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Preferential induction of apoptosis for primary human
leukemic stem cells
Monica L. Guzman, Carol F. Swiderski, Dianna S. Howard, Barry A. Grimes, Randall M. Rossi, Stephen J. Szilvassy,
and Craig T. Jordan*
Blood and Marrow Transplant Program, Markey Cancer Center, Division of Hematology兾Oncology, University of Kentucky Medical Center,
Lexington, KY 40536
Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved October 22, 2002 (received for review August 2, 2002)
Acute myelogenous leukemia (AML) is typically a disease of stem兾
progenitor cell origin. Interestingly, the leukemic stem cell (LSC)
shares many characteristics with normal hematopoietic stem cells
(HSCs) including the ability to self-renew and a predominantly G
0
cell-cycle status. Thus, although conventional chemotherapy reg-
imens often ablate actively cycling leukemic blast cells, the prim-
itive LSC population is likely to be drug-resistant. Moreover, given
the quiescent nature of LSCs, current drugs may not effectively
distinguish between malignant stem cells and normal HSCs. None-
theless, based on recent studies of LSC molecular biology, we
hypothesized that certain unique properties of leukemic cells could
be exploited to induce apoptosis in the LSC population while
sparing normal stem cells. In this report we describe a strategy
using treatment of primary AML cells with the proteasome inhib-
itor carbobenzoxyl-
L-leucyl-L-leucyl-L-leucinal (MG-132) and the an-
thracycline idarubicin. Comparison of normal and leukemic speci-
mens using in vitro culture and in vivo xenotransplantation assays
shows that the combination of these two agents induces rapid
and extensive apoptosis of the LSC population while leaving
normal HSCs viable. Molecular genetic studies using a dominant-
negative allele of inhibitor of nuclear factor
B(I
B
␣
) demonstrate
that inhibition of nuclear factor
B (NF-
B) contributes to apoptosis
induction. In addition, gene-expression analyses suggest that ac-
tivation of p53-regulated genes are also involved in LSC apoptosis.
Collectively, these findings demonstrate that malignant stem cells
can be preferentially targeted for ablation. Further, the data begin
to elucidate the molecular mechanisms that underlie LSC-specific
apoptosis and suggest new directions for AML therapy.
A
cute myelogenous leukemia (AML) is a serious and often
lethal form of hematologic cancer. Although the develop-
ment of better chemotherapy regimens has improved remission
induction and overall survival, relapse remains a common prob-
lem, especially among older patients and兾or patients with poor
prognosis cytogenetics (1). In recent years the clinical charac-
teristics of AML have become better understood in light of
studies elucidating the biological origins of the disease. Several
lines of evidence clearly indicate that AML is a disease of stem
or progenitor cell origin, and that the leukemic stem cell (LSC)
stands apart from more mature leukemic cells with its own set
of unique biological properties (2–9). Thus, although chemo-
therapeutic agents effectively ablate leukemic blast cells in a
majority of patients, the efficacy of LSC targeting is not known.
Indeed, it is attractive to speculate that failure to sustain durable
remission may be due to a drug-refractory兾resistant malignant
stem cell population. Given the potentially critical role of stem
cells in both the genesis and perpetuation of AML, recent studies
have attempted to better characterize LSC properties (10–12).
Notably, although the immunophenotype of LSCs is similar to
normal hematopoietic stem cells (HSCs; CD34
⫹
, CD38
⫺
, HLA-
DR
⫺
), there are at least three antigens with expression that is
known to vary in malignant cells: CD90, CD117, and CD123
(7–9). Thus, it has been possible to prospectively identify and
isolate enriched LSC populations, which has allowed subsequent
studies to analyze the cell-cycle status and gene-expression
characteristics of normal vs. malignant stem cells (13–15). A
somewhat counterintuitive finding from cell-cycle studies is that
the LSC population seems mostly quiescent, which seems to be
true despite the often aggressive characteristics of leukemic
disease. Thus, the LSC should not be preferentially susceptible
to cycle-active chemotherapeutic agents.
Because standard chemotherapy approaches may not effec-
tively target the LSC population, we have attempted to charac-
terize molecular properties of the LSC that could be useful for
apoptosis induction. Data from our laboratory has shown re-
cently that nuclear factor
B (NF-
B) is constitutively activated
in the majority of primary AML specimens (15). Although
activation of NF-
B is a relatively common feature of many
cancers (16–18), a surprising aspect of our studies was the finding
that NF-
B is active also in quiescent LSC populations. Thus,
strategies to inhibit NF-
B, and thereby block growth and
survival pathways regulated by NF-
B, may represent a useful
approach to more durable AML therapy. With this concept in
mind, we examined several agents that inhibit NF-
B. One such
class of drugs that is being widely explored for cancer therapy is
proteasome inhibitors (19, 20). Proteasome inhibition blocks
degradation of the NF-
B regulator I
B
␣
and results in loss of
NF-
B activity. Initial studies of primary AML cells have
demonstrated that treatment with the proteasome inhibitor
carbobenzoxyl-
L-leucyl-L-leucyl-L-leucinal (MG-132) causes
rapid inhibition of NF-
B and strongly induces apoptosis (15).
Thus, proteasome inhibition seems to be a promising strategy for
ablation of leukemic cells.
Another well documented mechanism of apoptosis induction
is mediated by activation of specific p53-regulated genes such as
Bax, GADD45, and p21
WAF/CIP
(21–23). This mechanism is
typically activated in response to DNA-damaging agents includ-
ing the anthracycline family of chemotherapy drugs (24, 25),
which are widely used for AML-induction therapy. Interestingly,
anthracyclines have also been shown to induce NF-
B activity
(26, 27), presumably as a cellular survival mechanism to resist
toxic effects of the drug. Therefore, we reasoned that anthra-
cycline treatment would increase cellular dependency on NF-
B
activity and thus further sensitize cells to the loss of NF-
B
activity induced by proteasome inhibitors. Consequently, in the
present study an analysis of proteasome inhibitors in combina-
tion with anthracyclines has been performed. The results of these
studies demonstrate that LSCs are extremely sensitive to a
combination of proteasome inhibition and anthracycline treat-
ment and undergo rapid and extensive apoptosis when briefly
cultured with these two agents. Importantly, normal HSCs show
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: AML, acute myelogenous leukemia; LSC, leukemic stem cell; HSC, hemato-
poietic stem cell; NF-
B, nuclear factor
B; I
B
␣
, inhibitor of NF-
B; CB, umbilical cord blood;
MG-132, carbobenzoxyl-
L-leucyl-L-leucyl-L-leucinal; IDR, idarubicin; NOD, nonobese dia-
betic; SCID, severe combined immunodeficient; EMSA, electrophoretic mobility-shift assay;
I
B-SR, I
B superrepressor.
*To whom correspondence should be addressed at: Markey Cancer Center, 800 Rose Street,
Room CC412, Lexington, KY 40536-0093. E-mail: cjordan@uky.edu.
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little to no effect when treated with the same agents. Thus,
apoptosis can be induced in noncycling malignant stem cells
while sparing the normal HSC population.
Materials and Methods
Cell Isolation and Culture. AML cells, normal bone marrow, and
umbilical cord blood (CB) cells were obtained with informed
consent and processed as described (9, 15). In some cases,
marrow and CB cells were also obtained from the National
Disease Research Interchange (NDRI). Samples were subjected
to density-gradient separation to isolate mononuclear cells fol-
lowed by cryopreservation. As needed, samples were thawed and
used immediately. For in vitro studies, cells were cultured in
serum-free medium (28) for 1 h before the addition of MG-132
(Calbiochem) and兾or idarubicin (IDR, Pharmacia–Upjohn).
When IDR and MG-132 where used in combination, cells were
incubated with IDR for 15 min prior the addition of MG-132.
Nonobese Diabetic (NOD)兾Severe Combined-Immunodeficient (SCID)
Mouse Assays.
NOD兾SCID (NOD.CB17-Prdkdc Scid兾J) mice
(The Jackson Laboratory) were exposed to 300 rad of
␥
irradi-
ation from a
137
Cs source 1 day before transplantation. Cells to
be assayed were resuspended in 0.2 ml of PBS (GIBCO) with 2%
albumin and injected via the tail vein (5–10 million cells per
recipient). After 6–8 weeks, animals were killed, and bone
marrow was analyzed for the presence of human cells by using
flow cytometry.
Flow Cytometry. Cell-cycle and apoptosis analyses were per-
formed as described (15, 29). To analyze NOD兾SCID mice,
marrow cells were blocked with the anti-Fc receptor antibody
2.4G2 and 25% human serum followed by labeling with human-
specific CD34-FITC and CD45-PE monoclonal antibodies
(PharMingen). Control samples consisted of marrow cells from
nontransplanted mice. To distinguish HLA-A2
⫹
and HLA-A2
⫺
populations, cells were labeled by using FITC-conjugated mono-
clonal MA2.1 (kindly provided by John Yannelli, Division of
Hematology兾Oncology, University of Kentucky Medical
Center). Adenovirus-infected AML cells were labeled with
CD34-PE (PharMingen), and CD34
⫹
兾GFP
⫹
cells were isolated
by fluorescence-activated cell sorting (⬎94% purity).
Adenovirus Vector Construction and Infection. Adenovirus vectors
used in this study were based on the AdBM5GFP vector from
Quantum Biotechnologies (Montreal). Details of the vector
construction will be described elsewhere. Briefly, the vector was
digested with AflII ⫹ BglII to remove the major late promoter,
which then was replaced with a cytomegalovirus promoter. A
mutant allele of I
B
␣
(serine to alanine at positions 32 and 36)
was then cloned immediately downstream of the cytomegalo-
virus promoter by using the BglII site. Virus was generated by
homologous recombination in 293 cells and purified by using
standard procedures (30). Infection of primary AML cells was
performed exactly as described by Howard et al. (31).
Electrophoretic Mobility-Shift Assay (EMSA), Immunoblot, and RNA
Analysis. EMSA analysis was performed as described (15). For
immunoblots, cells were harvested and analyzed as described by
Jordan et al. (9). Blots were probed with anti-p53 (clone DO-1)
from Santa Cruz Biotechnology and antiactin from Sigma (clone
AC-15). RNA was prepared by using Trizol (Invitrogen) per
manufacturer instructions. Five micrograms of RNA per sample
were analyzed by using the RiboQuant kit and the h-stress-1
multiprobe set (PharMingen) for RNase protection assays. Gels
were scanned by PhosphorImager (Molecular Dynamics), and
bands were quantitated by using Kodak
1D software.
Results
Proteasome Inhibition Preferentially Ablates Primitive AML Cells
in
Vitro
. To assess the effects of proteasome inhibition on primitive
quiescent cells, studies were performed initially by using multi-
parameter flow cytometry. In Fig. 1A, cell-cycle analysis shows
that primitive cells of both normal and leukemic origin (defined
by the immunophenotype CD34
⫹
兾CD38
⫺
兾CD123
⫺
and
CD34
⫹
兾CD38
⫺
兾CD123
⫹
, respectively) are predominantly in G
0
.
This is evident based on the lack of labeling with nuclear antigen
Ki-67, which is a common marker for entry into G
1
(32). Based
on our studies (15), tests were performed first by using the
proteasome inhibitor MG-132 at 1.0
M for 12 h. This treatment
readily induced apoptosis for AML cells, whereas normal
CD34
⫹
cells were almost entirely unaffected (as shown by
annexin-V labeling, Fig. 1B). Analysis of the 13 AML specimens
described in Table 1 showed an average viability of 26 ⫾ 18%
after 12 h of culture in MG-132. In contrast, parallel studies of
four normal CD34
⫹
cell specimens showed an average viability
of 89 ⫾ 5% when analyzed by using the same conditions. All
cultures were performed in serum-free medium and in the
absence of any exogenous cytokines.
Fig. 1. (A) Cell-cycle profiles for normal and leukemic CD34
⫹
兾CD38
⫺
speci-
mens. BM, bone marrow. (B) Flow-cytometric profile of normal (Left) and
leukemic (Right) CD34
⫹
cells stained with annexin-V FITC and 7AAD after 12 h
of treatment with 1
M MG-132. Numbers indicate the percentage of viable
cells. DAPI, 4⬘,6-diamidino-2-phenylindole.
Table 1. AML specimens
Case FAB type Cytogenetics
1 M4 46,XY,del(7)
2 M2 46,XY
3 M5 46,XX
4 M4 48,XY,⫹8,⫹13
5M1兾M2 46,XY
6 M4 46,XX
7 M5 45,X,⫺Y
8 M5 46,XX
9 MDS兾AML 46,XY
10 M1 46,XX
11 M4 47,XY,⫹9
12 M2 46,XY,t(9;19);t(8;21)
13 M4 46,XY,del(7)
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Because MG-132 demonstrated toxicity to phenotypically
primitive AML cells in vitro, we sought to further investigate
toxicity to the LSC compartment when proteasome inhibition
was combined with the commonly used chemotherapeutic an-
thracycline IDR. Studies have indicated that anthracyclines
induce NF-
B in malignant cells (27). Thus, we hypothesized that
anthracycline treatment might sensitize leukemic cells to the
effects of proteasome inhibition (i.e., loss of NF-
B activity).
Moreover, anthracyclines have been shown to increase protea-
some activity in leukemic cells, which also could increase sen-
sitivity to proteasome inhibition. To test this theory, both
MG-132 and the anthracycline IDR were first titrated to rela-
tively nontoxic doses of 0.25
M and 15 ng兾ml, respectively (data
not shown). Each specimen listed in Table 1 then was cultured
for 12 h in low-dose MG-132 (0.25
M) and兾or IDR (15 ng兾ml).
MG-132 treatment alone was only mildly toxic to the cells (mean
viability ⫽ 73 ⫾ 9%). Similarly, treatment with IDR alone
also showed only a small effect on cell viability (mean viability ⫽
88 ⫾ 9%). However, the combination of both drugs synergized
strongly to induce robust apoptosis (mean viability ⫽ 11 ⫾ 5%).
Fig. 2A shows a representative example of an AML specimen
(Upper) vs. a normal CB specimen (Lower) treated with the
low-dose MG-132兾IDR combination. As observed for MG-132
alone, the AML specimen was extremely sensitive to treatment
with MG-132兾IDR, whereas the normal specimen showed only
a slight loss in viability.
To examine effects on the most primitive cells more specifi-
cally, four AML and normal specimens were assayed for apo-
ptosis induction in the CD34
⫹
兾CD38
⫺
population. As shown in
Fig. 2B, the primitive AML compartment was also affected
dramatically by the combination of MG-132兾IDR, whereas the
normal CD34
⫹
cells were almost entirely resistant. These data
demonstrate that MG-132 in combination with IDR induces
apoptosis specifically in primitive AML cells, whereas normal
primitive cells seem largely unaffected.
Treatment with MG-132 and IDR Destroys LSCs Able to Engraft
NOD兾SCID Mice. To determine whether stem cells defined by
in vivo functional assays were impaired by treatment with
MG-132兾IDR, normal and leukemic specimens were analyzed
by using transplantation into immune-deficient NOD兾SCID
mice. NOD兾SCID xenogeneic models have been used to assess
both human HSC and LSC activity (33–35). This model can be
used therefore to identify therapies that affect either the LSC or
HSC population. Initially, cells from primary CB and AML
samples were treated with 0.25
M MG-132 and 15 ng兾ml IDR
for 12 h and then injected into NOD兾SCID mice. After 6–8
weeks, marrow was analyzed for the presence of donor cells by
using a human-specific antibody for CD45. Each triangle in Fig.
3A Left represents the percent engraftment for one animal
transplanted with untreated or MG-132兾IDR-treated CB cells
(15 animals in each group using CB from six independent
specimens). The data show that drug treatment does not impair
the capacity of normal HSCs to proliferate in NOD兾SCID mice.
Further, analysis of human cells in animals receiving treated vs.
untreated CB cells showed no difference with respect to levels of
CD33 (myeloid), CD19 (B lymphoid), and CD34 (primitive)
cells, thereby indicating that the differentiation potential of
MG-132兾IDR-treated CB cells was not impaired (data not
Fig. 2. (A) Flow-cytometric analyses of primary AML cells (Upper) compared
with normal CB cells (Lower) after 18 h of treatment with or without 0.25
M
MG-132 ⫹ 15 ng兾ml IDR. Numbers indicate the percentage of viable cells.
(B) Percentage of viable CD34
⫹
兾CD38
⫺
cells from primary AML (Left) and
normal CB (Right) cell samples after an 18-h treatment with 0.25
M MG-132
⫹ 15 ng兾ml IDR. Viability levels for each sample are normalized to untreated
control specimens.
Fig. 3. (A) Engraftment of NOD兾SCID mice with CB cells after 12 or 18 h of
culture ⫾ 0.25
M MG-132 and 15 ng兾ml IDR (six independent CB specimens
were analyzed at each time point). (B) Engraftment of AML cells after 12 or
18 h of culture ⫾ MG-132 and IDR (four independent AML samples were
analyzed after 12 h, and the two showing the highest levels of engraftment
were reanalyzed after 18 h of culture). Each triangle or circle represents a
single animal analyzed 6 – 8 weeks posttransplant for the percentage of
human cells in marrow. The median level of engraftment is indicated by the
horizontal bars.
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shown). In some cases, CB samples lost their ability to engraft
after 12 h of culture without drugs. Notably, when the same CB
samples were treated with the MG-132兾IDR combination, they
often retained engraftment ability. Hence, the median engraft-
ment efficiency actually increased for drug-treated specimens
(median indicated by horizontal bars). A possible explanation for
this surprising result might be that the proteasome inhibitor
prevents degradation of proteins involved in homing. Alterna-
tively, the drug treatment may enhance survival or inhibit
terminal differentiation of normal stem兾progenitor cells. Irre-
spective of the reason for this observation, the data indicate that
engraftment of normal cells is not impaired by MG-132兾IDR
treatment and supports the in vitro data suggesting the drugs are
not toxic to normal stem cells. In contrast, when AML samples
were treated in the same fashion and transplanted into NOD兾
SCID animals, engraftment efficiency was reduced dramatically.
Fig. 3B Left shows percent donor engraftment after 12 h of
culture ⫾ drug treatment (25 animals in each group using AML
cells from four independent specimens). Two AML specimens
showed no detectable engraftment, and two specimens displayed
only low-level engraftment (3.9–7.5%) after they were exposed
to MG-132兾IDR. In an attempt to eradicate residual AML cells
more completely, we extended the drug exposure to 18 h for the
two specimens that showed engraftment at 12 h (Fig. 3B Right).
Longer exposure resulted in a further decrease in engraftment
but did not eliminate AML cells completely (0.1–2.8% engraft-
ment, seven animals in each group). Finally, to determine
whether the few remaining AML cells retained self-renewal
potential, marrow from primary animals was transplanted into
secondary NOD兾SCID recipients (in the absence of any further
drug treatment). At 6 weeks posttransplant, no detectable
human cells were found in the marrow of secondary recipients,
thereby suggesting that the original drug treatment had ablated
all NOD兾SCID engrafting activity completely. Control experi-
ments using normal CB samples were also performed by using
the 18-h MG-132兾IDR regimen (Fig. 3A Right). Although
overall engraftment of the CB samples was affected by 18 h of
culture, treatment with MG-132兾IDR did not reduce engraft-
ment efficiency further when compared with untreated controls
(15 animals per group).
Because treatment with MG-132兾IDR strongly induced apopto-
sis in the total AML cell population, it is possible that LSCs that
survive drug treatment might fail to engraft because of the presence
of excess dead cells. To address this possibility, an experiment was
performed in which CB and AML cells were treated with the
combination of MG-132兾IDR and coinjected into NOD兾SCID
animals (n ⫽ 6). If apoptotic cells induce an inhibitory effect on the
engraftment of viable stem cells, one would predict reduced en-
graftment when the MG-132兾IDR-treated cells are injected to-
gether. The specimens used for this experiment expressed disparate
HLA-A2 loci such that normal vs. AML cells could be distinguished
by flow cytometry. After 6–8 weeks, the animals were killed, and
the bone marrow was analyzed for engraftment of human cells. Fig.
4 shows a representative example of bone marrow cells stained with
human-specific CD45 and HLA-A2 antibodies. The total percent-
age of CD45
⫹
cells in both treated and untreated samples was very
similar (73–79%). However, labeling with the HLA-A2 antibody
shows strong AML cell engraftment in the untreated controls (74%
of the CD45
⫹
population) but virtually no leukemic engraftment in
MG-132兾IDR-treated samples (0.5% of the CD45
⫹
population).
These data indicate that the inability of treated AML samples to
engraft in the animals is not due to an inhibitory effect caused by
apoptotic cells present in the transplanted sample. Taken together,
the data in Figs. 2–4 strongly suggest that the combination of
MG-132 and IDR effectively eradicates human LSCs while sparing
the normal HSC population.
Inhibition of NF-
B Contributes to Apoptosis Induced by MG-132兾IDR
Treatment.
To investigate the mechanisms that underlie LSC-
specific apoptosis, a series of molecular studies were performed.
Previously we showed that NF-
B is constitutively activated in
primary LSC populations but is not detected in normal CD34
⫹
cells (15). Further, these studies showed that treatment with 1.0
M MG-132 was sufficient to inhibit NF-
B activity. Thus,
EMSA analysis was used in the present studies to determine the
activity of NF-
B in AML cells treated with 0.25
M MG-132
and 15 ng兾ml IDR. To assess molecular changes occurring at
early times posttreatment (i.e., before the onset of overt apo-
ptosis), cells were isolated after6hofdrugexposure. As shown
in Fig. 5A, treatment with MG-132 ⫹ IDR induced strong
inhibition of NF-
B in comparison to untreated controls in five
independent AML specimens. This observation suggests that
NF-
B might mediate survival in primary AML cells. To address
this possibility directly, an adenovirus vector was constructed
that encodes both a mutant version of I
B
␣
and the GFP gene.
The I
B
␣
gene used for this vector encodes an allele known as
the I
B superrepressor (I
B-SR). This gene is mutated at serines
32 and 36 such that it cannot be phosphorylated and hence is not
degraded by normal proteasomal mechanisms. Thus, I
B-SR
expression exerts potent inhibition of NF-
B activity as a result
of stabilized I
B
␣
protein levels. Three primary AML specimens
were transduced with the Ad-I
B-SR vector (Ad-I
B) or a
control virus encoding GFP alone (Ad-GFP) as described (31).
Infected populations then were monitored over time to assess
viability of CD34
⫹
blast cells. The data in Fig. 5C show that
although Ad-GFP-infected cells maintained 100% viability over
a 36-h period relative to uninfected controls, cells infected with
Ad-I
B-SR decreased to ⬇50% of control values (52 ⫾ 4%
viable cells). EMSA analysis demonstrated that expression of
I
B-SR by the vector completely inhibited NF-
B activity in
transduced AML cells (Fig. 5D). These data indicate that NF-
B
activity is important for survival of primary AML cells, but that
inhibition of NF-
B alone is not sufficient to mediate the very
rapid and extensive apoptosis observed after treatment with
MG-132兾IDR.
Activation of a p53-Regulated Mechanism During Apoptosis Induced
by MG-132兾IDR.
To further characterize molecular mechanisms
related to apoptosis induction, pathways commonly associated
with cell death were examined. Recent studies by Tergaonkar et
al. (36) have shown that treatment with the anthracycline
doxorubicin induces a p53-mediated apoptosis response. More-
over, this report showed that loss of NF-
B activity resulted in
Fig. 4. AML and CB cells were cultured for 12 h ⫾ 0.25
M MG-132 and 15
ng兾ml IDR and injected into sublethally irradiated NOD兾SCID mice. Bone
marrow was analyzed 6 weeks later with anti-human CD45-PE and HLA-A2-
FITC to detect engraftment of human cells. The histograms indicate the
percent of human AML vs. CB cells.
Guzman et al. PNAS
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a stronger induction of apoptosis after activation of p53. Thus,
we reasoned that the IDR used in our studies might elicit a
similar response. To test this hypothesis, total protein and RNA
were prepared from cells cultured for6hintheabsence or
presence of 0.25
M MG-132 and 15 ng兾ml IDR. Immunoblots
(Fig. 5B) showed that levels of p53 protein were increased
strongly by drug treatment in six independent AML specimens.
This observation suggests that p53-mediated changes in gene
expression might be involved in apoptosis induced by MG-132兾
IDR treatment. To examine this question in more detail, RNA
was isolated from five primary AML specimens after6hof
culture and expression of the p53-regulated genes Bax, GADD45,
and p21
WAF/CIP
were analyzed by RNase protection assays. These
three genes are strongly associated with p53-induced apoptosis
in a variety of different cell types (21–23). As shown in Fig. 5E,
RNA levels of Bax, GADD45, and p21
WAF/CIP
all are increased
by treatment with MG-132兾IDR. Interestingly, the antiapoptotic
protein Bcl-xL was reduced markedly in cells treated with
MG-132 ⫹ IDR. Bcl-xL is an NF-
B-regulated gene; thus the
observed decrease in Bcl-xL mRNA is consistent with the block
to NF-
B activity seen in Fig. 5A. Collectively, these data suggest
that the combination of MG-132 and IDR induces apoptosis
through activation of p53-regulated genes and simultaneous
inhibition of NF-
B.
Discussion
Recent studies clearly indicate a central role for LSCs in the
pathogenesis of AML and emphasize the need to develop
treatment strategies that specifically target this rare population
(12). Equally important, it is necessary to identify approaches
that minimize toxicity to the normal HSC, which is required to
sustain all blood-cell lineages. Consequently, we sought to
investigate drug-treatment regimens that would target the LSC
population specifically and preferentially. To this end, we have
demonstrated that low concentrations of MG-132 (0.25
M) in
combination with IDR (15 ng兾ml) are sufficient to induce a
strong apoptotic response in primary AML specimens during a
short in vitro culture period (⬇12 h). More importantly, by using
flow-cytometric analysis of phenotypically primitive cells and
functional assays in NOD兾SCID mice, the data indicate that
MG-132 ⫹ IDR also effectively ablates the LSC population. In
contrast, normal HSCs are almost entirely refractory to the same
treatment. These findings formally demonstrate that with the
appropriate stimulus quiescent LSCs are preferentially suscep-
tible to apoptosis induction. Thus, it should be possible to
develop treatment regimens that specifically target the LSC
population. Moreover, recent clinical evaluation of pharmaceu-
tical-grade proteasome inhibitors (37) may soon provide an
opportunity to examine the therapeutic potential of such drugs
in combination with anthracyclines.
Going forward, it will be important to develop a detailed
profile of the molecular events that mediate LSC-specific apo-
ptosis. Studies in this report investigate this phenomenon by
analyzing unique molecular characteristics of the LSC popula-
tion. Previously it was shown that primitive AML cells exhibit
substantial NF-
B activity (15). Given the known role of NF-
B
in the growth and survival of many tumor types (16), we
speculated that inhibition of this factor might be a key step
toward inducing LSC apoptosis. Interestingly, although our
EMSA data show that NF-
B is inhibited potently by MG-132兾
IDR treatment, subsequent studies using a dominant-negative
allele of I
B
␣
showed that down-regulation of NF-
B activity
alone was not sufficient to mediate the same degree of rapid and
extensive apoptosis in AML cells as was observed for MG-132兾
IDR. This finding indicates that other effects of the drug
treatment must be contributing to AML cell death. In seeking to
characterize LSC apoptosis further, we hypothesized that pro-
teasome inhibition might be stabilizing proteins relevant to
apoptosis induction such as p53 (38–40). Moreover, the activity
of IDR was expected to activate p53-mediated apoptosis mech-
anisms. Immunoblot studies confirmed that p53 was increased in
drug-treated cells. Further investigation by RNase protection
assay showed that several p53-regulated genes known to be
mediators of apoptosis were up-regulated in the MG-132兾IDR-
treated cells. These data indicate that activation of a p53-
dependent mechanism is likely to be one component of the
overall apoptosis process. Notably, p53 is only mutated in ⬇9%
of AML specimens (41); thus a strategy that relies on functional
p53 for ablation of leukemic cells should be applicable to a
majority of patients. Future studies may directly address the role
of p53 by expressing a dominant-negative allele of the gene in
Fig. 5. (A) NF-
B EMSA of nuclear extracts from five primary AML specimens
after6hofculture (UNT., untreated; MG兾IDR, 0.25
M MG-132 ⫹ 15 ng兾ml
IDR). Numbers above each lane indicate the specimen number. (B) Immuno-
blot analysis of p53 for each of the specimens shown in A. Each blot was
stripped and reprobed with actin (lower panel of each p53 blot). (C) Three
primary AML specimens were infected with Ad-GFP or Ad-I
B-SR and cultured
for 36 h. The graph indicates the average percentage of viable GFP
⫹
cells
where Ad-GFP is defined as 100%. (D) EMSA analysis of NF-
B in sorted GFP
⫹
or GFP
⫺
cells at 12 h postinfection with Ad-GFP or Ad-I
B-SR (lane 1, NF-
B
consensus probe; lane 2, probe ⫹ 100-fold excess unlabeled consensus probe).
(E) Relative change in expression level as determined by RNase protection
assay for five primary AML specimens after6hofculture in 0.25
M MG-132
⫹ 15 ng兾ml IDR. Untreated specimens were assigned an arbitrary level of 1.0
(shown by the horizontal bar). Analysis of each specimen was performed in
triplicate (standard deviation is shown by error bars). Loading was normalized
by using L32 ribosomal and glyceraldehyde-3-phosphate dehydrogenase
probes as controls.
16224
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.252462599 Guzman et al.
AML cells and determining whether loss of p53 activity is
sufficient to block apoptosis induced by MG-132兾IDR.
Another important issue in understanding the biology of LSCs
is determining how NF-
B is activated. Our studies indicate that
NF-
B plays an important role in the survival of LSCs and
represents a potentially useful target for therapeutic interven-
tion. One mechanism for NF-
B activation may be related to
mutation of the Flt3 gene. Constitutive activation of Flt3 has
emerged recently as the most commonly known aberration in
AML (42). Studies have shown that signaling via Flt3 can
stimulate Ras (43), which in turn is a known activator of NF-
B
(16). Thus, pathways leading from Flt3 to NF-
B seem to exist.
Moreover, several recent studies have begun to examine the use
of Flt3 inhibitors for AML therapy (44). Consequently, direct
analysis of the degree to which Flt3- and NF-
B-regulated
pathways overlap are now feasible. Interestingly, the kinetics and
degree of primary AML cell death reported for Flt3 inhibitors
(45, 46) appear very similar to data in this report using expres-
sion of I
B-SR (i.e., 40–50% cell death over 36–72hof
treatment). This similarity may indicate a substantial degree of
overlap between strategies that target Flt3 and NF-
B and
suggests that Flt3 inhibitors and proteasome inhibitors may be
functionally equivalent in the context of leukemic cell biology. If
this concept is true, then based on findings in this study, we
suggest that combining Flt3 inhibition with IDR treatment may
yield a more rapid and robust induction of apoptosis, as was
observed for MG-132 ⫹ IDR. This approach may be attractive
for clinical use, because Flt3 inhibitors are expected to have less
nonspecific toxicity than proteasome inhibitors. Further, we
suggest that combination of Flt3 inhibitors with agents such as
IDR may increase the likelihood that they will target quiescent
LSC populations effectively.
We thank Drs. Katherine Borden, Gary Van Zant, Hartmut Geiger, and
Deborah Echlin for critical evaluation of the manuscript. We also thank
Dr. Marty Mayo for helpful discussions and for providing the I
B-SR
gene. We gratefully acknowledge the generous support of The Markey
Cancer Center Foundation and The Donatina Colachicco Cancer Re-
search Fund. This work was supported by American Cancer Society
Grant RPG-99-206-01-LBC (to C.T.J.) and National Institutes of Health
Grant R01-CA90446 (to C.T.J.). D.S.H. is a fellow of the Abraham J. and
Phyllis Katz Foundation.
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Guzman et al. PNAS
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December 10, 2002
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vol. 99
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no. 25
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MEDICAL SCIENCES