Human Cancer Biology
DAPK1 in Acute Myeloid Leukemia
Rajasubramaniam Shanmugam1,4, Padmaja Gade5, Annique Wilson-Weekes1,4, Hamid Sayar1,
Attaya Suvannasankha1,4, Chirayu Goswami2, Lang Li2, Sushil Gupta1, Angelo A. Cardoso1,
Tareq Al Baghdadi1, Katie J. Sargent3, Larry D. Cripe1, Dhananjaya V. Kalvakolanu5, and H. Scott Boswell1,4
certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced
Experimental Design: Heterogeneous primary AMLs were screened to identify a subgroup with
Flt3ITD in which repression of DAPK1, among NF-kB–and c-Jun–responsive genes, was studied. RNA
interference knockdown studies were carried out in an Flt3ITDþcell line, MV-4-11, to establish genetic
epistasis in the pathway Flt3ITD–TAK1–DAPK1 repression, and chromatin immunoprecipitations were
carried out to identify proximate effector proteins, including TAK1-activated p52NF-kB, at the DAPK1
DAPK1transcriptsnormalized totheexpressionofc-Jun,atranscriptional activatorofDAPK1,ascompared
with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic
gene signature was measured as control. These Flt3ITDþAMLs overexpress relB, a transcriptional repressor,
which forms active heterodimers with p52NF-kB. Chromatin immunoprecipitation assays identified
p52NF-kB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6
activation of p52NF-kB.
Conclusions: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-kB to suppress DAPK1
in association with HDACs, which explains DAPK1 repression in Flt3ITDþAML. Clin Cancer Res; 18(2);
360–9. ?2011 AACR.
Recent evidence indicates that attenuation of the unfold-
ed protein response (UPR)—an endoplasmic reticulum
(ER)–dependent stress response—may explain therapeutic
failure of acute myeloid leukemia (AML; refs. 1–3). Down-
duplication (ITD) in AML may impose such a status by
regulating expression of effectors that control stress-depen-
dent apoptosis. For example, the ratio of ER levels of bcl-2
other effectors may determine this output at a defined
DAPK1 is a calcium-calmodulin–dependent serine–thre-
onine protein kinase, which suppresses tumor cell survival
and metastasis via autophagy and apoptosis. It plays a
central role in ER stress–dependent apoptosis (7). DAPK1
We previously showed the existence of a Flt3–JNK1–c-Jun
Authors' Affiliations: Departments of
logy Division) and
University Melvin and Bren Simon Cancer Center, Indiana University
School of Medicine;
Affairs Medical Center, Indianapolis, Indiana; and5Department of Micro-
biology and Immunology, Greenebaum Cancer Center, University of
Maryland School of Medicine, Baltimore, Maryland
2Biostatistics and Computational Biology, Indiana
3Indiana University Health Systems;
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Current address for R. Shanmugam: Regional Medical Center (ICMR),
P. Gade and A. Wilson-Weekes contributed equally to this work.
Corresponding Author: H. Scott Boswell, Indiana University Melvin and
Bren Simon Cancer Center, R3-C-312, 980 W. Walnut Street, Indianapolis,
IN 46202. Phone: 317-274-0528; Fax: 317-274-0396; E-mail:
?2011 American Association for Cancer Research.
Clin Cancer Res; 18(2) January 15, 2012
pathway in Flt3ITDþAML (9). c-Jun is known to drive the
expression of not only bcl-2, but also DAPK1 (10, 11).
However, the latter circumstance would be antagonistic to
the progression of poor-prognosis Flt3ITDþAML. On the
AML is known (12–15). We hypothesized that a resistance
to apoptosis in certain AMLs occurs via severe repression of
DAPK1, through recruitment of p52NF-kB to the putative
NF-kB site at ?134bp of its promoter (12, 13). Indeed,
expression of DAPK1 is lost in a number of human cancers,
of DAPK1 is well reported, the upstream mechanisms that
contribute to tumor promotion via the recruitment of
epigenetic apparatus to the DAPK1 promoter are not
terminal kinase 1 (JNK1) and IkB kinase (IKK)/NF-kB may
beinvolvedinconcerted regulation ofantiapoptotic aswell
as proapoptotic genes to achieve an antiapoptotic/proa-
poptotic effector balance (e.g., bcl-2/DAPK1) to permit
higher aggressiveness in Flt3ITDþAML (4, 6, 14, 20). This
postulation was tested in context of prior functional and
cohort analyses of AML blasts carried out in our laboratory
that linked Flt3 phosphorylation/activation to JNK1 phos-
phorylation (9) and with regard to the known role for
c-Jun1/AP-1 in DAPK1 and bcl-2 expression (10, 11). In
addition, we inferred that a conserved dual-activation
mechanism, which relies upon TAK1, for JNK1–c-Jun and
IKK–NF-kB may exist (21) to promote the optimal anti-
apoptotic/proapoptotic effector balance. This hypothesis
was given emphasis by the recognition that TAK1 is among
the most highly expressed genes in a leukemic stem cell
(LSC) signature of poor-risk AML in which DAPK1 repres-
sion coexists (22, 23).
In this study, we showed that TAK1 activated p52NF-kB,
binds at the tandem NF-kB and CRE sites of DAPK1, and
recruits certain transcriptional repressors, belonging to the
histone deacetylase (HDAC) family (12). Because p52NF-
a de-repression of DAPK1 to contribute toward enhanced
apoptosis in Flt3 ITDþAMLs. Finally, we propose a thera-
peutic model for the rational combination of Flt3- and
HDAC-inhibitors for suppressing AML growth.
Materials and Methods
The human leukemic cell lines, HL-60 and MV-4-11
(derived from a biphenotypic leukemia), were obtained
from the American Type Culture Collection (ATCC) in
McCoy’s andRPMImedia, respectively,supplemented with
10% fetal calf serum. The MV-4-11 cell line overexpresses
ing the MLL gene (9). Blast cells from the bone marrow of
patients with AML were obtained at the time of diagnosis,
after informed consent. The buoyant fraction was isolated
over Ficoll–Hypaque, and then washed with PBS before
processing. The cohort of AMLs subjected to gene expres-
sion analysis showed mean 88 ? 10% blasts (Supplemen-
and cytoplasmic fractions using the NE-PER Extraction Kit
(Pierce Biotechnology). For Western blot analysis, bone
marrow samples with ?70% blast cells in the purified
aspirate were used.
Transfections and reporter assays
MV-4-11 cells were electroporated using the Amaxa sys-
tem and then placed in McCoy’s medium supplemented
with 10% FBS. Cells were transfected with a luciferase
reporter (13) driven by the DAPK1 promoter (1.2 kB)
harboring either wild-type or mutated CRE site (?177 bp).
cotransfected to normalize for variations in transfection
Western blot analysis
Cytosolic or nuclear proteins were subjected to Western
blotting with indicated antibodies as described previously
(9). Densitometry was carried out to quantify specific
bands, and data were normalized to either cytoplasmic
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or
nuclear (Sp1) internal controls, depending on the case.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were
conducted as previously described (13). In brief, DNA–
protein complexes were cross-linked by incubating
Acute myeloid leukemia (AML) is a group of complex
and heterogeneous diseases, which can be classified
according to cytogenetic/genotypic features of the blast
cells and activated signaling pathways. These signaling
pathwayscooperatetopromote blastcellsurvivaland to
prevent tumor suppressor-induced senescence. In this
article, we report a tyrosine kinase (Flt3ITD)-initiated
noncanonical NF-kB signaling pathway in a subset of
AMLs, where p52NF-kB, in association with certain
histone deacetylases (HDAC), represses the tumor sup-
pressor gene DAPK1. DAPK1 is an essential player in
endoplasmic reticulum (ER)–stress-induced apoptosis,
and is implicated in poor outcome of AML by its repres-
sion. The mechanism for repression of DAPK1 by
p52NF-kB and HDACs, influenced by Flt3ITD, was
found to involve the MAP3K, TAK1. Because TAK1 is
one among the most highly expressed genes in a leuke-
mic stem cell signature for poor-risk AML, these studies
focus attention on the interface between signal trans-
duction and epigenetic remodeling in AML.
p52NF-kB and DAPK1 Repression in Flt3ITD AML
www.aacrjournals.orgClin Cancer Res; 18(2) January 15, 2012
26. Mineva ND, Rothstein TL, Meyers JA, Lerner A, Sonenshein GE. CD40
ligand-mediated activation of the de novo RelB NF-kappaB synthesis
27. Compagno M, Lim WK, Grunn A, Nandula SV, Brahmachary M, Shen
in diffuse large B-cell lymphoma. Nature 2009;459:717–21.
28. Ninomiya-Tsuji J, Kishimoto K, Hiyama A, Inoue J, Cao Z, Matsu-
moto K. The kinase TAK1 can activate the NIK-I kappaB as well as
the MAP kinase cascade in the IL-1 signalling pathway. Nature
29. Liu H, Deng X, Shyu YJ, Li JJ, Taparowsky EJ, Hu CD. Mutual
regulation of c-Jun and ATF2 by transcriptional activation and sub-
cellular localization. EMBO J 2006;25:1058–69.
30. Ludwig D, Hicklin D, Witte L, Li Y, Small D. FLT3 ligand causes
autocrine signaling in acute myeloid leukemia cells. Blood 2004;103:
31. Keedy KS, Archin NM, Gates AT, Espeseth A, Hazuda DJ, Margolis
DM. A limited group of class I histone deacetylases acts to repress
human immunodeficiency virus type 1 expression. J Virol 2009;83:
32. Bradbury CA, Khanim FL, Hayden R, Bunce CM, White DA, Drayson
MT, et al. Histone deacetylases in acute myeloid leukaemia show a
distinctive pattern of expression that changes selectively in response
to deacetylase inhibitors. Leukemia 2005;19:1751–9.
33. Xu X, Xie C, Edwards H, Zhou H, Buck SA, Ge Y. Inhibition of
histone deacetylases 1 and 6 enhances cytarabine-induced apo-
ptosis in pediatric acute myeloid leukemia cells. PLoS One 2011;6:
34. Govindan MV. Recruitment of cAMP-response element-binding
protein and histone deacetylase has opposite effects on gluco-
corticoid receptor gene transcription. J Biol Chem 2010;285:
et al. Runx2 (Cbfa1, AML-3) interacts with histone deacetylase 6 and
represses the p21(CIP1/WAF1) promoter. Mol Cell Biol 2002;22:
36. Bali P, Pranpat M, Bradner J, Balasis M, Fiskus W, Guo F, et al.
Inhibition of histone deacetylase 6 acetylates and disrupts the chap-
erone function of heat shock protein 90: a novel basis for antileukemia
activity of histone deacetylase inhibitors. J Biol Chem 2005;280:
37. Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray
SJ, Greenman CD, et al. A comprehensive catalogue of somatic
mutations from a human cancer genome. Nature 2010;463:191–6.
38. Pleasance ED, Stephens PJ, O'Meara S, McBride DJ, Meynert A,
Jones D, et al. A small-cell lung cancer genome with complex signa-
tures of tobacco exposure. Nature 2010;463:184–90.
39. Ley TJ, Mardis ER, Ding L, Fulton B, McLellan MD, Chen K, et al. DNA
sequencing of a cytogenetically normal acute myeloid leukaemia
genome. Nature 2008;456:66–72.
Recurring mutations found by sequencing an acute myeloid leukemia
genome. N Engl J Med 2009;361:1058–66.
DNMT3A mutations in acute myeloid leukemia. N Engl J Med 2010;
42. Figureroa ME, Abel-Wahab O, Lu C, Ward PS, Patel J, Shih A, et al.
Leukemic IDH1 and IDH2 mutations result in a hypermethylation
phenotype, disrupt TET2 function, and impair hematopoietic differen-
tiation. Cancer Cell 2010;18:553–67.
43. Figueroa ME, Lugthart S, Li Y, Erpelinck-Verschueren C, Deng X,
Christos PJ, et al. DNA methylation signatures identify biologically
distinct subtypes in acute myeloid leukemia. Cancer Cell 2010;17:
44. Lugthart S, Figueroa ME, Bindels E, Skrabanek L, Valk PJ, Li Y, et al.
Aberrant DNA hypermethylation signature in acute myeloid leukemia
directed by EVI1. Blood 2011;117:234–41.
45. Laurenzana A, Petruccelli LA, Pettersson F, Figueroa ME, Melnick A,
Baldwin AS, et al. Inhibition of DNA methyltransferase activates tumor
necrosis factor alpha-induced monocytic differentiation in acute mye-
loid leukemia cells. Cancer Res 2009;69:55–64.
46. Claus R, Hackanson B, Poetsch AR, Zucknick M, Sonnet M, Blagitko-
Dorfs N, etal. Quantitativeanalyses of DAPK1 methylation inAML and
MDS. Int J Cancer 2011 Sep 14. [Epub ahead of print].
47. Cedar H, Bergmann Y. Linking DNA methylation and histone modifi-
cation: patterns and paradigms. Nat Rev Genet 2009;10:295–304.
48. Morioka S, Omori E, Kajino T, Kajino-Sakamoto R, Matsumoto K,
Ninomiya-Tsuji J. TAK1 kinase determines TRAIL sensitivity by mod-
ulating reactive oxygen species and cIAP. Oncogene 2009;28:
49. Herrero-Mart? ?n G, Høyer-Hansen M, Garc? ?a-Garc? ?a C, Fumarola C,
Farkas T, L? opez-Rivas A, et al. TAK1 activates AMPK-dependent
cytoprotective autophagy in TRAIL-treated epithelial cells. EMBO J
50. Xia ZP, Sun L, Chen X, Pineda G, Jiang X, Adhikari A, et al. Direct
activation of protein kinases by unanchored polyubiquitin chains.
51. Skaug B, Jiang X, Chen ZJ. The role of ubiquitin in NF-kappaB
regulatory pathways. Annu Rev Biochem 2009;78:769–96.
52. Wulf GM, Ryo A, Wulf GG, Lee SW, Niu T, Petkova V, et al. Pin1 is
overexpressed in breast cancer and cooperates with Ras signaling in
increasing the transcriptional activity of c-Jun towards cyclin D1.
EMBO J 2001;20:3459–72.
53. Pulikkan JA, Dengler V, Peer Zada AA, Kawasaki A, Geletu M, Pasalic
Z, et al. Elevated PIN1 expression by C/EBPalpha-p30 blocks C/EBP
alpha-induced granulocytic differentiation through c-Jun in AML.
prolyl isomerase activity and cellular function. Mol Cell 2011;42:
p52NF-kB and DAPK1 Repression in Flt3ITD AML
www.aacrjournals.orgClin Cancer Res; 18(2) January 15, 2012