the primary tumor, which suggests that
CLV dilation may have a far more central
role in the metastatic process than hith-
erto appreciated. Interestingly, Etodolac
also diminished metastatic burden in the
lung. These results suggest that a level
of control over the lymphatic and sys-
temic dissemination could potentially be
achieved by administration of relatively
safe anti-inflammatory agents.
This provocative study adds an impor-
tant dimension to the process that might
be viewed as vascular system ‘‘condi-
tioning’’ for cancer metastasis. While the
focus of the present study is on CLV dila-
tion, others observed lymphangiogenesis
within lymph nodes prior to their meta-
static colonization (Tobler and Detmar,
2006), a process that may be attributed
to remote influences of growth factors
or exosomes (Hood et al., 2011). Analo-
gous pre-metastatic niches were also
described at sites of blood borne metas-
tases (Kaplan et al., 2005).
located outside of a growing tumor is not
restricted to CLVs. Similar increases in
diameter are often observed in the case of
which is also apparent from some of the
images included in the study by Karnezis
et al., (2012). Although this is a commonly
logical process has thus far attracted
minimal attention (Yu and Rak, 2003). In
contrast to angiogenesis, which occurs at
the level of microscopic capillaries(Carme-
liet and Jain, 2011), formation of larger
tumor-feeding blood vessels may involve
such mechanisms as dilation, similar to
that occurring in CLVs, or circumferential
growth (‘‘tumor arteriogenesis’’) (Yu and
Rak, 2003). Whether such macroscopic
changes control tumor microenvironment,
growth, or hematogenous metastasis (by
analogy to CLVs) remains to be studied.
The novel and fascinating link between
CLV dilation and lymphatic metastasis
described by these authors raises several
important questions. For example, how
does CLV dilation promote metastasis? Is
this merely a wider conduit (‘‘plumbing’’)
effect, or does it involve more subtle regu-
latory mechanisms (e.g., tumor-LEC inter-
increase inprostaglandinlevelsis detected
indicative of impending lymphatic metas-
tasis in the clinic?How early inprogression
glandins occur, and how discrete, how
detectable, would this event be? What
systemic consequences may be associ-
ated with VEGF-D-induced increase in
prostaglandins in blood, e.g., for the
vascular system? What turns on lymphan-
giogenic growth factors in metastatic
cancers, and is there a link between onco-
genic pathways and CLV dilation?
logical blockade of the pathological CLV
dilation and metastasis could be achieved
with already available agents (VEGF/
VEGFR3/2 inhibitors and NSAIDs). How-
could interfere withthe lymph outflowfrom
the primary tumor mass leading to a build
up of interstitial fluid pressure (IFP)?
drug delivery and could result in vascular
compression, hypoxia, and perhaps in
hematogenous metastasis. It is unclear if
apeutic interference with CLV dilation.
Indeed, the work of Karnezis et al., (2012)
opens up several new lines of inquiry
and a new domain in the field of lymphan-
giogenesis and cancer progression.
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aSIRTing Control over Cancer Stem Cells
Takahiro Ito,1Bryan Zimdahl,1,2and Tannishtha Reya1,*
1Department of Pharmacology, University of California San Diego School of Medicine, La Jolla, CA 92093, USA
2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
Cancer stem cells lie at the root of chronic myelogenous leukemia (CML) and mediate its continued growth.
Li et al. identify SIRT1 as a new target for eliminating CML cancer stem cells.
Chronic myelogenous leukemia (CML) is
a cancer that begins in hematopoietic
stem cells. Triggered by the BCR-ABL
translocation (Melo and Barnes, 2007),
progression from a slow-growing chronic
mutationscan induce its
Cancer Cell 21, February 14, 2012 ª2012 Elsevier Inc.
phase to a more aggressive and undiffer-
entiated blast crisis phase. The discovery
of the kinase inhibitor imatinib mesylate
revolutionized the treatment of CML.
Over the years, however, it has become
clear that while kinase inhibitors can
hold CML at bay, they are unable to erad-
icate the disease, leading to a life-long
dependence on the drug and an in-
creased risk of relapse and progression.
In addition, kinase inhibitors are ineffec-
tive against drug-resistant and advanced
stage disease. Although such patients
may not form a large group in developed
countries, the global face of CML is very
different, and many patients are not diag-
nosed until the disease is at an advanced
stage. Insight into the limitations of tar-
geted kinase therapy came from an
understanding that CML is composed of
differentiated cells as well as a more
undifferentiated pool of cancer stem cells
that have the capacity to propagate the
disease (Wang et al., 1998). Emerging
evidence suggests that differentiated
CML cells are addicted to ABL and can
be eliminated by kinase inhibitors, while
cancer stem cells can become ABL inde-
pendent and thus persist despite therapy
(Graham et al., 2002; Corbin et al., 2011).
required for CML cancer stem cell growth
and renewal is critical for effectively tar-
geting the disease. In this issue of Cancer
Cell, Li et al. (2012) identify SIRT1, the
founding member of the Sirtuin family of
proteins, as an exciting new target for
eradicating CML cancer stem cells and
thereby stopping CML growth.
Sirtuins, mammalian homologs of the
yeast protein silent information regulator
2, represent a unique subclass of histone
include both histones and non histone
proteins, and unlike other HDACs, they
act in an NAD-dependent manner (Haigis
ful influence on a wide array of cellular
processes including DNA repair, cell
survival, metabolism, and aging in diverse
organisms (Haigis and Sinclair, 2010).
In this study, the authors use a combi-
nation of genetically engineered mouse
models and primary leukemia xenografts
to assess the role of SIRT1 in mouse and
human CML growth. The authors first
examined the expression of SIRT1 in
normal and CML cells, focusing on the
stem cell enriched CD34+population.
SIRT1 was expressed at higher levels in
human CML CD34+cells than in normal
CD34+cells. Moreover, knockdown of
SIRT1 in CD34+CML cells led to reduced
proliferation, enhanced apoptosis, and
impaired colony-forming ability. Impor-
tantly, SIRT1 knockdown had less of an
effect on proliferation and apoptosis of
cells, suggesting that
CML and normal stem cells display
a differential dependence on SIRT1.
Further, the combined use of SIRT1 inhi-
bition together with imatinib led to an
increase in cell death, suggesting that
suppression of SIRT1 could cooperate
with imatinib to more effectively block
CML stem cells (Figure 1).
To test if the dependence of CML on
SIRT1 could be useful in a therapeutic
context, the authors used the small mole-
cule Tenovin 6 (TV-6), which blocks the
activity of sirtuin family proteins (Lain
Figure 1. SIRT1 Inhibition Effectively Targets CML Cancer Stem Cells
Chronicmyelogenousleukemia (CML)iscomposedofdifferentiatedcells (blue andpurple)aswell asamore primitive poolofcancerstemcells (red) thathavethe
capacity to propagate the disease (left). The kinase inhibitor Imatinib can eliminate differentiated CML cells but cannot effectively target cancer stem cells
(middle). Though insensitive to Imatinib, cancer stem cells remain dependent on SIRT1. Thus, the combined use of the SIRT1 inhibitor Tenovin 6 and Imatinib
effectively removes residual cancer stem cells and may block CML at its root (right).
Cancer Cell 21, February 14, 2012 ª2012 Elsevier Inc.
et al., 2008). In vitro treatment with TV-6,
and to a greater extent with TV-6 and im-
atinib, reduced colony formation and
in vivo engraftment more effectively than
imatinib alone, highlighting the potential
utility of SIRT inhibition in the context of
While the experiments involving ex vivo
exposure suggested that pharmacologic
blockade of SIRT1 was effective against
CML, it was critical to assess whether
the drug could affect disease in a physio-
logical context. To test this, the group iso-
lated leukemic cells from an inducible
BCR-ABL transgenic mouse and trans-
planted them into irradiated recipients.
These mice were subsequently treated
with imatinib, TV-6, or the combination
daily for 21 days. Although imatinib alone
impaired leukemia growth, it failed to
target CML stem cells. In contrast, TV-6
alone, and to a greater extent TV-6 and
imatinib, led to a very significant loss of
CML stem cells. Consistent with this,
showed improved survival, with reduced
numbers of residual leukemic cells in
the bone marrow after discontinuation of
treatment. Although the changes in sur-
vival were perhaps not as dramatic as
the drop in cancer stem cell content may
have predicted, it is important to note
that the drug was discontinued after 3
weeks; thus, continued treatment, modi-
fied dosing or the use of alternate inhibi-
tors might show further benefits in vivo.
In a key experiment, the authors also
tested the effect of TV-6 on mice xeno-
grafted with an imatinib-resistant blast
crisis CML patient sample and found
that it led to a significant reduction in
engraftment at multiple sites of leukemia
growth. This suggests that targeting
SIRT1 may be effective against both
chronic phase and in imatinib-resistant
advanced stage disease. More broadly,
this work identifies Sirtuins as an impor-
tant control point for cancer stem cells
and provides a strong rationale for
considering SIRT1 inhibitors for treatment
of myeloid leukemias and perhaps other
malignancies that display activation of
How does SIRT1 inhibition eliminate
CML cancer stem cells? SIRT1 has previ-
ously been shown to deacetylate p53 and
thereby regulate its transcriptional activity
(Haigis and Sinclair, 2010). In support of
ylated and total p53 levels in both chronic
and blast crisis phase CML CD34+cells,
triggering a rise in p53 target genes. Loss
of function studies indicated that TV-6
with the fact that p53 activation can effec-
tively target CML. This suggests that
consideration of SIRT1 as a target should
take into account a patient’s p53 status,
since the 30% of blast crisis patients
whose disease display p53 mutations are
unlikely to respond to this strategy (Melo
and Barnes, 2007).
In the last few years, basic and transla-
tional work has identified several path-
ways that are critical for CML stem cell
function and renewal, including promye-
locytic leukemia protein (PML), b-catenin,
Alox5, and Smoothened (reviewed in
Chen et al., 2010). These studies shed
light on the molecular mechanisms that
protect and sustain CML cancer stem
cells, allowing them to evade imatinib.
Some have been of immediate transla-
tionalinterestbecause theycan bereadily
targeted; this is true in particular for PML
and Smoothened, which can be inhibited
by arsenic trioxide and by Hedgehog
pathway antagonists (Dierks et al., 2008;
Ito et al., 2008; Zhao et al., 2009). Both
strategies are currently being tested in
trials of myeloid leukemia, and it will be
of great interest to see how effective and
durable they turn out to be. But consid-
ering the fact that kinase inhibitors can
hold CML at bay in many patients, the
bar for a new therapeutic in this disease
may be high. At this stage, it is not unrea-
cancer stem cells and an ability to discon-
tinue therapy without relapse. Perhaps
the blockade of SIRT1 will allow us to
finally assert control over CML cancer
stem cellsand accelerate
toward this goal.
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