AbstractAbstractHistone deacetylase inhibitors (HDACi) have recently emerged as promising anticancer

agents. However, the mechanisms by which HDAC inhibitors arrest proliferation and induce

apoptosis in tumor cells is far from clear. Activation of the stress MAP kinases (MAPK) and

induction of DNA damage by different HDACi have been reported, however, the potential

role of the p38-MAPK in the antitumor activity of these drugs has not been described. P38 is

known to be activated independently or downstream of DNA damage.

The purpose of this study was to elucidate the mechanisms by which the hydroxamic acid

HDACi vorinostat (Zolinza®) triggers apoptosis in acute myeloid leukemia (AML) cells and to

assess the role of p38-MAPK.

We have shown that DNA damage as well as activation of p38 occurs relatively early in AML

cell lines after treatment with vorinostat. Using comet assays, we detected direct evidence

of early DNA damage, and western blotting revealed induction of gH2AX as well as the DNA

damage response proteins ATM and Chk2. Cell cycle analysis revealed that, before inducing

apoptosis, vorinostat causes cells to exit G1 and accumulate in the G2-M phase of the cell

cycle. Notably, downregulation of p38 by shRNA or inhibition of p38/ activity by the

pharmacological inhibitor SB203580 significantly decreased both G2-M accumulation and

apoptosis induced by vorinostat, indicating a pro-apoptotic p38 function.

Interestingly, several other HDACi tested induced p38 activation but, depending on the

HDACi, this activation was found to be either pro- or anti-apoptotic. Like vorinostat, the

short-chain fatty acid sodium butyrate requires p38 for induction of apoptosis, while

inhibition of p38 has no effect on apoptosis induced by LBH589 (panobinostat, another

hydroxamic acid). Moreover, inhibition of p38 stimulates apoptosis induced by the benzimide

MGCD0103.

In conclusion, we have shown that vorinostat-induced apoptosis in AML cells is preceded by

generation of DNA damage and accumulation of cells in the G2-M phase of the cell cycle.

Further, G2-M arrest and apoptosis induction (but not DNA damage) by vorinostat requires

activation of the p38-MAPK, which is not the case for all HDACi. Therefore, a better

understanding of the role of p38-MAPK in the action of specific HDACi may help in the

development of rational combination therapies including these agents.

INTRODUCTIONINTRODUCTIONSeveral HDACis, including suberoylanilide hydroxamic acid (Vorinostat, Zolinza™), have been tested in

early clinical trials in leukemia patients, with some encouraging results [1-5]. From in vitro studies, it is well

established that HDACis induce both growth arrest, differentiation and activation of apoptotic pathways,

and interestingly, normal cells have been reported to be significantly more resistant to these effects than

tumor cells [6-9].

Acetylation of histones plays a crucial role in regulation of transcription and for that reason, the

mechanism of anti-tumor action of HDACi was initially suggested to be linked mainly to the re-activation of

tumor suppressor genes that may be silenced in tumor cells through epigenetic mechanism. While HDACi

can indeed activate transcription by increasing acetylation of both histones and transcription factors [10],

additional mechanisms of action clearly contribute to their biological effect. A recent study quantified

global acetylation changes in response to vorinostat, and identified 3600 lysine acetylation sites on 1750

proteins, mainly part of large macromolecular complexes involved in various cellular processes, including

chromatin remodeling, cell cycle progression, nuclear transport and others [11]. HDACi thus affect a

variety of pathways and the full mechanism by which they exert their anti-tumor effects is still far from

defined.

In this study, we investigated the role of DNA damage and p38 activation in vorinostat-induced

apoptosis in AML cells. We found evidence of early DNA damage as well as p38 activation. Following DNA

damage, cells accumulate in the G2-M phase of the cell cycle, and subsequently undergo apoptosis.

Notably, p38 activation is required for accumulation of cells in G2-M as well as induction of apoptosis in

the presence of vorinostat. However, when comparing a variety of other HDACis, we found that p38 plays

very different roles in the induction of apoptosis.

MATERIALS AND METHODSMATERIALS AND METHODSReagents : Suberoylanilide Hydroxamic Acid (vorinostat) was obtained from Merck Frost. SB203580

were purchased from Calbiochem.

Cell lines. Acute promyelocytic leukemia NB4 cells and acute myelocytic leukemia U937 cells were

maintained in RPMI supplemented with 10% fetal bovine serum at 37 0C with 5% CO2.

Propidium iodide staining. NB4 cells were seeded at 2 x 105 cells/mL in 24-well plates and treated

as described. Cells were washed in buffer (PBS/ 5% FBS/ 0.01 M NaN3) at 4ºC, pelleted, and

resuspended in 0.5 ml of hypotonic solution containing 50 µg/ml propidium iodide, 0.1% sodium citrate,

and 0.1% Triton X-100. Fluorescence was detected using a FACS Calibur flow cytometer, and analysis

carried out using CellQUEST software (Becton Dickinson, San Jose, CA, USA). Cells undergoing DNA

fragmentation and apoptosis were defined as events with propidium iodide fluorescence weaker than

the G0-G1 cell cycle peak.

Caspase 3/7 assay. Purchased from Promega and used according to specifications.

Comet assay : Comet Assay was performed under alkaline conditions. Briefly, isolated tumor cells

(5,000 per slide in duplicates) were imbedded in agarose at a final concentration of 1:10 and layered on

glass slides. Slides were equilibrated to 4°C and submerged in cold lysis buffer overnight. Slides were

then incubated for 1 hour at room temperature in alkaline electrophoresis solution (pH >13). Comet

tails were generated by a 40-minute electrophoresis at 20 V, 4°C. Slides were then washed in

neutralizing buffer and fixed with ice cold methanol. Slides were stained for 5 minutes with 1 mg/ml

ethidium bromide. Comet Tail Moment (TM) was calculated using CometScore 1.5 (TriTek, Sumerduck,

VA, USA) for 100 randomly selected nuclei in duplicate experiments. TM correlates with the level DNA

breaks and alkali-labile sites.

Western blotting. Whole cell extracts were prepared in RIPA lysis buffer, run on SDS-polyacrylamide

gels, and blotted onto nitrocellulose membranes. Membranes were blocked and subsequently probed

with rabbit-anti-H2AX (Upstate, # 07-164, 1:1000), rabbit-anti-phospho-p38 (Cell signaling, #9211,

1:1000), rabbit-anti-phospho(thr68)-Chk2 (Cell Signaling, #26615, 1:1000). Membranes were probed

with donkey-anti-rabbit or sheep-anti-mouse (Amersham, 1:2000), followed by visualization by

enhanced chemiluminescence (ECL, Amersham).

Protein detection by flow cytometry : NB4 cells were seeded at 2 x 105 cells/mL in 24-well plates

and treated as described. Cells were washed in PBS and fixed overnight in 0.5% paraformaldehyde.

Cells were permeabilized in PBS with 0.1% Triton X-100 for 15 minutes and stained with anti-phospho-

ATM(thr1981) (Rockland, # 200-302-400) for 1 hour followed by washing, incubation with CY5-

conjugated anti-mouse antibody (Upstate), washing, and resuspension in PBS. Fluorescence was

detected using a FACS Calibur flow cytometer, and analysis carried out using CellQUEST software.

Stable cell lines : NB4 cells (5x106 cells/transfection) were rinsed in serum-free OPTI-MEM (Invitrogen)

and transfected by electroporation with 10 μg of the pSUPER RETRO vector (Oligoengine Inc.)

containing scrambled or p38 shRNA. Transfected cells were replenished with media and grown for 5

days. Then, puromycine was added to media at a concentration of 2 μg/ml and cells were maintained in

culture for 2 weeks. ShRNA sequences are available upon request.

Vorinostat resistant cell lines: U937 cells were maintained in 0.5 μM vorinostat until they started

growing and reached confluency. Cells were re-plated at 0.3x106 cells/ml and exposed to 1 μM

vorinostat. The procedure was repeated until the concentration reached 4 μM and resistance was

confirmed by PI staining as described above.

Acknowledgements : This work was supported by grants to WHM from the Canadian Institute of Health Research and Merck.

REFERENCES1.Kuendgen, A., et al., The histone deacetylase (HDAC) inhibitor valproic acid as monotherapy or in combination with all-trans retinoic acid in patients with acute myeloid leukemia. Cancer, 2006. 106(1): p. 112-9.2.Giles, F., et al., A phase I study of intravenous LBH589, a novel cinnamic hydroxamic acid analogue histone deacetylase inhibitor, in patients with refractory hematologic malignancies. Clin Cancer Res, 2006. 12(15): p. 4628-35.3.Garcia-Manero, G., et al., Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes. Blood, 2008. 111(3): p. 1060-6.4.Gojo, I., et al., Phase 1 and pharmacologic study of MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias. Blood, 2007. 109(7): p. 2781-90.5.Petrie, K., N. Prodromou, and A. Zelent, Histone Deacetylase Inhibitors in APL and Beyond, in Acute Promyelocytic Leukemia. 2007. p. 157-203.6.Xu, W.S., R.B. Parmigiani, and P.A. Marks, Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene, 2007. 26(37): p. 5541-52.7.Bolden, J.E., M.J. Peart, and R.W. Johnstone, Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov, 2006. 5(9): p. 769-84.8.Minucci, S. and P.G. Pelicci, Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer, 2006. 6(1): p. 38-51.9.Glozak, M.A. and E. Seto, Histone deacetylases and cancer. Oncogene, 2007. 26(37): p. 5420-32.10.Pandolfi, P.P., Transcription therapy for cancer. Oncogene, 2001. 20(24): p. 3116-27.11.Choudhary, C., et al., Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science, 2009. 325(5942): p. 834-40.

Figure 3: The MAPK p38 activity is induced by vorinostat and promotes G2-M accumulation and apoptosis. A. Western blot analysis of NB4 cells show early and sustained phosphorylation of p38. B. Inhibition of p38 activity impede accumulation of cells in G2-M. Left panel: NB4 cells were pretreated or not for 1 hour with 10 µM SB203580, an inhibitor of p38, then treated with 1 µM vorinostat for the indicated times and submitted to PI staining followed by flow cytometry. C. Inhibition of p38 activity prevents vorinostat induced cell death in AML cell lines. Left panel: NB4 and U937 cells were pretreated or not for 1 hour with 10 µM SB203580, an inhibitor of p38β and p38α, then treated with 1 or 1.5 µM vorinostat for 48h and submitted to PI stain. Percentage of cells with fragmented DNA was evaluated by flow cytometry. Right panel: NB4 and U937 cells were treated for 24h before caspase 3/7 activity was assessed. D. P38 is not induced by vorinostat in U937 cells resistant to vorinostat. Left panel: Western blot analysis of U937-vorinostat resistant (VR) cells and U937 parental cells treated or not with 2 µM vorinostat for 16h. Right panel: PI staining of U937 parental and VR cells

2 µM vorinostat - + - +

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Figure 4: P38-MAPK can promote or suppress HDAC inhibitor-induced apoptosis. A. Left panel: Western blot analysis of U937 cells following 24h exposure to vorinostat, LBH589 (panobinostat), MGCD0103, sodium butyrate (NaB) or valproic acid (VPA) as indicated. Right panel : PI staining of U937 cell samples showing similar levels of sub-G0 cells after exposure to the different HDAC inhibitors. B. HDAC inhibitors show different dependency on p38 for induction of apoptosis. NB4 cells were pretreated with 10 µM SB203580 for 1 h and then with the HDAC inhibitor indicated for 24h or 48 h and submitted to PI staining followed by flow cytometry for quantification of sub-G0 cells. C. P38 downregulation selectively suppresses apoptosis induced by Vorinostat. Left panel : NB4 cells stably transfected with a vector expressing shRNA against p38α or a scrambled sequence, were treated with 1 vorinostat or 15 nM panobinostat for 24h and 48h, and submitted to PI staining as in B. Right panel : Western blot analysis using an antibody specific to p38α confirmed downregulation of p38α protein level in NB4 cells. Antibody specific to β-actin was used as a loading control.

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Pro-apoptotic Pro-apoptotic vsvs. pro-survival functions of HDAC inhibitor-induced p38-MAPK in AML cells. pro-survival functions of HDAC inhibitor-induced p38-MAPK in AML cells..

Filippa Pettersson, Daphné Dupéré-Richer, Luca Petruccelli, and Wilson H. Miller, Jr.Filippa Pettersson, Daphné Dupéré-Richer, Luca Petruccelli, and Wilson H. Miller, Jr.

Lady Davis Institute and the Segal Cancer Centre of the SMBD Jewish General Hospital, McGill University, Montreal, Canada.Lady Davis Institute and the Segal Cancer Centre of the SMBD Jewish General Hospital, McGill University, Montreal, Canada.

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Figure 1 : Vorinostat induces early DNA damage in AML cell lines. A. Left panel: alkaline comet assays show induction of DNA damage after 3h exposure to 1.5 µM vorinostat in NB4 cells. Graphs show quantification of tail moment. Pictures show examples of comets obtained from non-treated cells (ctl) and cells treated for 12h with vorinostat. Right panel: Blast cells isolated from 2 different AML patients and treated 12h ex-vivo with 2 and 3 µM vorinostat were submitted to comet assay. Graphs illustrate average of tail moment. B. Vorinostat induces early oxidative DNA damage in NB4. Quantification of 8-oxoguanine detected by flow cytometry in NB4 exposed to 1.5 µM vorinostat for the indicated times. C. Vorinostat induces double strand break in NB4 as shown by induction of H2AX. Western blot analysis using antibody specific to H2AX, H2AX, H2A and -actin as a loading control. D. Vorinostat induces the DNA damage checkpoint proteins ATM and Chk2. Left panel: NB4 cells exposed to 1.5 µM vorinostat for 6h were stained with antibodies specific to phospho-ATM and ATM, and analysed by flow cytometry. Exposure of NB4 cells to UV light for 1h was performed as a positive control for ATM induction. Right panel: western blot analyses were performed using antibodies specific to phospho-chk2, chk2 and -actin as a loading control in NB4 cells treated 1.5 μM for the indicated times.

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CONCLUSIONCONCLUSION

Vorinostat induces DNA damage and cell cycle arrest in the G2-M phase in AML cells, along with activation of the DNA damage associated proteins ATM Chk2, and H2AX

Vorinostat and a variety of HDACi (NaB, VPA, Panobinostat , MGCD0103) induce the MAPK p38.

G2-M arrest and apoptosis induced by vorinostat depend on p38 activation.

vorinostat

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Apoptosis

.

?

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