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Expression of leukemia associated fusion proteins increases sensitivity to histone deacetylase inhibitor induced DNA damage and apoptosis. Luca A Petruccelli1, Filippa Pettersson1, Sonia V del Rincón1, Cynthia Guilbert1, Jonathan D Licht2, Wilson H Miller Jr.1 1Lady Davis Institute for Medical Research, Segal Cancer Center, Jewish General Hospital, McGill University, Montréal, Canada;2Division of Hematology/Oncology, Northwestern University, Robert H. Lurie Comprehensive Cancer Center, Chicago, IL. Running Title: AML fusion proteins enhance DNA damage by HDAC inhibitors Keywords:

1. Acute Myeloid Leukemia 2. Leukemia Associated Fusion Proteins 3. Histone Deacetylase Inhibitors 4. DNA damage 5. Apoptosis

Funding:

• L.A Petruccelli is supported by a student fellowship from the McGill Systems Biology Training Program.

• J.D Licht is funded by the Samuel Waxman Cancer Research Foundation. • W.H Miller Jr. is funded by a grant MOP-12863 from the Canadian

Institute of Health Research and the Samuel Waxman Cancer Research Foundation.

Corresponding Author: Dr. Wilson H Miller Jr. Lady Davis Institute 3755 Côte Ste-Catherine Rd Montréal, Québec H3T 1E2 Tel: 514-340-8222 ext.4365 Fax: 514-340-7574 Email: [email protected] Conflict of Interest Statement: The authors have no conflicts of interest to disclose. Word Counts Abstract: 236 words Manuscript: 5653 words References: 50 Figures: 5 (print all black and white) Supplemental Figures: 7 (print all black and white)

on September 17, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 27, 2013; DOI: 10.1158/1535-7163.MCT-12-1039

http://mct.aacrjournals.org/

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Abstract

Histone deacetylase inhibitors (HDI) show activity in a broad range of

hematological and solid malignancies, yet the percentage of patients in any given

malignancy who experience a meaningful clinical response remains small. In this

study, we sought to investigate HDI efficacy in acute myeloid leukemia (AML)

cells expressing leukemia associated fusion proteins (LAFPs). HDI have been

shown to induce apoptosis in part through accumulation of DNA damage and

inhibition of DNA repair. LAFPs have been correlated with a DNA repair deficient

phenotype, which may make them more sensitive to HDI-induced DNA damage.

We found expression of the LAFPs PLZF-RAR�, PML-RAR� and RUNX1-ETO

(AML1-ETO) increased sensitivity to DNA damage and apoptosis induced by the

HDI vorinostat. The increase in apoptosis correlated with an enhanced down-

regulation of the pro-survival protein BCL2. Vorinostat also induced expression of

the cell cycle regulators p19INK4D and p21WAF1, and triggered a G2-M cell cycle

arrest to a greater extent in LAFP expressing cells. The combination of LAFP and

vorinostat further led to a greater down-regulation of several base excision repair

(BER) enzymes. These BER genes represent biomarker candidates for response

to HDI-induced DNA damage. Notably, repair of vorinostat-induced DNA double

strand breaks was found to be impaired in PLZF-RAR� expressing cells,

suggesting a mechanism by which LAFP expression and HDI treatment

cooperate to cause an accumulation of damaged DNA. These data support the

continued study of HDI based treatment regimens in LAFP-positive AMLs.




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Introduction

The pathogenesis of acute myeloid leukemia (AML) involves activating

mutation(s) resulting in a proliferative and/or survival advantage and loss of

function mutation(s) resulting in a differentiation arrest (1). Leukemia associated

fusion proteins (LAFPs), such as PLZF-RAR�, PML-RAR� and RUNX1-ETO

(referred to as AML1-ETO henceforth), are generated from chromosomal

translocations and are involved in blocking differentiation, thus directly driving the

malignant phenotype. They do so by recruiting histone deacetylase (HDAC)

containing co-repressor complexes to the promoters of differentiation genes,

resulting in their transcriptional silencing (2, 3). HDACs remove acetyl groups

from the lysine residues of histones and other proteins. In the case of histones,

deacetylation results in the re-organization of chromatin into a closed

conformation that obstructs transcription (4). Targeting the HDAC component of

these co-repressor complexes with small molecule HDAC inhibitors (HDIs) has

proven to be an effective strategy in re-sensitizing leukemic cells to differentiating

stimuli (5, 6). Indeed, preclinical and clinical studies have found HDIs to display

activity in a broad range of hematologic and solid malignancies, yet the portion of

patients with a given malignancy that experiences a meaningful therapeutic

response remains small (4). Thus, HDIs have presently only gained FDA

approval for the treatment of cutaneous T-cell lymphoma (7), and it is clear that

our understanding of the mechanisms of response to HDIs is still lacking.




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In addition to their inhibitory effects on differentiation, multiple groups have

reported that expression of LAFPs results in a DNA repair deficient phenotype (8,

9). Expression of PML-RAR�, PLZF-RAR� or AML1-ETO has been shown to

down-regulate genes implicated in base excision repair (BER), resulting in

increased DNA damage (8). Thus, LAFPs may also contribute to the leukemic

phenotype by promoting genetic instability and the accumulation of DNA

mutations. Importantly, DNA repair capacity has been linked to response to DNA

damaging agents (10). Therefore, LAFP expressing AMLs may represent a group

that will respond favorably to DNA damaging agents. Our lab and other groups

have shown that one mechanism by which HDIs arrest growth and induce

apoptosis in malignant cells is through the accumulation of DNA damage, which

can occur through induction of reactive oxygen species (ROS) (11), down-

regulation of DNA repair proteins (12-14), replication fork stalling (15) and

impairment of DNA repair protein function (12, 16). Interestingly, various HDIs

have shown promising activity in LAFP expressing AMLs. For example,

expression of AML1-ETO in U937 cells increased sensitivity to apoptosis induced

by the HDI ITF2357 (17). Moreover, in a Phase II clinical trial, romidepsin

showed greater efficacy in AML patients expressing AML1-ETO and other core

binding factors (18). Despite these observations, the role of DNA damage

mechanisms remains to be ascertained.

The aim of this study was therefore to investigate the effect of LAFP

expression on sensitivity to HDI-induced DNA damage and apoptosis. We have

evaluated the impact of HDI treatment on cell lines with inducible expression of




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PLZF-RAR�, PML-RAR� or AML1-ETO. LAFP expression was found to increase

sensitivity to HDI-induced DNA damage and apoptosis. LAFP expression and

vorinostat treatment alone or in combination were found to significantly down-

regulate BER genes and correlated with impaired DNA repair. These data

demonstrate that the DNA damage capabilities of HDI are greater in LAFP

expressing cells, and suggest possible biomarkers for response to HDI-induced

DNA damage.

Materials and Methods

Reagents and cell lines

Vorinostat was obtained courtesy of Merck & Co. LBH58 and MGCD0103 were

obtained courtesy of Novartis AG and MethylGene. Sodium butyrate (NaB) and

trichostatin A (TSA) were purchased from Sigma. Romidepsin was purchased

from Selleckchem. Chemical structures of HDIs are shown in Figure 6. NB4,

Kasumi-1 and U937 cells were obtained from ATCC and cultured in RPMI 1640

(Wisent) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2.

U937 is a p53 functionally null histiocytic lymphoma cell line (19). The

PLZFRAR�3 cell line is based on the U937T auto-regulatory tetracycline-off

system. Cells were cultured as previously described (20) and PLZF-RAR�

expression was induced by washing cells with phosphate-buffered saline (PBS)

and culturing without tetracycline (Sigma). B412, PR9 and U937-A/E cells

expressing PLZF-RAR�, PML-RAR� or AML1-ETO respectively, under the




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control of a Zn-inducible promoter, were obtained from Dr. Martin Ruthardt and

Dr. Myriam Alcalay (8, 21). SN4 cells (U937 cells stably transfected with empty

pSG-MtNeo) were used as a control. B412, PR9, U937-A/E and SN4 cells were

treated with 100�M ZnSO4 (Sigma) to induce expression of their respective

fusion protein. Authentication of NB4, Kasumi-1, PLZFRAR�3, B412, PR9 and

U937-A/E was conducted by assaying for expression of their respective

endogenous or inducible LAFPs. Authentication of U937 and SN4 has not been

carried out within the last 6 months. NU7026 (Tocris), an ATP-competitive small

molecule inhibitor of DNA-PKcs (22) was dissolved in DMSO. N-acetyl-L-cysteine

(NAC) was purchased from Sigma and dissolved in DMEM (Wisent).

Doxorubicin, etoposide and cisplatin were purchased from Sigma. Z-VAD-FMK

was purchased from Promega. Chemical structures for Z-VAD-FMK, NAC,

doxorubicin, etoposide, cisplatin and NU7026 are shown in Figure 7. Irradiation

of cells was done at room temperature using a Theratron T-780 Cobalt Unit

located in the Department of Radiation Oncology at the Jewish General Hospital

(Montréal, Québec, Canada). Radiation was delivered at a rate of 0.66 Gy/min.

The cells were returned to an incubator after irradiation and maintained at 37°C

with 5% CO2 until further analysis.

Growth assay

PLZFRAR�3 cells were seeded at 5x104 cells/mL in the presence or absence of

tetracycline. Viable cells were counted using trypan blue exclusion at Day 2 and

Day 5.




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Propidium iodide staining

Propidium iodide staining was done as previously described (11).

Caspase activity assay

Caspase activity was assayed as previously described (11).

Protein quantification

Protein quantification by western analysis was done by first preparing whole cell

extracts from pelleted cells lysed in RIPA buffer (150mM sodium chloride, 1.0%

Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris pH 8.0).

Western blot was then performed to detect protein levels of BCL2, p21WAF1,

p19INK4D, Pol�2, FEN1, ETO (Santa Cruz Biotechnology), apurinic/apyridimic

endonuclease 1 (Ape1), �H2AXser139, pChk2 T68, Chk2 (Cell Signaling) or

PML-RAR� (Abcam). �-Actin (Sigma) was used to confirm equal protein loading.

Protein quantification of pATM S1981 and pDNA-PKcs T2056 (Abcam) was done

by flow cytometry. Cells were fixed in 1% paraformaldehyde and then

permeabilized by 1% Triton X-100 dissolved in PBS. Permeabilized cells were

incubated with primary antibody against pATM S1981 or pDNA-PKcs T2056 at

room temperature. Afterwards, cells were washed with PBS and incubated with

secondary Alexa Fluor® 488 antibody (Invitrogen). Fluorescence was measured

by flow cytometry and analyzed using FCS Express v3.0. Ten thousand events

were recorded per sample.




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COMET assay

Single cell gel-electrophoresis (COMET assay) was performed under alkaline

conditions as previously described (11, 23). COMETs were scored using

CometScore (TriTek) for Tail Moment (TM). TM is presented in arbitrary units

(A.U.).

Fast Micromethod

The fast micromethod for single strand break detection was performed as

previously described (24). Briefly, cells were incubated with PicoGreen for 15

minutes and then exposed to alkaline conditions to induce DNA unwinding.

PicoGreen preferentially binds to double stranded DNA but not to single-stranded

DNA. The loss of fluorescence caused by DNA unwinding is measured every

minute for 30 minutes. DNA strand breaks are expressed as strand scission

factor (SSF) multiplied by -1 after 20 minutes of denaturation.

Quantitative real-time PCR

RNA was isolated from cells using the Absolutely RNA® Miniprep Kit (Agilent).

cDNA was generated from 1�g total RNA using iScript cDNA synthesis kit (Bio-

Rad Laboratories). mRNA levels for CDKN2D, BCL2, CDKN1A, APEX1, FEN1,

POLD2, POLD3, OGG1, BRCA1, HUS1, NBS1 and BLM were assessed by

quantitative real-time PCR (QPCR) analysis using Power SYBR green master

mix (Applied Biosystems). mRNA levels for 18S were assessed by QPCR




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analysis using Taqman hybridization probe and Taqman Fast Master Mix

(Applied Biosystems). Relative mRNA levels were determined using the ��CT

method and 18S served as the endogenous control. QPCR was performed using

the 7500 Fast Real-time PCR system (Applied Biosystems). Primer sets used are

presented in Supplementary Figure 1.

Chromatin Immunoprecipitation Assay

Chromatin Immunoprecipitation (ChIP) assay was performed as previously

described (25). Briefly, PLZF-RAR� non-expressing and expressing

PLZFRAR�3 cells were treated with 1.5�M vorinostat for 6h, fixed with 1%

formaldehyde and then whole-cell lysates were prepared. Protein lysates (1mg)

were subject to ChIP with histone H3 (acetyl-K9) (Millipore), followed by DNA

purification and QPCR with the indicated primer sets (Supplementary Figure 1).

DNA damage recovery assay

DNA damage was induced in PLZFRAR�3 cells by treating PLZF-RAR�

expressing or non-expressing cells with 1.5�M vorinostat for 6h. Next, vorinostat

was washed off using PBS and cells were replated in fresh vorinostat-free media

and allowed to recover for 24h. Reduction in �H2AX (DNA double strand break

marker) was used to assess DNA repair competency. Levels of �H2AX were

assayed by western blot.

Statistics




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Significance was determined by ANOVA followed by Newman-Keuls post-tests

using Prism version 4.0 (GraphPad).

Results

Expression of the leukemia associated fusion protein PLZF-RAR�

enhances sensitivity to HDAC inhibitors

To test the effects of LAFP expression on sensitivity to vorinostat-induced cell

death, we used PLZFRAR�3 cells, which are U937 cells stably transfected with

PLZF-RAR� cDNA under the control of a tetracycline-off system (20). When

cultured in the absence of tetracycline, PLZFRAR�3 cells express PLZF-RAR�

(Figure 1A). Expression of PLZF-RAR� did not affect proliferation of PLZFRAR�3

cells within 48h of induction. However, the proliferation rate of PLZF-RAR�

expressing PLZFRAR�3 cells began to slow down after 48h (Supplementary

Figure 2A). Therefore, vorinostat-induced cell death was examined within 48h of

PLZF-RAR� expression. We have previously shown that vorinostat induces

significant cell death in U937 cells beginning at 24 hours (11). To assay for cell

death, PLZF-RAR� expressing and non-expressing cells were treated with

vorinostat and subsequently stained with propidium iodide (PI) in order to

quantify fragmented DNA by flow cytometry. Expression of PLZF-RAR� caused a

striking increase in vorinostat-induced cell death in PLZFRAR�3 cells after 48h

(Figure 1B). Tetracycline had no effect on vorinostat-induced cell death in the




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parental U937 cells (Supplementary Figure 2B). Similar results were also

obtained with B412, a ZnSO4-inducible model for PLZF-RAR� expression (21)

(Supplementary Figure 2C). Next, we assessed caspase involvement using a

caspase-3/7 activity assay. Expression of PLZF-RAR� enhanced vorinostat-

induced caspase-3/7 activity (Figure 1C). An enhanced activation of both

caspase-8 (extrinsic apoptosis) and caspase-9 (intrinsic apoptosis) activity was

also observed in response to vorinostat in the PLZF-RAR� expressing cells

(Supplementary Figure 2D). To determine the contribution of caspases, we pre-

treated PLZF-RAR� expressing and non-expressing cells with the pan-caspase

inhibitor Z-VAD-FMK for 1h, followed by vorinostat for 48h. Caspase inhibition

resulted in decreased vorinostat-induced cell death in both PLZF-RAR�

expressing and non-expressing cells (Figure 1D), confirming that the cells were

dying by apoptosis. The increase in caspase activity led us to investigate the

effect of PLZF-RAR� and vorinostat on the pro-survival apoptosis regulator BCL2,

which was previously shown to be negatively regulated by HDI (26). We

confirmed that vorinostat reduced BCL2 mRNA and protein levels and,

furthermore, these reductions were enhanced by PLZF-RAR� expression (Figure

1E and Supplementary Figure 2E). To test whether PLZF-RAR� increases

sensitivity to HDIs other than vorinostat, PLZF-RAR� expressing and non-

expressing cells were treated with a panel of HDI and assayed for cell death by

PI stain. Expression of PLZF-RAR� increased sensitivity to LBH589-, TSA-, NaB-,

MGCD0103- and romidepsin-induced cell death (Figure 1F). Previously, we

showed that vorinostat induces reactive oxygen species (ROS) in leukemia cells




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and that pre-treatment with the antioxidant N-acetyl cysteine (NAC) could reduce

vorinostat-induced cell death (11). Pre-treatment with NAC for 1h, followed by

vorinostat for 48h reduced cell death equally in both PLZF-RAR� expressing and

non-expressing cells (Figure 1G). Consistent with this, vorinostat induced

hydrogen peroxide to an equal extent in PLZF-RAR� expressing and non-

expressing cells (Supplementary Figure 2F), suggesting that enhancement of

HDI toxicity by PLZF-RAR� is not dependent on ROS.

Expression of PLZF-RAR� enhances vorinostat-induced DNA damage

We and other groups have shown that vorinostat induces DNA damage (11).

LAFPs have also been shown to induce a DNA repair deficient phenotype (8).

Thus, to determine if PLZF-RAR� expression affects vorinostat-induced DNA

damage, we performed single-cell gel electrophoresis (COMET assay) on

PLZFRAR�3 cells. The COMET assay was performed under alkaline conditions,

allowing detection of DNA single strand breaks, DNA double strand breaks

(DSBs) and alkali-labile sites. As previously observed, expression of PLZF-RAR�

resulted in an increased COMET Tail Moment (8). Moreover, treatment with

vorinostat resulted in a greater COMET Tail Moment in PLZF-RAR� expressing

cells versus non-expressing cells (Figure 2A). Doxorubicin was used as a

positive control and also induced a greater Tail Moment in PLZF-RAR�

expressing cells (Figure 2A). Next, we performed the fast micromethod to

specifically assay for single strand breaks (SSBs) (24). This confirmed that




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PLZF-RAR� expression and vorinostat treatment alone induced SSBs (Figure

2B), while PLZF-RAR� expression combined with vorinostat treatment resulted in

the greatest induction of SSBs (Figure 2B). Phosphorylation of H2AX was then

measured by western blot, as a measure of DNA DSBs. The phosphorylated

form of H2AX (�H2AX) localizes to DSBs within minutes of their formation,

making it a sensitive marker for this lesion (27). We observed no increased

�H2AX in untreated cells expressing PLZF-RARα, but the combination of PLZF-

RAR� expression and vorinostat induced �H2AX to a greater extent than

vorinostat alone (Figure 2C). Supporting our findings, an increased Tail Moment

and �H2AX induction by vorinostat in cell expressing PLZF-RAR� was also

observed in B412 cells (Supplementary Figure 3A and Supplementary Figure 3B).

We also tested the ability of the only other FDA-approved HDI, romidepsin, to

induce �H2AX in PLZFRAR�3 cells expressing PLZF-RAR� or not. Interestingly,

romidepsin was observed to induce �H2AX to the same extent in both PLZF-

RARα expressing and non-expressing cells (Figure 2C), despite the fact that cell

death was enhanced (Figure 1F).

Induction of DNA damage in leukemic stem cells expressing LAFP, or

following HDI treatment, has been described to correlate with induction of the cell

cycle regulator CDKN1A (p21WAF1) (28-30). HDI and DNA damage have also

been shown to induce the cell cycle regulator CDKN2D (p19INK4D) (31, 32).

Consistent with these findings, we found that both PLZF-RAR� and vorinostat

induced p21WAF1 and p19INK4D at the mRNA (Figure 2D) and protein level (Figure

2E and Supplementary Figure 3C). We found that the combination of PLZF-




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RAR� and vorinostat resulted in a greater induction of both p21WAF1 and p19INK4D.

While the increase in p19INK4D was transient, p21WAF1 remained high for at least

24h (Figure 2D and 2E). We also assessed expression of the cell cycle regulator

CDKN1B (p27KIP1), which has previously been shown to be induced in response

to PLZF-RAR� expression (20). We confirmed that PLZF-RAR� increases

CDKN1B mRNA levels, but vorinostat was observed to down-regulate CDKN1B

(Supplementary Figure 3D).

To determine if the enhanced induction of p21WAF1 correlated with increased

histone acetylation, we performed ChIP analysis using an antibody specific to

histone H3 (acetyl-K9). As expected, vorinostat caused a substantial increase in

histone H3 (acetyl-K9) enrichment at the p21WAF1 gene, however expression of

PLZF-RARα did not enhance this effect (Supplementary Figure 3E).

To assess whether PLZF-RAR� expression could increase sensitivity to other

DNA damaging stimuli we tested PLZF-RAR� expressing and non-expressing

cells against a panel of such stimuli. Indeed, PLZF-RAR� increased sensitivity to

cell death induced by gamma radiation (IR) and the cytotoxic agents doxorubicin,

etoposide and cisplatin (Figure 2F).

Expression of PLZF-RAR� enhances the DNA damage checkpoint response

to vorinostat

DNA damage results in the phosphorylation and activation of phosphoinositide

3-kinase related kinases like ATM and DNA-PKcs. These kinases function to




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detect DNA damage and signal for a cell cycle arrest to allow for proper repair of

DNA lesions (33). We used flow cytometry to measure phosphorylation of ATM at

serine 1981 (pATM S1981) and DNA-PKcs at threonine 2056 (pDNA-PKcs

T2056). We found that although expression of PLZF-RAR� did not induce

phosphorylation of either ATM or DNA-PKcs, it enhanced vorinostat-induced

phosphorylation of both proteins (Figure 3A). Activated ATM and DNA-PKcs

phosphorylate and activate downstream checkpoint proteins, including Chk2 (34,

35). Thus, to validate ATM and DNA-PKcs activation, we assayed for Chk2

phosphorylation at threonine 68 (pChk2 T68) by western blot. Vorinostat alone

can induce Chk2 phosphorylation, however, expression of PLZF-RAR� enhances

this induction (Figure 3B). Additionally, pre-treatment of cells with NU7026, a

DNA-PKcs small molecule inhibitor (22), greatly reduced Chk2 phosphorylation

(Figure 3B). To determine if activation of checkpoint kinases correlated with a cell

cycle arrest, we stained cells with PI and measured their DNA content by flow

cytometry. Vorinostat induced a G2-M arrest and this was enhanced by PLZF-

RAR� expression (Figure 3C). While pre-treatment with NU7026 did not affect

the cell cycle arrest induced by vorinostat alone, it greatly reduced the

percentage of cells arresting in the G2-M cell cycle phase in response to

vorinostat combined with PLZF-RARα expression (Figure 3C).

Of note, romidepsin also induced a G2-M cell cycle arrest in PLZFRAR�3 cells,

but expression of PLZF-RARα did not augment this arrest (Supplementary Figure

4). This is consistent with the fact that PLZF-RARα did not increase romidepsin-

induced �H2AX (Figure 2C).




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Other leukemia associated fusion proteins also increase cell death and

DNA damage in response to vorinostat

Vorinostat induces significant cell death in cell lines that endogenously

express LAFPs like NB4 (PML-RAR�) and Kasumi-1 (AML1-ETO)

(Supplementary Figure 5A). To further investigate whether these LAFPs can

increase sensitivity to vorinostat, similar to PLZF-RAR�, we used U937 clones

that conditionally express PML-RAR� (PR9) or AML1-ETO (U937-A/E). These

cells are stably transfected with PML-RAR� or AML1-ETO cDNA under

transcriptional control of a Zn-inducible mouse metallothionein promoter (8)

(Supplementary Figure 5B). A U937 clone (SN4) containing the empty cloning

vector was used as a control. Cell death was assayed by PI stain after LAFP

expressing and non-expressing cells were treated with vorinostat. Expression of

PML-RAR� and AML1-ETO both increased vorinostat-induced cell death (Figure

4A). To assess whether PML-RAR� and AML1-ETO had an effect on vorinostat-

induced DNA damage, we performed an alkaline COMET assay. As with PLZF-

RAR� expression, induction of PML-RAR� or AML1-ETO increased vorinostat-

induced DNA damage (Figure 4B). Doxorubicin was used as a positive control

and also induced a greater Tail Moment in LAFP expressing cells (Figure 4B).

The effect of PML-RAR� and AML1-ETO on vorinostat-induced DSB formation

was then assayed by �H2AX western blot. Expression of PML-RAR�, AML1-ETO

or vorinostat treatment alone induced �H2AX and the combination of PML-RAR�




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or AML1-ETO expression and vorinostat treatment resulted in a greater induction

of �H2AX (Figure 4C).

Vorinostat and leukemia associated fusion proteins down-regulate DNA

repair proteins and inhibit repair

A previous study established LAFP expression down-regulates mRNA levels

of base excision repair (BER) genes and inhibits DNA repair (8). To determine

the combined effect of LAFP expression and vorinostat on repair gene

expression, we performed QPCR for PLZFRAR�3 (PLZF-RAR� inducible) and

U937-A/E (AML1-ETO inducible) cells treated with 1.5�M vorinostat for 18h. As

previously reported, expression of LAFP resulted in decreased mRNA levels for

the BER genes APEX1, POLD2, POLD3 and FEN1 (Figure 5A, 5B). We found

OGG1 mRNA levels to be reduced with AML1-ETO but slightly increased by

PLZF-RAR� expression (Figure 5A, 5B). Vorinostat treatment alone reduced

mRNA levels of all BER genes in both cell line models (Figure 5A, 5B). While the

combination of PLZF-RAR� and vorinostat resulted in a further decrease in BER

gene mRNA levels (with the exception of OGG1), this was not observed when

combining AML1-ETO expression and vorinostat (Figure 5A, 5B). To better

understand the mechanism of repression of the BER genes by HDI and LAFP,

we assessed the level of histone H3 (acetyl-K9) associated with the APEX1 gene.

Interestingly, vorinostat mediated down-regulation of APEX1 mRNA correlated

with an overall enrichment of histone H3 (acetyl-K9) at all sites of the APEX1




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gene assessed, except near the transcriptional start site (-78kb). Expression of

PLZF-RARα did not significantly alter the level of histone H3 (acetyl-K9) at this

gene (Supplementary Figure 6A). We further performed western blot analysis to

validate the changes in BER enzyme mRNA levels observed at the protein level.

Expression of PLZF-RAR� alone reduced protein levels of Ape1 (APEX1) and

FEN1 but had no effect on Pol �2 (POLD2). The combination of PLZF-RAR� and

vorinostat enhanced the down-regulation of Ape1 and Pol �2 but did not further

reduce FEN1 protein levels (Figure 5C). Expression of AML1-ETO alone reduced

Ape1 protein levels but had little effect on Pol �2 or FEN1. Vorinostat treatment

alone also induced a down-regulation of Ape1 and FEN1 protein, while little

change in Pol �2 was observed (Figure 5C).

As HDI have also been shown to down-regulate DSB repair genes and

vorinostat induced �H2AX, a marker of DSB, to a greater extent in LAFP

expressing cells (Figure 2C, Figure 4C), we investigated the effect of LAFP and

HDI on the mRNA levels of DSB repair enzymes. Vorinostat and romidepsin

were found to down-regulate mRNA levels for BRCA1, HUS1, NBS1 and BLM in

PLZFRAR�3 and U937-A/E cells (Supplementary Figure 6B and 6C), as well as

in NB4 and Kasumi-1 (Supplementary Figure 6D). We found that induced

expression of PLZF-RAR� down-regulated HUS1 and NBS1 but did not enhance

the effect of the HDI (Supplementary Figures 6B and 6C). AML1-ETO down-

regulated NBS1 and BLM but did not enhance the effect of HDI (Supplementary

Figure 6C).




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To assess functional DNA repair capacity, PLZF-RAR� non-expressing and

expressing cells were treated with 1.5�M vorinostat for 6h to induce DNA DSBs.

Next, cells were washed, replated in vorinostat-free media and given 24h to

recover. Changes in �H2AX levels were evaluated by western blot to measure

DNA repair. After 6 h with vorinostat, �H2AX was increased, consistent with

previous data (11). Importantly, in cells lacking PLZF-RAR�, �H2AX levels were

reduced after 24 hours, indicating efficient DNA repair. In contrast, cells

expressing PLZF-RAR� displayed increased �H2AX after the 24h recovery

period (Figure 5D). The presence of PLZF-RAR� alone also increased �H2AX

levels over time, indicating an accumulation of DNA damage in these cells

(Figure 5D). To ensure changes in �H2AX levels were not due to changes in cell

death, we performed PI stain on cells taken from each condition in parallel. We

observed no cell death in any of the conditions (data not shown).

Discussion

LAFPs have been shown to down-regulate DNA repair genes resulting in a

DNA repair deficient phenotype (8). HDIs have been shown to increase levels of

DNA damage by numerous mechanisms (11-15). In this work, we examined

whether expression of LAFPs would improve response to HDI treatment. We

demonstrate for the first time that expression of the LAFPs PLZF-RAR�, PML-

RAR� and AML1-ETO increases HDI-induced DNA damage (Figure 2 and 4) and

apoptosis (Figure 1). We show that increased levels of DNA damage correlate




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with a down-regulation of DNA repair proteins by LAFPs and/or vorinostat, and

reduced DNA repair capacity (Figure 5). Interestingly, we found differences

between the effects of vorinostat and romidepsin. While romidepsin also induced

greater cell death in PLZF-RAR� expressing cells (Figure 1F), this did not

correlate with an enhanced cell cycle arrest (Supplementary Figure 4) or

increased levels of DNA damage (Figure 2C). We found that romidepsin down-

regulated BCL2 protein to a greater extent in PLZF-RAR� expressing cells

(Supplementary Figure 7A). This suggests that while the enhanced cell death

induced by romidepsin is independent of DNA damage, it may still be mediated

by an enhanced effect on apoptotic mediators.

DNA damage is often accompanied by an induction of cell cycle inhibitor

proteins and a cell cycle arrest to allow for proper DNA repair (36). Consistently,

we observed an enhanced G2-M arrest (Figure 3C). The additional DNA damage

induced by vorinostat in PLZF-RAR� cells correlated with activation of DNA-PKcs

and a greater G2-M arrest. While pre-treatment with KU7026 blocked Chk2

phosphorylation, it did not completely block the vorinostat-induced G2-M arrest.

This suggests that activated pATM S1981 may compensate by activating a

different downstream cell cycle arrest mediators such as Chk1. We also

observed an enhanced induction of the cell cycle inhibitor proteins p21WAF1 and

p19INK4D in PLZF-RAR� positive cells treated with vorinostat (Figure 2D and 2E).

Of interest, Pellici’s group recently showed that expression of LAFPs in mouse

hematopoietic stem cells (HSCs) induces DNA damage and p21WAF1 (28).

Activated p21WAF1 arrested the cell cycle, allowing for DNA repair and protection




� ��

of the leukemic HSCs. This raises the concern that in an in vivo or HSC setting,

despite the potential for vorinostat to induce greater amounts of DNA damage

(and apoptosis) in LAFP expressing cells, leukemic stem cells may remain

protected due to increased p21WAF1 activation. The development of drugs or

strategies to target p21WAF1 or p21WAF1-mediated cell cycle arrest may offer an

approach to further improve HDI efficacy in LAFP-positive AML cells and LAFP-

positive leukemic HSCs. Indeed, a recent study by Bug’s group investigating the

effects of HDI on leukemic hematopoietic stem cells (HSCs) supports this

concern (37). They found that HDI treatment of PLZF-RAR� or AML1-ETO

expressing murine leukemic HSCs impaired their self-renewal potential as

assayed by serial replating experiments. However, residual colony formation was

still observed at later replatings in PLZF-RAR� expressing leukemic HSCs

treated with vorinostat (37).

It is noteworthy that the enhanced up-regulation of p19INK4D in PLZF-RAR�

expressing cells treated with vorinostat dissipates over time and that vorinostat

down-regulates PLZF-RAR� induced p27KIP1 (Figure 2D, 2E and Supplementary

Figure 3D). Previous studies have shown p19INK4D and p27KIP1 to induce a G1

arrest thereby protecting cells from DNA damage (31, 38). In our model,

vorinostat induced a G2-M arrest (Figure 3), suggesting p19INK4D is insufficient to

mount a G1 arrest or that the G2-M arrest response is stronger. Interestingly,

vorinostat eventually abrogates p19INK4D protein expression compared to vehicle-

treated cells expressing PLZF-RAR� (Figure 2E). Given the findings of Pelicci’s

group (28), the induction of a cell cycle regulator is not a desirable event in the




� ��

context of leukemogenesis or leukemic HSC clearance. Therefore, it is tempting

to speculate that the eventual suppression of PLZF-RAR�-induced p19INK4D and

p27KIP1 may represent a mechanism by which vorinostat inhibits a cell cycle

regulator that could potentially protect leukemic HSCs.

The DNA damage we observed is dependent on two factors; DNA damage

induced by LAFPs and/or vorinostat, and reduced DNA repair capacity. Our

group and others have previously shown that vorinostat may induce DNA

damage through reactive oxygen species production (11, 14). Several studies

have also shown HDI to have radiosensitizing effects in a variety of cancer

models (13, 39). However, PLZF-RAR� ��on did not lead to

increased ROS production in response to vorinostat in our cells, suggesting the

enhanced DNA damage is independent of ROS. Expression of LAFPs was

previously shown to down-regulate a number of base excision repair (BER)

genes (8). HDI have been shown to down-regulate a number of DNA repair

enzymes including enzymes implicated in BER and DSB repair (14, 40, 41).

Therefore, we sought to investigate the effect of vorinostat on these same genes

in the presence or absence of PLZF-RAR� or AML1-ETO. Interestingly,

vorinostat alone was found to down-regulate all BER and DSB repair genes

tested at the mRNA level (Figures 5A, 5B and Supplementary Figure 6C). In

PLZFRAR�3 cells, the combination of PLZF-RAR� expression and vorinostat

treatment resulted in a greater decrease in BER gene mRNA levels than either

alone (Figure 5A). In U937-A/E cells, however, the combination of AML1-ETO

expression and vorinostat only caused a greater reduction in Pol �2 mRNA levels




� ��

(Figure 5B). The effects on BER enzyme protein levels were not as dramatic as

observed at the mRNA level and were observed only at later time points (18h-

24h, Figure 5A-C). The fact that the accumulation of DNA damage is observed

earlier (12h, Figure 2B) thus suggests the presence of additional mechanisms of

either enhanced DNA damage or DNA repair suppression. However, the later

down-regulation of BER enzymes is still significant as it likely contributes to the

continued or accelerated accumulation of DNA damage. Moreover, while we

observed overlap in the DNA repair genes down-regulated by LAFPs and

vorinostat, the literature suggests that there are possibly other repair genes and

pathways uniquely regulated by either as well (12, 14, 15, 28). Indeed, we found

that several DSB repair genes were down regulated by HDI, LAFP, or both

(Supplementary Figure 6B and 6C). This may help explain why the greatest

amount of DNA damage is observed when LAFP expression is combined with

vorinostat treatment (Figure 2).

Of note, Ape1 protein levels were strongly reduced in both PLZF-RAR� and

AML1-ETO expressing cells compared to non-expressing cells (Figure 5C). The

combination of vorinostat treatment and expression of either PLZF-RAR� or

AML1-ETO expression also resulted in a greater reduction of Ape1 protein levels

(Figure 5C). Ape1 is critical to the BER pathway, implicated in the repair of 95%

of all apurinic/apyridimic sites (42). Ape1 also possess a redox domain, which

allows it to alter transcription factor DNA binding and therefore gene expression.

Through its redox function, Ape1 has been shown to regulate the p53, AP-1 and

HIF-1� transcription factors, and expression of their downstream target genes




� ��

implicated in homologous recombination repair, mismatch repair and global

genome repair (43). Accordingly, Ape1 knockout mice die early in embryonic

development (44), while heterozygous Ape1 mice experience increased

spontaneous mutagenesis (45). This suggests that Ape1 down-regulation may be

integral to the enhanced DNA damage observed in LAFP expressing cells

treated with vorinostat. Generally, Ape1 overexpression has been correlated with

aggressive proliferation and poor prognosis (43, 46, 47). Decreasing Ape1 levels

via knockdown or chemical inhibition has been shown to reduce cancer cell

growth and sensitize cells to DNA damaging agents (48, 49). We also found that

vorinostat alone down-regulates Ape1 in Kasumi-1 cells (Supplementary Figure

7B). This suggests that vorinostat or other HDI may represent a novel strategy to

target Ape1. Alternatively, knocking-up Ape1 may reduce HDI efficacy thereby

validating Ape1 as an HDI-target and marker of HDI response or resistance. Our

data also provides a rationale to future testing of HDI in combination with Ape1

inhibitors.

In the clinic, HDI have shown promising results in LAFP-positive AML patients.

A small Phase II trial by Stock’s group showed HDI had improved anti-leukemic

activity in AML patients with LAFPs (18). Twenty patients were separated into

two groups based on the absence or presence of LAFPs and treated with the

HDI romidepsin. While there was no response to HDI by the LAFP-negative

cohort, HDI displayed anti-leukemic activity in 3/5 patients from the LAFP-

positive cohort. However, disease progression eventually occurred in all patients.

Our findings suggest that response may have been linked to romidepsin-induced




� ��

DNA damage and regulation of apoptosis and DNA repair genes. For future trials,

evaluation of DNA damage and DNA repair gene expression may lead to

predictive biomarkers for the response to HDI in LAFP-positive AMLs.

In summary, we present evidence that HDI display enhanced activity in LAFP

expressing AML cells. We show that vorinostat treatment leads to a greater

accumulation of DNA damage in LAFP expressing cells and correlates with a

down-regulation of DNA repair enzymes and impaired DNA repair. Presently,

HDI have not been approved for the treatment of AML, as results from the clinic

have been mixed (18, 50, 51). Our data show that HDI treatment may be more

relevant in LAFP-positive patients and support the continued clinical evaluation of

HDI in this setting.

Acknowledgments

We would like to thank Dr. Myriam Alcalay for her gift of U937-A/E cells. We

would like to thank Dr. Martin Ruthardt for his gift of B412 cells.

Grant Support

This study was supported by the Samuel Waxman Cancer Research Foundation

(W.H Miller Jr., J.D Licht), a grant MOP-12863 from the Canadian Institute of

Health Research (W.H Miller Jr.) and the McGill Systems Biology Training

Program (L.A Petruccelli).




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Figure Legends

Figure 1. Expression of the leukemia associated fusion protein PLZF-RAR�

enhances sensitivity to HDAC inhibitors. A PLZFRAR�3 cells cultured in the

absence of tetracycline express PLZF-RAR�.�-Actin was used as a loading

control. B PLZFRAR�3 (PLZF-RAR� inducible) cells were cultured in the

presence or absence of tetracycline and treated with 1.0�M or 1.5�M vorinostat

for 48h. Cells were stained with PI and analyzed by flow cytometry. Cell death

was quantified as the percentage of cells with DNA content below that of the G0-

G1 peak (Sub-G0). C PLZF-RAR� non-expressing and expressing PLZFRAR�3

cells were treated with 1.5�M vorinostat for 48h. Cells were counted and then

assayed for caspase-3/7 activity using a luciferase-based assay, where reactive




� ��

light units (RLU) are normalized to number of cells and reported as fold change

over vehicle treated control. D PLZF-RAR� non-expressing and expressing

PLZFRAR�3 cells were pre-treated with 50�M Z-VAD-FMK for 1h followed by

1.5�M vorinostat for 48h. Cells were stained with PI and analyzed by flow

cytometry for cell death. E PLZF-RAR� non-expressing and expressing

PLZFRAR�3 cells were treated with 1.5�M vorinostat for 1h, 3h, 6h, 18h or 24h.

Protein levels for BCL2 were analyzed by western blot. �-Actin was used as a

loading control. F PLZF-RAR� non-expressing and expressing PLZFRAR�3 cells

were treated with 15nM LBH589, 200nM TSA, 2mM NaB, 1.5�M MGCD0103 or

5nM romidepsin for 48h. Cells were stained with PI and analyzed by flow

cytometry for cell death. G PLZF-RAR� non-expressing and expressing

PLZFRAR�3 cells were pre-treated with 2mM NAC for 1h followed by 1.5�M

vorinostat for 48h. Cells were stained with PI and analyzed by flow cytometry for

cell death. Asterisks indicate a significant difference and a non-significant

difference is indicated as “n.s” (* < 0.05 *** < 0.001).

Figure 2. Expression of PLZF-RAR� enhances vorinostat-induced DNA damage.

PLZF-RAR� non-expressing and expressing PLZFRAR�3 cells were treated with

1.5�M vorinostat or 5nM romidepsin for the indicated time points. A Cells were

assayed for DNA single strand breaks, double strand breaks and alkali-labile

sites by alkaline COMET assay. Doxorubicin was used as a positive control. At

least 100 nuclei were randomly selected and quantified for DNA damage,

represented as an increase in Tail Moment (A.U.). Representative images of




� ��

COMET tails obtained are presented in the panel to the right. B Cells were

assayed for single strand breaks by the fast micromethod. The extent of DNA

strand breaks is expressed as strand scission factor multiplied by -1 (SFF x -1).

C Induction of the DNA double strand break marker �H2AX was measured by

western blot. �-Actin was used as a loading control. D PLZF-RAR� non-

expressing and expressing cells were treated with 1.5�M vorinostat for 3h, 6h or

18h. Relative mRNA levels for CDKN1A (p21WAF1) and CDKN2D (p19INK4D) were

assayed by QPCR. E PLZF-RAR� non-expressing and expressing PLZFRAR�3

cells were treated with 1.5�M vorinostat for 1h, 3h, 6h, 18h or 24h. Protein levels

for p21WAF1 and p19INK4D were analyzed by western blot. �-Actin was used as a

loading control. F PLZF-RAR� non-expressing and expressing PLZFRAR�3 cells

were treated with 0.25�M doxorubicin, 0.5�M etoposide, 10�M cisplatin or 20Gy

gamma radiation (IR) for 48h. Cells were stained with PI and analyzed by flow

cytometry for cell death. Asterisks indicate a significant difference (* < 0.05 ** <

0.01 *** < 0.001).

Figure 3. Expression of PLZF-RAR� enhances the DNA damage checkpoint

response to vorinostat. PLZF-RAR� non-expressing and expressing

PLZFRAR�3 cells were treated with 1.5�M vorinostat for 18h. A Cells were

stained with antibody against either pATM (S1981) or pDNA-PKcs (T2056) and

analyzed by flow cytometry. B PLZF-RAR� non-expressing and expressing

PLZFRAR�3 cells were pre-treated with 10�M NU7026 followed by

1.5�Mvorinostat for 18h. Western blot was performed to measure




� ��

phosphorylation of Chk2 (T68) relative to total Chk2 protein levels. �-Actin was

used as a loading control. C Cell cycle distribution was assayed by staining cells

with PI and quantifying DNA content using flow cytometry. Asterisks indicate a

significant difference (* < 0.05 ** < 0.01 *** < 0.001).

Figure 4. Other leukemia associated fusion proteins also increase cell death and

DNA damage in response to vorinostat. A SN4 (mock transfected parental

U937cells), PR9 (PML-RAR� inducible) and U937-A/E (AML1-ETO inducible)

cells were cultured in the presence or absence of ZnSO4 and treated with 1.5�M

vorinostat for 48h. Cells were stained with PI and analyzed by flow cytometry for

cell death. B PR9 and U937-A/E cells were treated with 1.5�M vorinostat for 18h

and assayed for single strand breaks, DNA double strand breaks and alkali-labile

sites by alkaline COMET assay. Doxorubicin was used as a positive control. DNA

damage is represented as an increase in Tail Moment (A.U.). C PR9 (PML-RAR�

inducible) and U937-A/E (AML1-ETO inducible) cells were treated with 1.5�M

vorinostat for 18h and assayed for induction of the DNA double strand break

marker �H2AX by western blot. �-Actin was used as a loading control. Asterisks

indicate a significant difference and a non-significant difference is indicated as

“n.s” (* < 0.05 ** < 0.01 *** < 0.001).

Figure 5. Vorinostat and leukemia associated fusion proteins down-regulate

DNA repair proteins and inhibit repair. RNA was isolated from cells and

converted into cDNA to quantify relative mRNA levels of the base excision repair




� ��

(BER) genes: APEX1 (Ape1), POLD2 (Pol �2), POLD3, FEN1 and OGG1 by

QPCR. Relative mRNA levels (��CT) of BER genes were measured in A

PLZFRAR�3 (PLZF-RAR� inducible) and B U937-A/E (AML1-ETO inducible)

cells treated with 1.5�M vorinostat for 18h. C Changes in BER gene mRNA

levels were validated in PLZFRAR�3 and U937-A/E cells at the protein level by

western blot. �-Actin was used as a loading control. D DNA repair was assessed

using a DNA damage recovery assay in PLZFRAR�3 cells. Cells were treated

with 1.5�M vorinostat for 6h to induce DNA double strand breaks (DSBs).

Vorinostat was then washed off and cells were replated in vorinostat-free media

and allowed to recover for 24h. Western blot was used to evaluate any

reductions in the DNA DSB marker �H2AX, which reflects the amount of DNA

repair. �-Actin was used as a loading control. Asterisks indicate a significant

difference (** < 0.01 *** <0.001). Decimals indicate a significant difference versus

vehicle (no leukemia associated fusion protein) treated control (••• < 0.001).

Figure 6. Chemical structure of vorinostat, LBH589, trichostatin A, sodium

butyrate, MGCD0103 and romidepsin.

Figure 7. Chemical structure of Z-VAD-FMK, N-acetyl-L-cysteine, doxorubicin,

etoposide, cisplatin and NU7026.




Published OnlineFirst March 27, 2013.Mol Cancer Ther Luca A Petruccelli, Filippa Pettersson, Sonia V del Rincon, et al. damage and apoptosis.sensitivity to histone deacetylase inhibitor induced DNA Expression of leukemia associated fusion proteins increases

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