Download Mixed Lineage Leukemia Translocations and a Leukemia Stem Cell

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Mixed Lineage Leukemia Translocations and a Leukemia
Stem Cell Program
Joerg Faber and Scott A. Armstrong
Division of Hematology/Oncology, Children’s Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute,
and Harvard Medical School, Boston, Massachusetts
Abstract
Cancer stem cells (CSC) may provide the self-renewal capacity
required to sustain a tumor. One possibility is that CSC arise
from the stem cell counterparts in normal tissues. Alternatively, CSC may arise from more differentiated progenitor cells
found in certain tissues. In support of this idea, we showed
recently that mixed lineage leukemia fusion oncoproteins can
convert committed hematopoietic progenitors into leukemias,
which include leukemia stem cells expressing a self-renewal
associated program in the context of a differentiated myeloid
cell. The findings suggest a basis to understand the pathobiology of CSC and possible strategies to attack them to
undermine the self-renewal capacity of a tumor. [Cancer Res
2007;67(18):8425–8]
Stem cell self-renewal is the process by which a stem cell divides
and produces cells that possess identical developmental and
proliferative potential. The loss of self-renewal capabilities during
the transition between hematopoietic stem cells (HSC) and more
differentiated cells is a hallmark of hematopoietic development
that is likely shared by most tissues capable of regeneration. The
tight controls on self-renewal may provide a safeguard against
tumorigenesis as inappropriate self-renewal properties are found in
leukemias and other cancers (1). Cancer cells capable of indefinite,
dysregulated self-renewal and proliferation are often called cancer
stem cells (CSC) or cancer-initiating cells because they can initiate
and fully recapitulate all cellular characteristics of a primary cancer
in recipient organisms, most frequently immunodeficient mice
(2, 3). CSC represent a fraction of the total tumor and possess the
ability to infinitely self-renew and generate more differentiated
cells with limited proliferative capacity, features they share with
normal tissue stem cells (2–6). As a result of these characteristics,
CSC are crucial for tumor maintenance and regeneration, whereas
the majority of the malignant cells may have only limited
proliferative and self-renewal capacity. Cells with CSC properties
were first identified in acute myelogenous leukemia (AML) through
the seminal work of Dick et al. (4, 6). Subsequently, CSC have been
identified in solid tumors (7–12).
Accumulating evidence suggests that the eradication of CSC
such as leukemia stem cells (LSC) will be a prerequisite for the
development of successful targeted therapeutic approaches and
Requests for reprints: Scott A. Armstrong, Children’s Hospital Boston, Karp
Family Research Laboratories, 1 Blackfan Circle, Boston, MA 02115. Phone: 617-9192508; Fax: 617-730-0934; E-mail: [email protected].
I2007 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-0972
www.aacrjournals.org
treatments that fail may do so because CSC escape treatment.
Because normal stem cells and CSC most likely share features
related to their self-renewal capability, identification of mechanisms important for CSC survival that differ from those responsible
for their normal tissue counterparts may pinpoint programs or
pathways that can be targeted with a high therapeutic index. To
better understand the pathobiology of CSC formation, much recent
effort has focused on the question if CSC always arise from normal
tissue stem cells or if more differentiated progenitors can be
induced to express the properties of self-renewal and multipotency
(1). Because the LSCs identified in some human AMLs are
phenotypically related to normal HSC in that they express several
similar cell surface proteins (4), it is likely that some LSC originate
from normal HSC that have undergone oncogenic transformation.
Recent evidence suggests that this scenario is likely for some
oncogenes. Transduction of committed progenitor populations
with the BCR-ABL oncogene, which is found in chronic myelogenous leukemia (CML), did not impart self-renewal properties on
these populations, indicating that initial transformation by the
BCR-ABL oncogene in CML occurs in a multipotent cell such as an
HSC (13).
However, other recent studies in murine transduction/transplantation models have also shown that leukemia can be initiated
by at least some oncogenic fusion proteins found in human
leukemias when expressed in more differentiated hematopoietic
progenitor cells (13, 14). Transduction of committed hematopoietic progenitor populations with either the MLL-ENL or MOZTIF2 oncogenes leads to the reactivation of stem cell properties
in these cells in vitro and generated AML with high penetrance
when transplanted into mice (13, 14). These findings provided the
first evidence that stem cell properties can be imparted on more
differentiated committed hematopoietic progenitors to generate
leukemia.
We showed recently that expression of MLL-Af9, another
oncogenic fusion protein involving the mixed lineage leukemia
(MLL) locus on chromosome 11q23, is capable of initiating
leukemia in prospectively isolated granulocyte macrophage progenitors (GMP; ref. 15). Animals transplanted with MLL-Af9–
transduced GMP developed AML, similar to other MLL fusion
leukemia models (14, 16–18). Isolation of GMP-like leukemic cells
(L-GMP) and transplantation into secondary recipient mice
showed that this population is enriched for LSC as only four cells
were necessary to transfer the disease. In contrast, propagation
of L-GMP in liquid culture resulted in a loss of stem cell properties
as indicated by the induction of a more differentiated immunophenotype and a significantly reduced number of leukemiainitiating cells (<1:5,000). These findings show that cells with a
differentiated myeloid phenotype can possess LSC that both
initiate leukemia and produce more differentiated progeny
incapable of transferring the disease. To further characterize
changes that occur during transition from GMP to GMP-derived
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Figure 1. Model for development of LSC from granulocyte-macrophage progenitors (GMP ) through introduction of an Mll-Af9 fusion protein. Within 48 h of Mll-Af9
expression, an ‘‘immediate gene expression signature’’ is induced, which represents a subset of the complete ‘‘self-renewal associated signature’’ present in fully
developed L-GMP/LSC. Despite the acquisition of stem cell properties, the LSC retain a phenotype and global expression identity that is most similar to more
differentiated myeloid cells. Development of therapeutic strategies with activity against LSC through specific targeting of this ectopically expressed gene expression
program is an approach that warrants further exploration.
LSC and to address the question if global reprogramming (or
dedifferentiation) is required to generate a LSC from a committed
progenitor, the gene expression profiles of normal HSC, common
myeloid progenitors (CMP), normal GMP, and L-GMP were
assessed. This showed that L-GMP have a unique gene expression
program that is different from any of the HSC or progenitor
populations but globally more similar to the GMP from which they
arose than any other progenitor population. Further analysis
revealed a group of 363 genes that are highly expressed in normal
HSC, show decreased expression in committed progenitors, but are
reactivated in L-GMP. High-level expression of this gene set is
hence confined to populations with self-renewal potential (HSC
and L-GMP) and was termed a ‘‘self-renewal associated signature’’
(Fig. 1). Because the identified ‘‘self-renewal associated signature’’
represented only a subset of the 1,137 genes highly expressed in
normal HSC compared with committed progenitors, it seems that
the activation of only a small fraction of a stem cell program is
sufficient for the acquisition of leukemic self-renewal properties in
L-GMP. Conversely, a portion of the self-renewal associated
program was lost when LSC were forced to differentiate in liquid
culture and lost their self-renewal capabilities. Another recent
Cancer Res 2007; 67: (18). September 15, 2007
study analyzed the generation of LSC after introduction of MLL-Af9
into a population of cells that contained both stem and progenitor
cells and showed that LSC were most similar to differentiated
myeloid lineage cells, which have acquired partial properties of
normal HSC (19). Taken together, these studies show that transformation of myeloid progenitors activates ectopic expression of a
limited number of stem cell genes while still maintaining the global
identity of differentiated myeloid cells, thus generating an
abnormal hybrid cell that has characteristics of both stem cells
and more differentiated cells (Fig. 1). Therefore, transformed
progenitors do not fully dedifferentiate back to stem cells but
rather acquire circumscribed stem cell–like features enabling them
to self-renew and extensively proliferate.
The activation of a ‘‘self-renewal associated signature’’ in
committed myeloid progenitors prompts the question about the
nature of development of such a program. Is it immediately fully
activated by the translocation product or does it accumulate over
time suggesting a hierarchical organization and multistep development? Time course gene expression analysis after Mll-Af9
transduction showed that only a small subset of the signature
genes including multiple Homeobox A cluster genes is immediately
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A Stem Cell Program in MLL-Rearranged Leukemia
up-regulated after introduction of the MLL-Af 9 fusion (an
‘‘immediate signature’’), whereas the full signature evolves over
time probably as a summation of activation of various different
pathways or accumulation of subsequent genetic/epigenetic events
(Fig. 1). This supports a view that the architecture of the MLL
fusion-induced transcriptional program is hierarchical in nature
where certain HOX genes might play an important role as direct
proximate MLL fusion targets and important regulators of MLL
fusion-induced leukemogenesis.
Having identified a self-renewal associated signature after
introduction of Mll-Af9 in the murine model system, a key
question is whether the acquisition of stem cell properties in
committed progenitors and the accompanying expression of a selfrenewal associated signature is relevant and reproducible in
human disease. First evidence for the acquisition of stem cell–
like properties in human committed progenitors was provided by a
recent report assessing CML blast crisis where cells with a
phenotype similar to GMP were identified as LSC candidates and
transcriptional targets of h-catenin as potential mediators of selfrenewal (5). In our study, we showed significant overlap between
the murine self-renewal associated signature and a previously
defined human MLL-rearranged AML signature (20). This further
supports the relevance of the identified signature and suggests that
the mechanisms underlying leukemia formation seem to at least
partially overlap in murine leukemia model systems and primary
human disease. The finding that LSC do not necessarily have a
similar phenotype/gene expression program compared with
normal HSC but rather activate a limited self-renewal program in
the context of a committed myeloid cell has important implications for understanding how CSC develop and may aid in the
successful design of new therapeutic approaches.
Further evidence that somatic cells can be reprogrammed to a
pluripotent state through a few genetic manipulations comes from
a series of elegant studies, which have shown that fibroblasts
engineered to express only four transgenes (Klf4, Sox2, Oct4, and
c-Myc) acquire properties that are comparable with pluripotent
embryonic stem (ES) cells. These ‘‘inducible pluripotent stem’’ cells
resemble ES cells in morphology, proliferation, and teratocarcinoma formation including differentiated cell types of all three germ
layers and chimaera formation in adult mice when injected into
wild-type mouse blastocysts. Gene expression analysis revealed
activation of a transcriptional program that is similar but not
identical to multipotent ES cells (21–24). These important studies
provide further evidence that a limited number of genetic
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Mixed Lineage Leukemia Translocations and a Leukemia
Stem Cell Program
Joerg Faber and Scott A. Armstrong
Cancer Res 2007;67:8425-8428.
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