms
such as a block in apoptosis and/or differentiation, induces
leukemic self-renewal in any of the transduced cells. Future use
of well-defined highly purified cells as starting populations for
leukemogenic transformation will allow more. precise transcrip-
tional analysis of these early molecular events by RT-PCR or
microarray analysis and provide a deeper understanding of the
molecular basis of deregulated self-renewal in leukemic cells.
Conclusions
Emerging evidence indicates that leukemias are initiated by a
few LSCs that are heterogeneous in terms of their origin. They
can either be HSCs that have become leukemic as the result of
accumulated mutations or more restricted progenitors that have
reacquired the stem cell capability for self-renewal. Identifying
the LSCs for each given leukemia is therefore needed to fully
understand their specific biology. Leukemias are also heteroge-
neous in terms of phenotype, disease progression, prognosis, and
response to therapy, but there are general mechanisms under-
lying leukemic transformation that are starting to be well
understood. Future investigation of such deregulated mecha-
nisms in the newly identified LSCs will lead to a considerable
increase in our understanding of the molecular mechanisms and
signaling pathways that are affected in a given type of leukemia
and are likely to provide key insights into more efficient drug
design and therapy.
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Perspectives
LSC identification and prospective isolation would facilitate the
study of gene expression profiles of malignant progenitors
compared with their normal counterparts by using microarray
technology. The recent development of lentiviral vectors for
rapid and efficient transduction of highly purified populations of
cells, including the nonproliferating HSCs, will allow efficient
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HSCs. Purified LSCs will also provide a powerful substrate for
preclinical testing of the efficacy of newly developed chemo-
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The diagnostic evaluation of leukemia currently depends on
morphologic and flow cytometric determination of the blast
count, which in the light of LSCs being responsible for the
propagation of the leukemia, may represent an inaccurate
assessment of the disease status. If phenotypic markers were
available that facilitated the rapid identification of LSCs within
bone marrow or peripheral blood via flow cytometry, then
clinicians could more adequately assess the disease status and
respond with the most appropriate treatment, including chemo-
therapy, blood or bone marrow transplant, biologic response
modifier therapy, or molecularly targeted therapies. LSC phe-
notypic identification may also facilitate early diagnosis of
disease relapse postchemotherapy or hematopoietic cell trans-
plants and could allow the elimination of LSC contamina-
tion from hematopoietic cell transplants with cell sorting
technologies.
We are grateful to Thomas Serwold for helpful comments and critical
reading of this manuscript. E.P. is a fellow of the Jose Carreras Leukemia
Foundation, L.E.A. is a fellow of the Canadian Institutes of Health
Research, and C.H.M.J. is supported by a Yu-Bechmann fellowship for
Genomics and Oncology at the Center for Clinical Immunology at
Stanford. This work was supported by the National Institutes of Health
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PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 1 11849
Representative terms from entire chapter:
bone marrow
