Histone Deacetylase Inhibitors Activate NF-κB in Human Leukemia Cells through an ATM/NEMO-related Pathway*

Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-κB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (γ-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-κB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-κB activation (e.g. in IκBα super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-κB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-κB activation, blocked TNFα- but not HDACI-mediated NF-κB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-κB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, γ-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-κB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-κB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.

Chromatin structure and gene expression are regulated by reversible acetylation of lysine residues in histone tails, a process comprising a component of the histone code (1). Histone acetylation is regulated reciprocally by histone deacetylases (HDACs) 2 and histone acetylase transferases (2). Histone deacetylase inhibitors, a group of structurally diverse compounds, have shown encouraging activity in certain hematopoietic malignancies, including cutaneous T-cell lymphoma and acute leukemia (3,4). Numerous mechanisms have been proposed to account for HDACI-mediated lethality, including oxidative damage, up-regulation of death receptors or proapoptotic proteins (e.g. Bim), down-regulation of anti-apoptotic proteins, and more recently, DNA damage induction and/or interference with DNA repair proteins (3,5,6).
A novel pathway of NF-B activation, described recently, originates in the nucleus and is associated with DNA damage (20 -22). Double-stranded DNA breaks initiate signals that trigger SUMOylation of nuclear-localized NEMO, preventing its nuclear export (23). Concomitantly, these breaks activate ATM (ataxia-telangiectasia mutant), which phosphorylates SUMO-modified NEMO, promoting the removal of SUMO and enhancing NEMO ubiquitination (24). Ubiquitinated NEMO then translocates to the cytoplasm, where it phosphorylates IKK in cooperation with ATM and the ELKS (glutamate-, leucine-, lysine-, and serine-rich) protein leading to IB␣ phosphorylation and degradation, p65 nuclear translocation, and induction of p65-dependent prosurvival genes (20,25).
Although the contribution of HDACI-mediated acetylation to sustained p65 activation is well recognized (13,18,19), the mechanism by which HDACIs initially trigger IKK and p65 has not yet been elucidated. However, the recent description of the novel DNA damage/p65 activation pathway, as well as accumulating evidence that HDACIs trigger oxidative stress and DNA damage (5,26), raises the possibility that these processes might be related. To address this question, we have examined the roles of the components of the DNA damage response pathway, particularly ATM and NEMO, in p65 activation by HDACIs. The present findings identify the ATM/NEMO DNA damage pathway as a critical mediator of p65 activation in human leukemia cells exposed to HDACIs. They also indicate that in such cells, disruption of this pathway substantially lowers the threshold for HDACI-induced lethality.
Cell Cycle Analysis-Cell cycle analysis by flow cytometry was performed using a BD Biosciences FACScan flow cytometer and Verity WinList software (Verity Software, Topsham, ME) as described (32).
Assessment of Mitochondrial Membrane Potential (⌬ m )-Mitochondrial membrane potential loss was then determined by flow cytometry by using 40 nM DiOC 6 as we have described previously (30).
Measurement of Reactive Oxygen Species (ROS) Production-Cells were treated with either 20 M 2Ј,7Ј-dichlorodihydrofluorescein diacetate (H 2 DCFDA; Molecular Probes Eugene, OR) or 5 M dihydroethidium (Invitrogen) for 30 min at 37°C, and fluorescence was monitored by flow cytometry and analyzed with CELLQuest software as described previously (32).
Detection of Single Strand DNA Breaks by Flow Cytometry-Single-stranded DNA breaks were determined with the Apo ssDNA kit (Cell Technology, Mountain View, CA) and analyzed by flow cytometry as per the manufacturer's instructions.
Extraction of RNA and Real-time Reverse Transcriptase-Polymerase Chain Reaction-Total RNA was extracted using the RNeasy Isolation Kit (Qiagen, Valencia, CA). Real-time RT-PCR was performed in triplicate using the SensiMix One-Step SYBR Green solution (Bioline, Randolph, MA) and the corresponding QuantiTec primer assays (Qiagen). Results for the experimental gene were normalized to 18 S rRNA levels. Gene expression was compared according to the C T value.
Statistical Analysis-The significance of differences between experimental conditions was determined using either the Student's t test for unpaired observations or the analysis of variance test for multiple groups.

Induction of NF-B Activity and Regulation of ROS by
HDACIs-Previous studies have shown that HDACIs activate NF-B in diverse cell types (11,12,34). To characterize this phenomenon in greater detail, a time course analysis of NF-B activation was conducted (Fig. 1A) by ELISA (graph), electrophoretic mobility shift assay (inset, upper panel), and Western blot (p65/RelA translocation to the nuclear fraction; inset, lower panel). These studies demonstrated that exposure of human myeloid leukemia U937 cells to the pan-HDACI LBH-589 (20 nM) induced persistent NF-B activation between 4 and 16 h. Similar results were observed with other HDACIs (e.g. vorinostat, LAQ-824, and sodium butyrate; data not shown) and other human lymphoblastic (Jurkat) and promyelocytic (HL-60) leukemia cells, as well as primary acute myeloid leukemia specimens (supplemental Fig. 1).
Generation of ROS has been implicated in HDACI-mediated lethality (12,32,35). Consequently, detailed time course studies were performed to characterize the effects of HDACIs on oxidative injury more fully. Exposure of U937 cells to LBH-589 induced an early, transient increase in ROS, which returned to base-line levels by 8 h (Fig. 1B), presumably reflecting induction of the ROS scavenger Mn-SOD2 (12) (Fig. 1B, inset). Because the SOD2 gene is an NF-B target (36,37), the association among LBH-589-induced ROS generation, NF-B activation, and lethality was investigated in greater detail. To this end, ROS levels were monitored over time following LBH-589 (20 nM) treatment in empty vector-transfected U937 cells (U/EV) and in cells expressing an IB␣"super-repressor" (U/IB), which lacks the serine 32 and 36 phosphorylation sites required for proteasomal degradation (38). Although ROS levels returned to basal levels after 6 -8 h of LBH-589 exposure in control cells (U/EV; see Fig. 1B), they remained persistently elevated in U/IB cells, consistent with a lack of Mn-SOD2 induction at both the protein (Fig. 1B, inset) and mRNA levels (Fig. 1C, upper panel). In contrast, U/EV cells exposed to LBH-589 displayed robust Mn-SOD2 induction ( Fig. 1, B, inset (protein), and C, upper panel (mRNA)).
NF-B involvement in the regulation of Mn-SOD2 induction was further investigated in U937 cells treated with LBH-589 for 2 and 6 h by chromatin immunoprecipitation assay. Crosslinked DNA-protein complexes were immunoprecipitated using an anti-p65/RelA antibody followed by PCR analysis with primers recognizing SOD2 gene promoter NF-B proximal region (Ϫ1641/51) site 1, responsible for basal regulation, and NF-B enhancer distal region site 2 (Ϫ3326/34) (39,40). A timedependent increase in the association of p65/RelA with the SOD2 promoter was observed with primers corresponding to the NF-B site 2 (enhancer region), whereas no changes were observed in the region corresponding to NF-B site 1 (Fig. 1C, lower panel). As a control, the promoter region adjacent to site 1, which harbors the recognition site for AP-1, was also amplified and showed no changes. Template DNA obtained from a parallel chromatin immunoprecipitation assay using nonimmune IgG did not yield detectable PCR products. These findings may reflect the differential regulatory activity of both NF-B sites in which the proximal site (NF-B site 1) regulates basal expression, whereas the distal site (NF-B site 2) is responsible for NF-B-mediated inducible expression (39,40); they are consistent with SOD2 mRNA induction by LBH-589 in U/EV control cells (Fig. 1C). Significantly, the enhanced LBH-589 lethality observed in U/IB super-repressors was dependent upon sustained ROS production, in that co-incubation of cells with the ROS scavenger Mn-TBAP, which blocked LBH-589-induced ROS production (data not shown), abrogated the pronounced LBH-589-mediated apoptosis observed in these cells (Fig. 1D). Collectively, these results suggest that LBH-589mediated activation of NF-B plays an important functional role in protecting cells from ROS lethality through up-regulation of the NF-B-dependent antioxidant protein Mn-SOD2.
LBH-589-induced NF-B Activation Protects Cells from ROSmediated DNA Damage and Cell Death-HDACI-mediated DNA damage has been described previously, and recent findings raise the possibility that ROS generation or perturbations in the DNA repair machinery may be involved in this process (5,8,26,41). To characterize the relationship between these events in greater detail, evidence of oxidative DNA damage was monitored by confocal microscopic analysis of XRCC1, a component of the base excision repair system and early response protein recruited at the site of single strand breaks (SSBs), (42), as well as ␥-H2AX, a hallmark of DNA double strand breaks (DSBs). Increased XRCC1 fluorescence (green) was observed after 4 -8 h of exposure of cells to LBH-589, which declined by 16 h, coincident with the appearance of ␥-H2AX (red fluorescence), reflecting the transition from SSBs to DSBs ( Fig. 2A). Notably, LBH-589-mediated DNA damage was substantially diminished in cells co-incubated with Mn-TBAP, suggesting a functional link between HDACI-induced ROS generation and DNA damage ( Fig. 2A, lower panels). Furthermore, cells stably expressing siRNA directed against histone H1.2 ( Fig. 2B, inset, and supplemental Fig. 2), a key component of a chromatinderived signal linking nuclear DNA damage to mitochondrial injury and apoptosis (43), exhibited substantial resistance to apoptosis induced by LBH-589 ( Fig. 2B and supplemental Fig.  2) and other HDACIs (e.g. vorinostat; data not shown). Cells expressing the corresponding two-base mutated siRNA directed against histone H1.2 showed, as anticipated, no decrease in histone H1.2 expression and yielded results similar to those obtained with scrambled control siRNA oligonucleotidetransfected U937/siC cells (supplemental Fig. 2A).
To investigate the role of NF-B activation by LBH-589 in these events, the presence of DNA SSBs was monitored by flow cytometry using specific anti-DNA SSBs antibodies in control (U/EV) or U/IB␣ super-repressor cells exposed to LBH-589 in the presence or absence of Mn-TBAP. Shortly after the addition of LBH-589 (8 h), a modest increase in SSBs (e.g. to 116% of control values) was detected in U/EV control cells, whereas a pronounced increase (e.g. to 156% of controls) was observed in U/IB cells (Fig.  2C). Significantly, SSBs were abrogated in both control and U/IB cells by Mn-TBAP. U/IB cells monitored for the transition from DNA SSBs (XRCC1) to DNA DSBs (␥H2AX) by confocal microscopy showed that LBH-589 induced extensive DNA damage, reflected by early XRCC1 foci formation (4 h, green fluorescence) followed by a rapid transition to ␥-H2AX foci (8 h, red fluorescence; Fig. 2D). Notably, these effects were also abrogated by Mn-TBAP (Fig. 2D). Analysis of DNA damage at subsequent intervals (e.g. 16 -24 h) revealed that in the absence of NF-B activation (i.e. in U/IB cells), LBH-589-mediated DNA damage (␥-H2AX formation) was dramatically increased (Fig. 2E). In accord with the pronounced increase in cell death (Fig.  1D), LBH-589-mediated release of histone H1.2 into the cytosol was significantly increased in U/IB cells, accompanied by the pronounced conformational change and activation of the H1.2 target, the proapoptotic protein Bak (Fig. 2E). Finally, consistent with the attenuation of LBH-589-mediated cell death observed in U/IB cells exposed to Mn-TBAP (Fig. 1D), DNA damage (␥-H2AX), release of histone H1.2 into the cytosol, and activation of Bak were all significantly diminished by co-incubation of cells with Mn-TBAP (Fig. 2E). Collectively, these findings demonstrate that LBH-589-induced ROS generation plays an important functional role in triggering DNA damage, including induction of both DNA SSBs and DSBs as well as cell death. They also highlight the important cytoprotective role that NF-B activation plays in regulating LBH-589-mediated ROS generation, the resulting induction of DNA damage, and apoptosis in leukemic cells. LBH-589-mediated NF-B Activation Proceeds through a TNF␣and TRAF2-independent Process-To identify signaling pathways involved in LBH-589-mediated NF-B activation and to assess the involvement of the canonical, TNF␣-related pathway, cells were exposed to LBH-589 in the presence or absence of TNF-soluble receptor (100 ng/ml), which antagonizes TNF␣related activity (44). Whereas the TNF-soluble receptor completely blocked TNF␣-induced NF-B activity (Fig. 3A, left panel, TNF␣ ϩ SR), it had no effect on LBH-589mediated NF-B activation (Fig. 3A,  right panel). To extend these findings to other receptor-mediated stimuli (45,46), U937 cells expressing a siRNA directed against the adaptor and signaling protein TRAF2, a key intermediate in both the classical and alternative NF-B signaling pathways (47,48), were employed (Fig, 3B, inset, and supplemental Fig. 3). Consistent with the established cytoprotective role of TRAF2 in the canonical TNF␣ pathway (49), U937/siTRAF2 cells exposed to TNF␣ displayed significantly diminished NF-B activation ( HDACIs Induce NF-B Activation through an ROS-dependent Process-The preceding findings (e.g. Fig. 1B) indicate that NF-B activation played an important role in ROS regulation. On the other hand, previous studies have suggested a functional link between ROS generation and NF-B activation (45), prompting us to investigate whether HDACI-mediated ROS production might be related to the induction of NF-B. To this end, U937 cells were exposed to LHB-589 Ϯ Mn-TBAP, which in contrast to other antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate, known to Values represent percentage of cells displaying an increase in DNA SSBs. D, confocal microscopy of U937/IB cells exposed to LBH-589 (20 nM) Ϯ Mn-TBAP (400 M) for the indicated intervals. E, U/EV and U/IB cells were exposed LBH-589 (20 nM) and processed as needed (i.e. whole lysates, cytosolic S-100 fraction, and immunoprecipitation (IP)) to monitor protein levels (Western blot (WB)) of ␥-H2AX, histone H1.2, and conformationally changed Bak, respectively. For the latter, IgG was used to confirm equivalent loading and transfer; for the former, ␤-actin was employed. modulate NF-B activation directly, does not interfere with NF-B activity (50,51). Co-incubation of cells with 400 M Mn-TBAP, a concentration that blocked LBH-589-induced ROS production (data not shown), prevented HDACI-mediated NF-B activation, reflected by both p65/RelA ELISA (Fig.  4A, left panel) and p65 nuclear localization by confocal microscopy (Fig. 4A, right panels). In contrast, Mn-TBAP did not alter TNF␣-induced NF-B activation (Fig. 4B, left panel (p Ͼ 0.05)), nor did it affect LBH-589-mediated acetylation of histones H3 or H4 (Fig. 4B, right panel). Analysis of the NF-B activation cascade in lysates obtained from LBH-589-treated cells cultured in the absence of Mn-TBAP revealed increased expression of the phosphorylated forms of IKK␣/␤ within 4 to 8 h of exposure to drug (Fig. 4C). In marked contrast, Mn-TBAP-treated cells dis-played no change in phospho-IKK␣/␤ expression. The phospho-IKK target, IB␣, also exhibited pronounced phosphorylation following exposure to LBH-589 in the absence of Mn-TBAP (Fig. 4C, left panel) accompanied by modest reductions in the total levels of IB␣, presumably a consequence of ubiquitination and proteasomal degradation of the phosphorylated species (52). However, LBH-589-mediated phosphorylation of IB␣, as well as the reduction in total levels, was abrogated by Mn-TBAP (Fig. 4C). Activation of NF-B following LBH-589 exposure was also manifested by increased mRNA expression of its target, the NFKBIA (IB␣) gene (Fig.  4D). Significantly, cells cultured in the presence of Mn-TBAP exhibited no changes in NFKBIA mRNA levels (Fig. 4D, right panel). Together, these findings implicate early ROS production in the activation of the IKK/IB␣ cascade by HDACIs.
HDACIs Trigger NF-B through a NEMO-dependent Process in Association with ATM Activation-Given evidence that oxidative stress can trigger the DNA damage-associated NF-B response (16,53), the relationship between HDACI-induced DNA damage and NF-B activation was investigated. DNA damage-mediated NF-B activation is dependent upon interactions between the ATM kinase and IKK␥ (NEMO) (20,54). Phosphorylation of ATM, one of the initial kinases activated in response to DNA damage (55), was monitored in cells exposed to LBH-589. A rapid (i.e. 1 h) and sustained increase in the levels of phosphorylated ATM (pATM; Ser-1981) was observed by both confocal microscopy ( Fig. 5A upper panel) and Western blot analysis (Fig. 5A, lower panel). Concomitantly, nuclear NEMO accumulation, determined by confocal immunofluorescence, occurred within 2 to 4 h of the addition of LBH-589 (Fig. 5B, upper panel). Time course immunoprecipitation analysis of NEMO/ATM interactions revealed that although ATM association with NEMO was undetectable in untreated cells, co-immunoprecipitating ATM sharply increased within 1 h of addition of LBH-589, and although subsequent declines were noted, persisted throughout the 8-h treatment interval (Fig. 5B, lower  panel). Such findings suggest that as in the case of other genotoxic stimuli (16,53), HDACIs activate the ATM/NEMO DNA damage-related pathway. To investigate the functional role of ATM/NEMO interactions in NF-B pathway activation, U937 cells stably expressing NEMO siRNA were generated. Because only a small fraction of the total pool of NEMO is involved in the activation of the ATM/NEMO/NF-B pathway (20), two clones displaying only partial reductions in NEMO expression (Fig. 5C, inset (siN5 and  siN12), and supplemental Fig. 4B) were selected to minimize effects on non-DNA damage-related NF-B activity. Notably, siNEMO clones exhibited complete abrogation of LBH-589mediated NF-B activation compared with the responses of U937/siC control cells (Fig. 5C, left  panel, and supplemental Fig. 4B) (supplemental Fig. 4A: cells expressing a corresponding two-base mutated siRNA directed against NEMO exhibited no decrease in expression and yielded results similar to those obtained with scrambled control siRNA oligonucleotide-transfected U937/siC cells). Consistent with its established NEMO dependence (56,57), TNF␣-induced NF-B activation was attenuated in clones displaying partial reductions in NEMO levels; but in sharp contrast to the abrogation seen with HDA-CIs, these effects were very modest (Fig. 5C, right panel, and supplemental Fig. 4B). Such findings argue, albeit indirectly, against the possibility that knockdown of NEMO, the regulatory component of the IKK␣-IKK␤-IKK␥ complex (47), blocks HDACI-mediated NF-B activation solely or primarily by disabling IKK.
Consistent with the observation that knockdown of NEMO blocked HDACI-mediated NF-B activation, siNEMO clones exposed to LBH-589 exhibited pronounced attenuation of Mn-SOD2 protein and mRNA expression (Fig. 6A, left panel, inset and bar graph), accompanied by persistent LBH-589-induced ROS accumulation, compared with U937/siC control cells (Fig. 6A, right panel). NEMO knockdown cells also exhibited increased DNA damage, manifested by the early (8 h) appearance of DNA SSBs, the subsequent appearance of DNA DSBs (␥H2AX, 16 -24 h), and the release of histone H1.2 into the cytosol (Fig. 6B, left panels). Finally, siNEMO cells displayed a pronounced increase in LBH-589 lethality compared with controls (Fig. 6B, right panel). Collectively, these findings indicate that NEMO plays an important functional role in diminishing HDACI lethality by permitting NF-B activation and the resulting MnSOD2 induction, limiting ROS accumulation, and attenuating DNA damage.

NEMO SUMOylation Mutants Display Diminished NF-B Nuclear Translocation/Activation and Reduced NEMO Nuclear Accumulation and ATM Interactions in HDACI-treated
Cells-The rate-limiting step in NEMO-mediated DNA damage-related NF-B activation is the addition of SUMO residues to lysines 277 and 309, which prevents nuclear export of NEMO and permits ATM interactions (23,25). To gain insights into the role of these events in HDACI actions, we transfected cells with mutant NEMO in which SUMOylation sites lysine 277 and 309 were replaced by alanines, either as single (K277A and K309A) or double (K277/309A) mutants. Expression of SUMOylation site-mutated NEMO was confirmed by Western blot using anti-V5 tagged antibodies (Fig. 7A, inset). Single SUMOylation site mutations (i.e. K277A or K309A) resulted in partial abrogation of LBH-589-induced NF-B activation, whereas double mutant NEMO (U/K2-3) cells exhibited virtually complete NF-B inactivation (Fig. 7A, left  graph). In striking contrast, TNF␣induced NF-B activation (ELISA) was unimpaired in SUMOlation mutant cells (Fig. 7A, right panel). Analysis of p65/RelA subcellular localization by confocal immunofluorescence microscopy revealed that although administration of LBH-589 resulted in the timedependent nuclear translocation of p65/RelA in U/EV cells (i.e. 4 -16 h), this process was abrogated in double SUMOylation mutant NEMOexpressing cells (U/K2-3, Fig. 7B). Consistent with a requirement for NEMO SUMOylation in ATM interactions (20), NEMO appeared early (e.g. within 2 to 4 h) in the nucleus of control cells following treatment with LBH-589 but was undetectable in U937/K277-309A cell nuclei (Fig. 7C). Consistent with this observation, a dramatic reduction in ATM coimmunoprecipitation with NEMO was observed in mutant U937/ K277-309A (U/K2-3#1) cells exposed to LBH-589 compared with U/3.1EV control cells (Fig. 7D). Together, these findings indicate that SUMOylation plays an important functional role in NEMO nuclear translocation, ATM interactions, and NF-B activation in HDACI-treated human leukemia cells.
Investigation of the functional implications of SUMOylation in HDACI responses revealed that expression of SUMOylation mutant NEMO, which substantially attenuated or abrogated HDACI-mediated NF-B activation (Fig. 7A), markedly diminished expression of the NF-B target, Mn-SOD2 (Fig. 8A, left panel), and resulted in sustained ROS accumulation (Fig. 8A, right panel) analogous to the effects of siRNA NEMO knockdown (Fig. 6A). Whereas exposure of U/EV control cells to LBH-589 induced a progressive increase in XRCC1 (green fluorescence), reflecting DNA SSBs, over the 4 -8 h exposure interval, double mutant U/K277-309A cells displayed a very early (4 h) transition from DNA SSBs (XRCC1) to DNA DSBs (␥H2AX, red fluorescence; Fig. 8B) and markedly enhanced LBH-589-induced apoptosis  MARCH 26, 2010 • VOLUME 285 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 10071 (Fig. 8C). In contrast, expression of SUMOylation site mutated NEMO did not modify TNF␣-dependent NF-B activation or lethality (data not shown). Together, these findings indicate that SUMOylation plays an important functional role in NEMO nuclear translocation, ATM interactions, and NF-B activation in human leukemia cells exposed to HDACIs. They also provide evidence for a functional role for NEMO in the regulation of ROS generation and DNA damage responses in human leukemia cells exposed to HDACIs.

ATM/NEMO-dependent NF-B Activation by HDAC Inhibitors
ATM Contributes Functionally to HDACI-mediated NF-B Activation and Attenuation of Lethality-The preceding results strongly implicated the DNA damage/NEMO pathway in HDACI-mediated NF-B activation. To explore this pathway further, U937 cells stably expressing an siRNA directed against ATM (28) were generated (Fig. 9A, left panel, Fig. 6B) and protein levels (Fig. 9B, right panel) and persistent ROS accumulation (Fig. 9B, left panel). U/siATM cells exposed to LBH-589 also displayed early (8 h) evidence of enhanced DNA damage (e.g. DNA SSBs and DSBs; data not shown) compared with U/EV cells, as well as marked increases in cytosolic translocation of histone H1.2, Bak conformational change (Fig. 9C, left panel), and LBH-589induced apoptosis (Fig. 9C; p Ͻ 0.002). In accord with these findings, U/EV cells displayed a robust increase in the NF-B target gene NFKB1A (IkBa) mRNA in response to LBH-589, but this agent failed to increase IB␣ mRNA levels in either the U/siATM clone or U937/IB-SR cells used as controls (supplemental Fig. 6C, U/IB-SR). Taken together, these results highlight a critical functional role for the oxidative DNA damage/ ATM/NEMO pathway in initial NF-B activation, as well as attenuation of ROS-mediated DNA damage and lethality by HDACIs in human leukemia cells.

DISCUSSION
Inappropriate NF-B activation represents a hallmark of numerous malignancies (59), including those of hematopoietic origin, particularly multiple myeloma and leukemia (60). Consequently, components of the IKK/NF-B pathway have become the focus of interest as potential therapeutic targets (15,46). Members of the NF-B family are sequestered in inactive forms in the cytoplasm but are activated by diverse external stimuli (16,61). Three "outside-in" pathways of NF-B activation have been identified including the classical or canonical pathway, e.g. by TNF␣; the alternative or non-canonical pathway, e.g. by CD-40 ligand, B-cell-activating factor, or lympho-toxin-␤; and the atypical pathway, e.g. by UV light (16). In contrast, the recently described unorthodox DNA damage pathway operates through an inside-out mechanism in which genotoxic or oxidative stress signals originating in the nucleus activate NF-B (20,21). This involves nuclear export of two proteins, ATM and NEMO, which then activate cytoplasmic IKK complexes, leading to nuclear translocation of NF-B and transcription of cytoprotective genes; this allows cells to survive otherwise lethal insults, e.g. DNA DSBs (20,25). The present findings indicate that in addition to their cytoprotective actions in the face of genotoxic stress, components of the DNA damage pathway play important functional roles in the initial activation of NF-B by HDACIs.
In addition to acetylating histones, HDACIs acetylate diverse proteins including transcription factors E2F, YY-1, and NF-B (62). RelA acetylation plays an important role in regulating the degree and duration of NF-B activation (13,63) and is believed to play an important role in the sustained induction of this pathway by HDACIs (64,65). However, these events do not address the issue of how HDACIs initially trigger activation of NF-B. The present findings indicate that in certain human leukemia cells, initial RelA activation involves the ROSdependent induction of DNA damage and proceeds through the atypical, NEMO/ATM-dependent NF-B activation pathway. In support of this notion, the Mn-SOD2 mimetic,Mn-TBAP,blockedHDACImediated ROS generation, attenuated ROS-mediated DNA damage, and blocked activation of NF-B, reflected by diminished RelA nuclear transport and DNA binding activity. Although it is known that HDACI-mediated oxidative injury contributes to the lethality (32,35) and potentially the selectivity of these compounds (66), the present findings demonstrate that ROS generation also plays a central role in triggering the NF-B cascade by HDACIs.
The present observations also provide a connection between ROS-mediated DNA damage and the ATM/NEMO-dependent induction of NF-B by HDACIs. DNA DSBs activate ATM, which in turn phosphorylates multiple proteins involved in DNA damage/repair and checkpoint responses (67). Recently, NEMO has been identified as a novel ATM substrate linking DNA damage to NF-B stress responses through a complex and dynamic process (20,23,24). Genotoxic insults causing DNA DSBs induce SUMOylation of NEMO resident in the nucleus, blocking its export. Concomitantly, activated ATM allows removal of SUMO residues from NEMO, permitting NEMO ubiquitination (20,68). The ATM-ubiquitinated NEMO complex then migrates to the cytoplasm, where it activates the IKK complex, leading to RelA nuclear transport and culminating in the induction of NF-B-responsive genes. The identification of NEMO as an ATM substrate therefore provides a link between DNA damage responses and the cytoprotective NF-B pathway through a nuclear-to-cytoplasmic signaling cascade (20). Consequently, ATM, in addition to its nuclear activity (67), may exert important cytoplasmic functions. A corollary of his concept is that under some circumstances, nuclear rather than extracellular signals may initiate the NF-B activation cascade. The bulk of evidence indicates that in contrast to the cytokine TNF␣, HDACIs act primarily through the latter pathway to induce NF-B-dependent responses. This conclusion is based on evidence that knockdown of TRAF2, an important mediator of TNF␣-related IKK activation (47,48), markedly attenuated TNF␣-related NF-B signaling but had virtually no effect on that initiated by LBH-589. Although the dependence of TNF␣-induced NF-B activation on NEMO is well established (56,57), partial NEMO knockdown only modestly diminished NF-B activation by this cytokine, but it essentially abrogated activation by HDACIs. Furthermore, knockdown of ATM or transfection of cells with SUMOylationdefective NEMO mutant protein ablated HDACI-mediated NF-B activation and transcription of the NF-B target genes Mn-SOD2 and IB␣ but minimally affected TNF␣ responses. The finding that the loss of the NEMO SUMOylation signal accompanied by diminished association of NEMO with ATM specifically impaired HDACI-mediated NF-B activation argues that in the case of HDACIs, disruption of the atypical DNA damage pathway, rather than dysregulation of the IKK complex, is primarily responsible for attenuated NF-B responses.
ROS generation has been implicated in HDACI lethality in multiple earlier reports (32,35,66). Notably, in the present study, HDACI-induced ROS was clearly linked to the early appearance of XRCC1 complexes, indicating oxidative base damage, base excision repair, and DNA single strand breaks (42). Furthermore, HDACIs such as trichostatin A, SAHA, and MS-275 activate NF-B (11,12,69), an event that attenuates lethality by promoting transcription of antiapoptotic target genes including XIAP, Bcl-xL, and Mn-SOD2 (12,70,71). In this context, the NF-B-dependent induction of Mn-SOD2 attenuates TNF␣ (72) and HDACI lethality (11,12). It is therefore significant that genetic disruption of the atypical DNA damage activation pathway (e.g. by ATM/NEMO knockdown or mutation) mimicked pharmacologic (e.g. Mn-TBAP) or genetic (e.g. IB␣ super-repressor) interruption of NF-B cascade in blocking Mn-SOD2 induction, thereby promoting sustained ROS generation and DNA damage. Such findings argue that the initial induction of ROS by HDACIs and the resulting DNA damage are critical for NF-B activation, which, through induction of Mn-SOD2 and ROS elimination, limits further genotoxic stress and lethality. A corollary of this model is that interruption of HDACI-mediated NF-B activation and potentiation of lethality may occur at two separate levels: (a) interference with IKK activation and/or RelA acetylation (11,12); and (b) disruption of the ATM/NEMO DNA damage-related pathway (54, 60). engagement of the DNA repair machinery. The latter involves ATM activation and ATM-mediated phosphorylation of NEMO (20,67), which allows removal of SUMO residues promoting NEMO ubiquitination and ATM complex formation (20). NEMO-ATM complexes are then able to exit the nucleus and trigger IKK activation in the cytoplasm (20,23), resulting in IB␣ phosphorylation and proteasomal degradation (77). This leads in turn to the release and nuclear translocation of p65/ RelA and transcriptional activation of multiple NF-B-dependent genes, including the ROS scavenger Mn-SOD2 (78,79), which eliminates ROS and limits further DNA damage and cell death. Such a model may have implications for attempts to enhance the antileukemic activity of HDACIs. For example, it has previously been shown that in such cells, interference with IKK activation (e.g. by IKK inhibitors), by blocking NF-B activation, dramatically lowers the threshold for HDACI-mediated apoptosis (12). Interestingly, ATM and NEMO have recently been implicated in the constitutive NF-B activation characteristic of certain malignant hematopoietic cells (e.g. acute myeloid leukemia and myelodysplastic syndrome cells) (58,60). Thus, interference with ATM (e.g. by ATM inhibitors) (60) or other components of the atypical DNA damage-related NEMO pathway, by blocking NF-B activation at the nuclear level, may enhance HDACI activity in these disorders. Efforts to test this hypothesis are currently under way. Jurkat lymphoblastic leukemia and HL-60 promyelocytic leukemia cells were exposed to LBH-589 (20 nM) for the indicated intervals, after which nuclear extracts were prepared to monitor NF-κB activity using an ELISA-based procedure as described in Methods Figure 3. A, U937/siC and U937 cells stably expressing pSilencer with a 2-nucleotide mutated negative control sequence siRNA directed against TRAF2 (siTN, clones #6 and #16), and B, shC (stably expressing a negative scrambled control shRNA oligonucleotide) and shTRAF2 (expressing an alternative oligonucleotide directed against TRAF2, clones shT-5 and shT-8) were exposed to TNFα (10 ng/ml) for 2h or 24h and analyzed for NF-κB activity (ELISA) or cell death (annexin V/PI-positive cells) by flow cytometry, respectively. Insets: Western blot analysis of TRAF2 expression were performed by using whole cell lysates; each lane was loaded with 30 µg of protein; blots were stripped and re-probed with an antibody directed against actin to ensure equivalent loading and transfer. C, shC and shTRAF2 cells were treated with LBH-589 (20nM, 8h) after which NF-κB activity was monitored by ELISA Values represent Mean ± S.E.M. *, P<0.01. For all experiments, results represent the means ± S.D for three separate experiments performed in triplicate. Supplemental Figure 4. A, U937/siC and U937 cells stably expressing a 2-base mutated siNEMO negative control sequence directed against NEMO (siNN; clones #4 and #8), were exposed to either LBH-589 (20nM, 24h) or TNFa (10ng/ml, 2h) and analyzed for cell death (annexin V/PI-positive cells) by flow cytometry or NF-κB activity (ELISA), respectively. B, U937/shC (stably expressing a scrambled negative control sequence shRNA oligonucleotide) and U937/shN (expressing an alternative oligonucleotide directed against NEMO, clones #9 and #10) were exposed to either LBH-589 (20nM) or TNFα (10ng/ml) for 8h or 2 h, respectively and analyzed for NF-κB activity (ELISA); *, P<0.01. Western blot analyses were performed by using whole cell lysates; each lane was loaded with 30 µg of protein; blots were stripped and re-probed with an antibody directed against actin to ensure equivalent loading and transfer. For all experiments, results represent the means ± S.D. for three separate experiments performed in triplicate. Supplemental Figure 5. A, U937/siC and U937 cells stably expressing a 2-base mutated siNEMO negative control sequence directed against ATM (siAN; clones #44 and #46), and B, U937/shC cells (stably expressing a negative scrambled control shRNA oligonucleotide) and U937/shATM cells (expressing an alternative oligonucleotide directed against ATM, clones # 13 and # 17) were exposed to either LBH-589 (20nM) or TNFa (10ng/ml) for 8h or 2 h, respectively and analyzed for NF-κB activity (ELISA); *, P<0.01. Western blot analyses were performed using whole cell lysates; each lane was loaded with 30 µg of protein; blots were stripped and re-probed with an antibody directed against actin to ensure equivalent loading and transfer. For all experiments, results represent the means ± S.D. for three separate experiments performed in triplicate.