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. 2012 Jan 15;18(2):360-369.
doi: 10.1158/1078-0432.CCR-10-3022. Epub 2011 Nov 17.

A noncanonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia

Rajasubramaniam Shanmugam  1   2 , Padmaja Gade #  3 , Annique Wilson-Weekes #  1   2 , Hamid Sayar  1 , Attaya Suvannasankha  1   2 , Chirayu Goswami  4 , Lang Li  4 , Sushil Gupta  1 , Angelo A Cardoso  1 , Tareq Al Baghdadi  1 , Katie J Sargent  5 , Larry D Cripe  1 , Dhananjaya V Kalvakolanu  3 , H Scott Boswell  1   2
Affiliations

A noncanonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia

Rajasubramaniam Shanmugam et al. Clin Cancer Res. .

Abstract

Purpose: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis.

Experimental design: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD(+) cell 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-κB, at the DAPK1 locus.

Results: AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD(+) AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD(+) human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB-inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD(+) AMLs had selective nuclear activation of p52NF-κB.

Conclusions: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD(+) AML.

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Figures

Fig. 1
Fig. 1. Down regulation of DAPK1 expression in MV-4-11 cell line that bears Flt3ITD, and series of primary AML blasts with normal karyotypes and Flt3ITD, as compared to FltITD-ve AMLs
Western blot analyses were performed. A) Flt3ITD+ve AML 2797 and FltITD-ve AMLs-#2930 and 2993 demonstrated correlation of TAK1 phosphorylation levels with phosphorylation status of the secondary (TAK1-JNK1) downstream transcription factor target c-jun. Also, DAPK1 (a c-jun target gene) levels were analyzed in these samples: lower expression levels are found in the Flt3ITD+ve AML 2797 (denoted in two places by asterisk) (1.5-fold and >6-fold reduction) with strong TAK1 activation, when compared with FltITD-ve AMLs-#2930 and 2993 respectively. B) DAPK1 levels were quantified densitometrically in Flt3ITD+ AMLs and MV-4-11 cell line: for #2841, 3-to-4-fold higher compared with 2797 and 2854, respectively, as well as 1.5-fold higher vs. 2857). C) DAPK1 promoter activity is significantly augmented 2-fo l d b y c-jun (* p<0.004) in MV-4-11 cells. DAPK1 promoter vector with a mutated CRE site showed reduced activity (** p<0.03 ). Data represent mean ±SE replicate determinations, from 3 experiments. Results of Western blotting of MV-4-11 cells treated with RNAi for Flt3 or JNK1, compared to non-targeting control siRNA. Knockdown of Flt3 ITD reduces p-jun activation (by 96%) and DAPK1 and bcl-2 expression by 95% and 60%, respectively. RNAi-mediated knockdown of JNK1 (45%), reduces p-jun levels by 45%, and DAPK1 and bcl-2 levels by 40% and 45%, respectively,
Fig. 1
Fig. 1. Down regulation of DAPK1 expression in MV-4-11 cell line that bears Flt3ITD, and series of primary AML blasts with normal karyotypes and Flt3ITD, as compared to FltITD-ve AMLs
Western blot analyses were performed. A) Flt3ITD+ve AML 2797 and FltITD-ve AMLs-#2930 and 2993 demonstrated correlation of TAK1 phosphorylation levels with phosphorylation status of the secondary (TAK1-JNK1) downstream transcription factor target c-jun. Also, DAPK1 (a c-jun target gene) levels were analyzed in these samples: lower expression levels are found in the Flt3ITD+ve AML 2797 (denoted in two places by asterisk) (1.5-fold and >6-fold reduction) with strong TAK1 activation, when compared with FltITD-ve AMLs-#2930 and 2993 respectively. B) DAPK1 levels were quantified densitometrically in Flt3ITD+ AMLs and MV-4-11 cell line: for #2841, 3-to-4-fold higher compared with 2797 and 2854, respectively, as well as 1.5-fold higher vs. 2857). C) DAPK1 promoter activity is significantly augmented 2-fo l d b y c-jun (* p<0.004) in MV-4-11 cells. DAPK1 promoter vector with a mutated CRE site showed reduced activity (** p<0.03 ). Data represent mean ±SE replicate determinations, from 3 experiments. Results of Western blotting of MV-4-11 cells treated with RNAi for Flt3 or JNK1, compared to non-targeting control siRNA. Knockdown of Flt3 ITD reduces p-jun activation (by 96%) and DAPK1 and bcl-2 expression by 95% and 60%, respectively. RNAi-mediated knockdown of JNK1 (45%), reduces p-jun levels by 45%, and DAPK1 and bcl-2 levels by 40% and 45%, respectively,
Fig. 2
Fig. 2. A dichotomy exists in Meis1, c-jun, and relB expression (which are known to be c-jun-dependent) vs. DAPK1, (the dually-responsive c-jun/NF κB-dependent gene) in patient groups with normal karyotype Flt3ITD or MLL translocation
A). Expression levels of DAPK1, is significantly different in NK Flt3 ITD or MLL translocation AML's when compared with cases without Flt3ITD or nonrandom cytogenetic abnormalities. B) In normal karyotype Flt3ITD and the tMLL AML group the expression ratios of DAPK1 to c-jun were statistically less compared with other cytogenetic/genotype categories, but there was no difference in the ratios of Meis1 to jun.
Fig.3
Fig.3. In Flt3ITD+ve AMLs Flt3-to-TAK1 pathway is involved in establishing an effector apparatus involving p52NFκB for DAPK1 repression, whereas primary Flt3ITD-ve AMLs with high DAPK1 expression lack nuclear p52NFκB
Flt3 knockdown blocked TAK1 activating phosphorylation (78% and 53% by densitometry, respectively) and led to NIK degradation, which inhibits the activation of non- canonical NFκB (panel a). Panels b and c : (b) JNK1 and TAK1 knockdown lead to diminution of c-jun phosphorylation by 100% and 80%, respectively. (c): control MV-4-11 cells (NT siRNA x2), nuclear fraction is characterized by dominant expression levels of p52NFκB and relB, but not p65NFκB. Flt3, TAK1, or NIK knock down lead to diminution of nuclear (N) p52NFκB levels (by 46%, 73%, or 76%, respectively) (panel 1). (Panel 2), JNK1 knockdown led to diminution of p52NFκB levels (by 76%). Flt3 knockdown also reduced overall p52NFκB levels (by 50%) (panel 1). D) Nuclear translocation of p52NFκB is dominant among NKFlt3ITD+ve AML with DAPK1 repression ((#2797, 49%; #2854, 100%; #2857, 70%; #2874, 100%; #2969, 35%; #2999, 50%). Note that ITD+ve sample #2999 was exposed on the same blot with ITD-ve #2993 and 2930, with no nuclear p52NFκB).
Fig.3
Fig.3. In Flt3ITD+ve AMLs Flt3-to-TAK1 pathway is involved in establishing an effector apparatus involving p52NFκB for DAPK1 repression, whereas primary Flt3ITD-ve AMLs with high DAPK1 expression lack nuclear p52NFκB
Flt3 knockdown blocked TAK1 activating phosphorylation (78% and 53% by densitometry, respectively) and led to NIK degradation, which inhibits the activation of non- canonical NFκB (panel a). Panels b and c : (b) JNK1 and TAK1 knockdown lead to diminution of c-jun phosphorylation by 100% and 80%, respectively. (c): control MV-4-11 cells (NT siRNA x2), nuclear fraction is characterized by dominant expression levels of p52NFκB and relB, but not p65NFκB. Flt3, TAK1, or NIK knock down lead to diminution of nuclear (N) p52NFκB levels (by 46%, 73%, or 76%, respectively) (panel 1). (Panel 2), JNK1 knockdown led to diminution of p52NFκB levels (by 76%). Flt3 knockdown also reduced overall p52NFκB levels (by 50%) (panel 1). D) Nuclear translocation of p52NFκB is dominant among NKFlt3ITD+ve AML with DAPK1 repression ((#2797, 49%; #2854, 100%; #2857, 70%; #2874, 100%; #2969, 35%; #2999, 50%). Note that ITD+ve sample #2999 was exposed on the same blot with ITD-ve #2993 and 2930, with no nuclear p52NFκB).
Fig. 4
Fig. 4. ChIP analyses demonstrate that the tandem CRE and NF-κB sites of the proximal DAPK1 promoter are occupied by c-jun and to greater extent, by p52NFκBκB and HDAC2
A& B) Typical PCR patterns obtained in ChIP assays with DAPK1-specific primers in MV-4-11 cells were shown. For input control reactions, one-fifth of the soluble chromatin used for the ChIP analysis was employed. In each case thirty cycles of PCR were performed. Non specific IgG, HDAC1, HDAC2, p52 NF-κB and c-jun, as well as HDAC5, HDAC11, and HDAC6 IgGs were used at 5 μg each/reaction. Real-time PCR analysis of DAPK1 promoter fragments recovered in ChIP assays performed with the indicated antibodies. Each bar represents the mean abundance of DAPK1 promoter fragments for specific antibody when compared with non-specific IgG. SE of 6 separate reactions from 2 independent experiments were shown. ‘***’ p-value, <0.001, ‘**’ p-value <0.01 and ‘*’ p-value <0.05. RNAi-mediated knockdown of p52NFκB or NIK (panels c&d) upregulated DAPK1 expression. Flt3 or JNK1 knockdown reduce phospho-c-jun and DAPK1 levels by 60%, 40%, respectively (panels c, d, e).
Fig. 4
Fig. 4. ChIP analyses demonstrate that the tandem CRE and NF-κB sites of the proximal DAPK1 promoter are occupied by c-jun and to greater extent, by p52NFκBκB and HDAC2
A& B) Typical PCR patterns obtained in ChIP assays with DAPK1-specific primers in MV-4-11 cells were shown. For input control reactions, one-fifth of the soluble chromatin used for the ChIP analysis was employed. In each case thirty cycles of PCR were performed. Non specific IgG, HDAC1, HDAC2, p52 NF-κB and c-jun, as well as HDAC5, HDAC11, and HDAC6 IgGs were used at 5 μg each/reaction. Real-time PCR analysis of DAPK1 promoter fragments recovered in ChIP assays performed with the indicated antibodies. Each bar represents the mean abundance of DAPK1 promoter fragments for specific antibody when compared with non-specific IgG. SE of 6 separate reactions from 2 independent experiments were shown. ‘***’ p-value, <0.001, ‘**’ p-value <0.01 and ‘*’ p-value <0.05. RNAi-mediated knockdown of p52NFκB or NIK (panels c&d) upregulated DAPK1 expression. Flt3 or JNK1 knockdown reduce phospho-c-jun and DAPK1 levels by 60%, 40%, respectively (panels c, d, e).
Fig.5
Fig.5. A model for the repression of DAPK1 by Flt3ITD-induced signals
Whereas c-jun elicited by Flt3ITD/JNK1 drives DAPK1 transcription. This activity is blocked by p52NFκB which recruits HDAC2/HDAC6. Derepression of DAPK1 can be achieved by use of TKI/Flt3 inhibitor, especially in combination with HDAC inhibitor (eg. SAHA/Vorinostat) and/or Bortezomib as a proteasomal inhibitor of p52NF-κB
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