Improved understanding of the molecular pathways that suppress the genesis and maintenance of cancer stem cells will facilitate development of rationally targeted therapies. PU.1 is a transcription factor that is required for normal myelomonocytic differentiation in hematopoiesis, and reduced PU.1 activity has been associated with myeloid leukemogenesis in man and in mouse models. A recent study by Steidl et al. demonstrates that Junb and Jun, two AP-1 transcription factors, are critical downstream effectors of the tumor suppressor activity of PU.1, and that reduced expression of Junb, in particular, may be a common feature of acute myeloid leukemogenesis

Cleary, Michael L.

Somervaille, Tim C.P.

Elsevier - Publisher Connector 

PU.1 and Junb: Suppressing the formation of acute myeloid leukemia stem cells 

	 p r e v i e w s
Selected reading
Bao, S., Wu, Q., McLendon, R.E., Hao, Y., Shi, Q., 
Hjelmeland, A.B., Dewhirst, M.W., Bigner, D.D., 
and Rich, J.N. (2006). Nature. Published online 
October 18, 2006. 10.1038/nature05236. 
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Dimeco, F., and Vescovi, A. (2004). Cancer Res. 
64, 7011–7021.
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Sawaya, R.E., and Fuller, G.N. (2000). Nat. Genet. 
25, 55–57.
Hornsey, S. (1973). Strahlenschutz Forsch. Prax. 
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Marshman, E., Booth, C., and Potten, C.S. (2002). 
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13, 115–125.
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10, 1026–1028.
Rich, J.N., Sathornsumetee, S., Keir, S.T., Kieran, 
M.W., Laforme, A., Kaipainen, A., McLendon, 
R.E., Graner, M.W., Rasheed, B.K., Wang, L., et 
al. (2005a). Clin. Cancer Res. 11, 8145–8157.
Rich, J.N., Hans, C.H., Jones, B., Iverson, E.S., 
McLendon, R.E., Rasheed, B.K., Dobra, A., 
Dressman, H.K., Bigner, D.D., Nevins, J.R., and 
West, M. (2005b). Cancer Res. 65, 4051–4058.
Singh, S.K., Hawkins, C., Clarke, I.D., Squire, J.A., 
Bayani, J., Hide, T., Henkelman, R.M., Cusimano, 
M.D., and Dirks, P.B. (2004). Nature 432, 396–
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DOI 10.1016/j.ccr.2006.11.008PU.1 and Junb: Suppressing the formation of acute myeloid 
 leukemia stem cells
Improved understanding of the molecular pathways that suppress the genesis and maintenance of cancer stem cells will 
facilitate development of rationally targeted therapies. PU.1 is a transcription factor that is required for normal myelomonocytic 
differentiation in hematopoiesis, and reduced PU.1 activity has been associated with myeloid leukemogenesis in man and 
in mouse models. A recent study by Steidl et al. demonstrates that Junb and Jun, two AP-1 transcription factors, are critical 
downstream effectors of the tumor suppressor activity of PU.1, and that reduced expression of Junb, in particular, may be a 
common feature of acute myeloid leukemogenesis.Tissue-specific stem cells are considered 
fertile soil for some of the mutations that 
contribute to the development of human 
cancers. However, the molecular mecha-
nisms by which these mutations give rise 
to cancer stem cells, or otherwise lead to 
neoplastic disease, are less well defined. 
This issue is of major importance, since 
the implicated pathways may be targets 
for molecular therapies, particu-
larly if they are selectively involved 
in cancer versus normal stem cell 
maintenance.
Of the many molecular patholo-
gies associated with acute myeloid 
leukemia (AML), one recurrently 
implicated gene is PU.1 (SPI1; 
Sfpi1, for Spleen focus-forming virus 
proviral integration), which codes for 
a transcription factor that is essen-
tial for normal myelomonocytic dif-
ferentiation and consequently also 
functions as a tumor suppressor 
(reviewed in Koschmieder et al., 
2005). Repressed PU.1 transcrip-
tion has been reported in AMLs har-
boring PML-RARα (Mueller et al., 
2006) or FLT3-ITD mutations, and 
the AML-associated oncoprotein 
AML1-ETO functionally inactivates 
PU.1 through displacement of its 456	coactivator, JUN (Koschmieder et al., 2005). 
Heterozygous mutations of PU.1 have 
been observed in one series of patients 
with AML and are postulated to co-operate 
with reduced PU.1 activity induced by other 
mechanisms to promote leukemogenesis 
(Koschmieder et al., 2005). Consistent with 
these observations, reduced or abrogated 
PU.1 expression in mouse models results Figure 1.	PU.1	in	normal	and	leukemic	hematopoiesis
In	normal	hematopoiesis,	 PU.1	promotes	myelomonocytic	dif-
ferentiation	through	positively	regulating	expression	of	the	AP-1	
transcription	factors	Junb	and	Jun.	In	acute	myeloid	leukemia,	a	
variety	of	mechanisms	contribute	to	a	reduction	in	PU.1	activity,	
leading	to	reduced	Junb	and	Jun	expression,	with	consequent	
dysregulation	of	differentiation,	programmed	cell	death,	and	cel-
lular	proliferation.in AML (Rosenbauer et al., 2004; Metcalf 
et al., 2006). Thus, PU.1 activity appears to 
be a target of several oncogenic signaling 
pathways in AML (Figure 1). However, the 
downstream genes that are critical media-
tors of its leukemia-suppressive role have 
hitherto been undefined.
In an elegant series of experiments, 
Steidl et al. (2006) have recently solved a significant piece of this puzzle by 
identifying Jun and Junb, members 
of the activator protein-1 (AP-1) 
family of transcriptional regulators, 
as critical effectors of the PU.1 
tumor suppressor pathway. Their 
studies employed a PU.1 knock-
down (PU.1 KD) mouse model that 
develops highly penetrant AML as 
a consequence of reduced PU.1 
expression caused by deletion of 
a critical upstream regulatory ele-
ment in the PU.1 gene. Global tran-
scriptional analysis of an immature 
subfraction of bone marrow cells 
obtained from preleukemic PU.1 
KD mice identified Jun and Junb, 
in addition to a number of previ-
ously known PU.1 targets, to be 
downregulated compared with 
cells obtained from wild-type mice. 
The compared cell populations, cancer cell december	2006
	 p r e v i e w sso-called KSL cells (Kit+, Sca1+, Lin−), 
normally contain about 15% long-term 
hematopoietic stem cells (HSCs) as well 
as other transiently reconstituting multipo-
tent progenitors. When these cells were 
isolated from leukemic PU.1 KD donor 
mice and transplanted into immunocom-
promised recipient mice, the recipients 
developed AML. Thus, at least a propor-
tion of cells with this immunophenotype 
in leukemic PU.1 KD mice are leukemia 
stem cells (LSCs) that display significantly 
downregulated Jun and Junb expression 
by comparison with normal KSL cells.
Steidl et al. further demonstrated 
that Junb is a direct target gene for PU.1, 
which bound to and regulated expression 
of Junb through a conserved upstream 
DNA element. Interestingly, Junb itself has 
been shown to be a tumor suppressor in 
myelopoiesis. Mice lacking Junb expres-
sion in HSCs develop a myeloproliferative 
disease similar to human chronic myeloid 
leukemia (CML) (Passegue et al., 2004), 
and methylation-induced silencing of Junb 
occurs in cells from chronic phase and 
blastic transformation of CML (Yang et al., 
2003). Taken together, these observations 
suggest that direct regulation of Junb by 
PU.1 may constitute a critical transcrip-
tional circuit for suppression of myeloid 
leukemogenesis (Figure 1).
To directly test this possibility, Junb 
was forcibly expressed in PU.1 KD leuke-
mia cells in an effort to bypass the onco-
genic effects of reduced PU.1 expression. 
This antagonized the oncogenic proper-
ties of PU.1 KD AML cells as evidenced 
by reduced clonogenic potential, serial 
replating activity, and proliferation in liquid 
culture, whereas clonogenic potentials of 
normal bone marrow progenitor cells were 
unaffected. The effects were specific to 
Junb, because similar forced expression 
of Jun did not block AML cell proliferation. 
Furthermore, forced expression of Junb in 
PU.1 KD leukemia cells inhibited their abil-
ity to induce AML in secondary recipients, 
indicating that restoration of Junb expres-
sion was sufficient to abrogate LSC activ-
ity associated with PU.1 knockdown.
To test the relevance for human dis-
ease, the authors interrogated an AML 
global gene expression data set and dis-
covered that PU.1 and JUNB expression 
were very significantly correlated with each 
other, particularly in the AML-M4 and M5 cancer cell december	2006	subtypes. Prospective isolation of primitive 
hematopoietic progenitor cells (CD34+, 
CD38−, CD90low, Lin−) from AML patients 
showed that PU.1 and JUNB transcript lev-
els were also highly correlated in a phe-
notypic population reportedly enriched for 
human LSC activity. Furthermore, when 
compared with normal progenitor cells with 
a similar phenotype, JUNB transcript levels 
were lower in the LSC-enriched popula-
tions. However, the higher prevalence of 
reduced JUNB expression (17/20 cases) 
compared with reduced PU.1 expression 
(7/20 cases) in LSC-enriched populations 
compared with normal control populations 
also raised the intriguing possibility that a 
variety of mechanisms may account for 
impaired maintenance of JUNB expression 
other than reduced PU.1 activity. These 
data support a critical role for PU.1 and 
JUNB as myeloid lineage tumor suppres-
sors and suggest that dysregulation of this 
pathway may frequently occur in human 
leukemic stem cells.
The authors’ studies raise two interest-
ing questions. First, if loss of Junb expres-
sion is a critical downstream effect of PU.1 
knockdown, why do PU.1 KD mice develop 
AML rather than the myeloproliferative dis-
ease observed when Junb is inactivated in 
long-term HSCs (Passegue et al., 2004)? 
One likely explanation is that reduced PU.1 
expression has pleiotropic downstream 
effects. Consistent with this, Steidl et al. 
observed that in preleukemic PU.1 KD 
mice there is a block in terminal monocyte/
macrophage lineage differentiation, which 
is reversed by expression of Jun. It is pos-
sible, therefore, that impaired differentiation 
induced by Jun deficiency and increased 
proliferation induced by Junb deficiency 
collaborate to induce the observed disease 
morphology. Secondly, how might reduced 
Junb expression promote myeloid leuke-
mogenesis? Although not formally inves-
tigated in this study, Junb has previously 
been shown to repress cyclin D1, Bcl2, and 
Bcl-XL expression and to activate expres-
sion of the cyclin-dependent kinase inhibi-
tor p16INK4a (Passegue et al., 2001), which 
together may alter the apoptotic rheostat in 
favor of cell death as well as promoting cell 
cycle exit to facilitate normal myeloid dif-
ferentiation (Figure 1). Loss of Junb would 
favor the reverse processes.
Through their analysis of an interest-
ing murine genetic model of AML, Steidl et al. have defined an important transcrip-
tional pathway that may well be dysregu-
lated in human AML stem cells, which 
are the cells that must be eliminated by 
therapy in order to cure disease. These 
and other recent studies (for example, 
Somervaille and Cleary, 2006; Krivtsov 
et al., 2006) validate the use of murine 
models to further our understanding of 
LSCs and pertinent cellular pathways rel-
evant for human AML. Their studies also 
add further promise that the molecular 
pathways underlying LSC maintenance 
may be selectively targeted while sparing 
normal HSCs.
Tim	c.P.	Somervaille1	and	
michael	L.	cleary1,*
1department	of	Pathology,	Stanford	
University	School	of	medicine,	Stanford,	
california	94305	
*e-mail:	mcleary@stanford.edu
Selected reading
Koschmieder, S., Rosenbauer, F., Steidl, U., 
Owens, B.M., and Tenen, D.G. (2005). Int. J. 
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Wang, Y., Faber, J., Levine, J.E., Wang, J., Hahn, 
W.C., Gilliland, D.G., et al. (2006). Nature 442, 
818–822.
Metcalf, D., Dakic, A., Mifsud, S., Di Rago, L., Wu, 
L., and Nutt, S. (2006). Proc. Natl. Acad. Sci. USA 
103, 1486–1491.
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M.F., Asou, N., Buergi, U., and Tenen, D.G. (2006). 
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Passegue, E., Jochum, W., Schorpp-Kistner, M., 
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Somervaille, T.C., and Cleary, M.L. (2006). Cancer 
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Steidl, U., Rosenbauer, F., Verhaak, R.G., Gu, X., 
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Yang, M.Y., Liu, T.C., Chang, J.G., Lin, P.M., and 
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DOI 10.1016/j.ccr.2006.11.009457


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PU.1 and Junb: Suppressing the formation of acute myeloid leukemia stem cells

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