The Novel Deacetylase Inhibitor AR-42 Demonstrates
Pre-Clinical Activity in B-Cell Malignancies In Vitro and
David M. Lucas1,4, Lapo Alinari1, Derek A. West1, Melanie E. Davis1, Ryan B. Edwards1, Amy J. Johnson1,
Kristie A. Blum1, Craig C. Hofmeister1, Michael A. Freitas2, Mark R. Parthun3, Dasheng Wang4, Amy
Lehman5, Xiaoli Zhang5, David Jarjoura5, Samuel K. Kulp4, Carlo M. Croce2, Michael R. Grever1,4,
Ching-Shih Chen4, Robert A. Baiocchi1., John C. Byrd1,4*.
1Department of Internal Medicine, The Ohio State University, Columbus, Ohio, United States of America, 2Department of Molecular Virology, Immunology, and Medical
Genetics, The Ohio State University, Columbus, Ohio, United States of America, 3Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus,
Ohio, United States of America, 4College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America, 5Center for Biostatisics, Comprehensive
Cancer Center, The Ohio State University, Columbus, Ohio, United States of America
Background: While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to
date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or
unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell
Principal Findings: In mantle cell lymphoma (JeKo-1), Burkitt’s lymphoma (Raji), and acute lymphoblastic leukemia (697) cell
lines, the 48-hr IC50(50% growth inhibitory concentration) of AR-42 is 0.61 mM or less. In chronic lymphocytic leukemia (CLL)
patient cells, the 48-hr LC50(concentration lethal to 50%) of AR-42 is 0.76 mM. AR-42 produces dose- and time-dependent
acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of
stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through
reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models
of B-cell malignancy without evidence of toxicity.
Conclusions/Significance: Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses.
These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.
Citation: Lucas DM, Alinari L, West DA, Davis ME, Edwards RB, et al. (2010) The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell
Malignancies In Vitro and In Vivo. PLoS ONE 5(6): e10941. doi:10.1371/journal.pone.0010941
Editor: Alfons Navarro, University of Barcelona, Spain
Received February 10, 2010; Accepted May 13, 2010; Published June 3, 2010
Copyright: ? 2010 Lucas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the National Cancer Institute (P01 CA101956, P01 CA081534 and P50 CA140158) and a Specialized Center of Research grant
from the Leukemia and Lymphoma Society (www.lls.org). Laboratory support was also provided by the V Foundation (www.jimmyv.org), the D. Warren Brown
Foundation, and the Lymphoma Research Foundation (Mantle Cell Lymphoma Initiative) (www.lymphoma.org). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: MRG served as a consultant to Arno Therapeutics. CSC received royalty payments from Arno Therapeutics per the licensing agreement of
AR-42 between The Ohio State University Research Foundation and the company. This does not alter the authors’ adherence to all the PLoS ONE policies on
sharing data and materials.
* E-mail: email@example.com
. These authors contributed equally to this work.
Deacetylases (DACs) are a family of enzymes that catalyze the
removal of acetyl groups from lysine residues, and to date have
been extensively studied in the context of histone proteins.
Inhibitors of these enzymes were originally reported to relieve
transcriptional repression and subsequent epigenetic silencing
caused by histone deacetylation. It is now evident that the targets
of these enzymes also include a broad array of proteins such as
transcription factors, chaperones, signaling components, and
cytoskeletal proteins. Thus, the effects of DAC inhibitors are
diverse and incompletely understood, and likely vary by cell type
and context. Adding to the complexity of reported DAC inhibitor
activities is the different, but occasionally overlapping, effects on
class I and II DACs. Class I DACs (1, 2, 3 and 8) are primarily
found in the nucleus, although DAC3 is found in both the nucleus
and cytoplasm. Class II DACs (4, 5, 6, 7, 9 and 10) are generally
reported to shuttle in and out of the nucleus, depending on
intracellular signals. DAC6 is a cytoplasmic enzyme that
deacetylates tubulin , HSP90 [2,3], and likely additional
cytoplasmic proteins. Due to their broad effects on gene
transcription, cell growth and differentiation, inhibitors of DACs
have been shown to possess anti-cancer activity in a variety of
tumor cell models, in primary tumor cells, and in vivo [4,5,6].
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Clinical efficacy of this class of agents to date is perhaps best
exemplified by vorinostat (SAHA) and romidepsin (depsipeptide;
FK228) in cutaneous T-cell lymphoma, in which response rates of
approximately 30–35% are noted. However an enormous body of
evidence also supports the investigation of this class of agents in
tumors as diverse as prostate cancer, lung cancer and glioblastoma
Chronic Lymphocytic Leukemia (CLL) is immunophenotypi-
cally defined as a malignancy of CD5/CD19/CD23 positive,
CD20 and Ig dim B cells that manifests with bone marrow failure,
lymphadenopathy and infections as a consequence of disease-
associated immune suppression. While recent advances in
chemoimmunotherapy strategies have improved options for CLL
patients, the median overall survival for fludarabine-refractory
patients is just 13 months. Mantle cell lymphoma (MCL), an
aggressive B cell malignancy, is characterized by the abnormal
proliferation and accumulation of CD5/CD20/CD22 positive,
CD23 negative B cells in various hematopoietic tissues, with or
without peripheral blood involvement. While MCL comprises
approximately 8% of Non-Hodgkin lymphoma cases, it is
associated with a disproportionate number of deaths and a mean
survival of only three years . To date, therapeutic options for
these two B cell diseases are limited, and relapses are nearly
universal. Given the absence of effective therapies for these and
other B-cell malignancies, it is essential to explore new treatment
Multiple studies have demonstrated that DAC inhibitors
including romidepsin, entinostat (MS-275) and valproic acid can
alter histone acetylation status in CLL and lead to selective
cytotoxicity in these cells [10,11,12,13]. In preclinical studies done
by our group, the class I DAC inhibitor romidepsin induced
apoptosis in CLL cells via activation of caspase 3 and caspase 8,
with minimal alteration in caspase 9 activity . Caspase 8
activation occurred concomitantly with down-regulation of
c-FLIP, an inhibitory protein of caspase 8. The observation that
romidepsin operates via a caspase 8-mediated process is
significant, as this pathway is not typically activated by other
agents currently used in the treatment of CLL. Subsequent work
by our group has demonstrated that entinostat, also a class
I-specific DAC inhibitor, promotes apoptosis in CLL cells with
concurrent alteration in lysine acetylation of histones H3 and H4
. Whereas others report that entinostat and other DAC
inhibitors may mediate their cytotoxicity through generation of
reactive oxygen species, we demonstrated that this occurred later
in the process of CLL cell death, and was likely an effect rather
than a cause .
Clinical trials with class I DAC inhibitors N-acetyldinaline (CI-
994; J Byrd, personal communication), romidepsin  and
MGCD0103  have been performed in CLL, with the former
two demonstrating evidence of anti-tumor activity as supported by
improvement in lymphocyte counts and diminishment in lymph
node size. No significant clinical activity was observed with
MGCD0103 in CLL . In all three of these trials, significant
fatigue, anorexia, and other constitutional symptoms limited
compliance and patient willingness to continue therapy. To date,
clinical testing of class I/II DAC inhibitors in CLL has been
AR-42 (previously called OSU-HDAC42) is a novel hydro-
xamate-tethered phenylbutyrate derivative [16,17] with in vitro and
in vivo activity in multiple solid tumor models [18,19,20,21] and
more recently, in mouse and canine mast cells . AR-42 was
demonstrated to be more potent than the benchmark agent
vorinostat in inducing apoptosis and in causing reductions of
phospho-Akt, Bcl-xL, and survivin. In vivo, AR-42 suppressed PC-3
tumor xenograft growth by 67%, whereas vorinostat at the same
dose suppressed growth by 31% . Based on our previous
studies of class I DAC inhibitors in CLL and these encouraging
results, we tested AR-42 in vitro and in vivo using CLL and related
In vitro activity of AR-42
In MTT assays (figure 1A), the 50% growth inhibitory
concentration (IC50) of AR-42 at 48 hr was 0.61 mM (95%
CI=0.28, 1.33) in Raji Burkitt’s lymphoma cells, 0.22 mM (95%
CI=0.18, 0.26) in 697 acute lymphoblastic leukemia cells, and
0.21 mM (95% CI=0.17, 0.25) in JeKo-1 MCL cells (n=3 each).
In simultaneous assays, the IC50values of vorinostat were 3- to 6-
fold higher, consistent with results in prostate cancer cell lines .
In CLL patient cells, AR-42 exhibited a 48-hr LC50of 0.76 mM
(95% CI=0.55–1.05; n=11), similar to what we observed with
the class I DAC inhibitor entinostat (MS-275) . For the in vitro
work presented here, AR-42 was used at the LC50of 0.90 mM that
was initially calculated using a smaller number of CLL samples.
Although this LC50 was found to be moderately lower when
additional CLL samples were included, we continued to use the
initial LC50of 0.90 mM for consistency among experiments.
In washout experiments using CLL tumor cells, the 48-hr
cytotoxic effect of AR-42 was eliminated when the drug was
removed after 4 hr (n=5; p=0.266 relative to the untreated
control). However, cytotoxicity with a 16 hr exposure was similar
to that observed when samples were incubated continuously for
48 hr (n=5; p=0.115 relative to continuously treated cells)
(figure 1B). We previously observed that the cytotoxic activity of
the cyclin-dependent kinase inhibitor flavopiridol was substantially
reduced in medium containing human serum vs. fetal bovine
serum, with profound implications for effective clinical adminis-
tration . We therefore compared the cytotoxicity of AR-42
against 697 cells incubated in RPMI 1640 media supplemented
with 10% human serum or 10% fetal bovine serum. AR-42
showed no difference in cytotoxicity between these two serum
conditions (p$0.20 at 24 and 48 hr; figure 1C).
CLL tumor cells are known to receive a variety of survival
signals from the microenvironment, and cumulative evidence
clearly demonstrates the importance of such signaling in CLL cell
resistance to apoptosis and to chemotherapy [24,25,26]. We
therefore investigated the efficacy of AR-42 in the presence of
stromal protection using the human marrow-derived fibroblast cell
line HS-5 . HS-5 cells were seeded in tissue culture flasks one
day prior to treatment. CLL patient cells (n=8) were incubated
with or without AR-42 (0.90 mM) 16 hr before washing and
plating in flasks with or without HS-5 for a total of 48 hr. CLL
cells were then recovered by gentle pipetting and analyzed by flow
cytometry. Events due to non-adherent HS-5 cells were eliminated
by forward/side scatter characteristics as determined by evaluation
of HS-5 cells alone. Untreated CLL cells co-cultured with HS-5
cells showed dramatic reduction in apoptosis as measured by
annexin positivity relative to non-co-cultured cells, as noted by a
strong reduction the annexin-positive fraction (figure 2A). As
expected, cells treated with AR-42 without HS-5 co-culture
showed a substantial increase in annexin positivity at this time
point. However, the degree of HS-5 protection was significantly
different between untreated cells and cells treated with AR-42
(p=0.016), indicating that the pro-survival effect of HS-5 is unable
to effectively block AR-42-induced apoptosis. These results
provide important evidence that AR-42 may circumvent the
protective effects of the CLL cell microenvironment in vivo.
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We performed additional experiments to clarify events accom-
panying AR-42 mediated cell death. Caspase activation and
induction of the mitochondrial pathway of apoptosis are
documented effects of most DAC inhibitors [14,28]. However,
Mitsiades et al. reported that caspases were not activated following
vorinostat treatment in a myeloma cell line, nor did the broad
caspase inhibitor Z-VAD-fmk protect these cells from vorinostat
. We therefore investigated the role of caspase activation in
AR-42 mediated cell death in B-cell lymphoma lines. Cells were
incubated 24 hr with 0.90 uM AR-42, with or without the broad
caspase inhibitor Z-VAD-fmk (100 mM). AR-42-mediated apop-
tosis, as defined by annexin binding and processing of the caspase
substrate polyADP ribose polymerase (PARP) to its 85 kDa form,
was effectively abrogated by Z-VAD-fmk. Representative data
from JeKo-1 are shown in figure 2B; similar results were
obtained with 697 cells. We confirmed these results using CLL
tumor cells treated with AR-42 in the presence or absence of Z-
VAD-fmk. Relative to untreated controls, AR-42 caused a greater
than 60% decrease in live (annexin and PI negative) cells at 48 hr,
an effect that was nearly completely inhibited by Z-VAD-fmk
(figure 2C, n=5). AR-42 induced PARP cleavage in these same
samples at 24 hr, which also was effectively prevented by Z-VAD-
fmk (figure 2D, representative of 5 CLL patient samples).
Class-specific activity of AR-42
DAC inhibitory activity of AR-42 was assessed by examining
acetylation of multiple downstream targets in CLL patient cells. In
CLL patient cells, increased acetylation of class I DAC target
histone H3 and the class II target tubulin could be detected with
just 1 hr of exposure to 0.90 mM AR-42 (figure 3A, represen-
Figure 1. AR-42 exhibits potent cytotoxicity in B-leukemia/lymphoma cells. (A) Raji, 697 and JeKo-1 B-cell lines (n=3 each) or CLL patient
cells (n=11) were incubated 48 hr with or without AR-42. Growth inhibition (for cell lines) or viability (for non-proliferating CLL patient cells) was
assessed by MTT assay and calculated relative to time-matched untreated cells. Bars represent +/2 standard deviation. (B) CLL cells were incubated
with or without AR-42 (0.90 mM). At 4 hr or 16 hr, cells were replated in fresh media with or without drug. At 48 hr, viability was assessed by MTT
assay and is shown relative to time-matched, untreated controls (n=5 for 4 and 16 hr exposures; n=11 for 48 hr exposure and untreated controls).
Bars represent +/2 standard deviation. There was no significant difference in viability between untreated samples and samples with 4 hr AR-42
exposure. The difference in viability between untreated samples or those with 16 hr exposures was significant (p,0.0001). (C) 697 cells were
incubated 24 hr (solid bars) or 48 hr (striped bars) with or without AR-42, using RPMI 1640 supplemented either with 10% FBS (white bars) or 10%
human serum (grey bars) (n=3). Viability was assessed by MTT assay and calculated relative to the untreated samples. Bars represent +/2 standard
deviation. AR-42 cytotoxicity in media with human serum or FBS was similar (0.45 mM: p=0.20 and p=1.0 at 24 and 48 hr respectively; 1.35 mM:
p=0.21 and p=0.24 at 24 and 48 hr, respectively).
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tative of six patient samples). After a 24 hour exposure, still prior to
substantial cell death as determined by annexin/PI flow
cytometry, AR-42-mediated increases in acetylation of H3 and
tubulin in CLL cells were evident (figure 3B, representative of
seven patient samples). In contrast, the class I-specific DAC
inhibitor romidepsin produced no tubulin acetylation at this time
point, although it is important to note that romidepsin and
vorinostat concentrations were selected from previous work and do
not represent equitoxic doses. Thus immunoblot results with these
agents are presented for qualitative comparison only.
The DAC6-specific inhibitor tubacin  has been reported to
have multiple effects on lymphoid cells attributable to DAC6
inhibition in addition to inducing acetylation of tubulin and
HSP90. These include aggresome formation , motility ,
and cytotoxicity in EBV-positive lymphoma cells . We
therefore tested the effects of tubacin on CLL patient cells. No
significant effects on cell viability, as measured by MTT assay,
were noted at times up to 72 hr and concentrations up to 10 mM
(figure 3C), suggesting that the tubulin and/or HSP90 deacetyla-
tion activity of DAC6 is not by itself crucial for CLL cell survival.
However, these studies do not rule out a role for DAC6 inhibition
in combination with inhibition of other DACs in promoting CLL
AR-42 sensitizes CLL patient cells to apo2L/TRAIL
DAC inhibitors possessing class I inhibitory activity have shown
the potential to sensitize many types of tumor cells [33,34],
including CLL , to tumor necrosis factor-related apoptosis
inducing ligand (TRAIL). We therefore incubated CLL patient
cells with or without AR-42 and recombinant TRAIL and
examined the cells for apoptosis by annexin/PI flow cytometry.
F-ara A (active form of fludarabine) was used as a negative control.
AR-42 significantly increased the sensitivity of CLL cells to
TRAIL (figure 4A, n=6), as has been shown for class I DAC
inhibitors such as romidepsin [35,36]. We previously reported that
romidepsin resulted in reduction of the caspase-8 inhibitory
protein c-FLIP , potentially explaining sensitization to TRAIL
as described by MacFarlane et al. . We therefore investigated
Figure 2. AR-42 efficacy is independent of stromal survival factors but involves caspase activation. (A) CLL patient cells (n=8) were
incubated 16 hr with or without AR-42 (0.90 mM), then washed and cultured in the absence or presence of HS-5 stromal cells for a total of 48 hr. CLL
cells were collected and analyzed by flow cytometry. Data for each patient are shown individually (light dotted lines) as absolute percent annexin-
positive cells. Averages for each group are shown as a dark bar, connected by a solid line. The difference in HS-5 protection of CLL cells between
untreated and AR-42 treated was significant (p=0.016). (B) JeKo-1 cells (n=3) were incubated 24 hr with or without AR-42 (0.90 mM), romidepsin
(rom; 0.04 mM), F-ara A (5.0 mM) and Z-VAD-fmk (100 mM). Cells were analyzed by annexin/PI flow cytometry (top) or by immunoblot for PARP
(bottom). Results with 697 cells were similar. (C) CLL cells (n=5) were incubated with or without AR-42 (0.90 mM) and Z-VAD-fmk (100 mM). Cells were
analyzed by flow cytometry at 48 hr and percent live cells (annexin and PI negative) were calculated relative to time-matched untreated samples. Bars
represent +/2 standard deviation. In AR-42 treated cells, increase in live cells with Z-VAD-fmk was significant (p=0.0002). (D) Extracts from CLL cells
incubated 24 hr with or without AR-42 and Z-VAD-fmk were immunoblotted for PARP. Result is representative of seven CLL samples.
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the effect of AR-42 on c-FLIP in CLL patient cells. As seen with
romidepsin, AR-42 treatment of CLL cells resulted in notably
reduced levels of c-FLIP by 24 hr (figure 4B, representative of
seven CLL patient samples). This result was observed using a
c-FLIP monoclonal antibody from Enzo Life Sciences as used in
our earlier work , although no change in c-FLIP levels were
noted using a polyclonal c-FLIP antibody (#556567, BD
Pharmingen, San Diego CA) (data not shown). A similar
discrepancy was also reported by Inoue et al. . Therefore, in
addition to cell type and inhibitor differences, reagent differences
must also be considered when comparing these results with those
of other publications [34,36,37].
In vivo activity of AR-42
Given the promising pre-clinical data with AR-42 in CLL and
transformed B-leukemia cells, we sought to determine its in vivo
activity in this class of malignancies. Engraftment of the Raji
lymphoblastic lymphoma cell line into C.B-17 SCID mice
produces an aggressive disseminated B-cell lymphoma that results
in hind-limb paralysis requiring euthanasia approximately 15 days
post-inoculation . SCID animals received two million Raji
cells by tail vein injection, then were followed for three days prior
to initiating treatment with AR-42, vorinostat, or vehicle control
by oral gavage (figure 5A; n=11 for vehicle, n=5 for vorinostat,
and n=6 for AR-42 treatment). The median survival after the
initiation of therapy was 16 days (95% CI 15–18; CV=11%) for
mice treated with AR-42 (75 mg/kg Mon-Wed-Fri), vs. 12 days
(95% CI 11–14; CV=10%) for the control group, resulting in a
33% increase in median survival (p=0.001). In contrast, treatment
with the maximum tolerated dose of vorinostat in this model
(50 mg/kg every day) produced no survival benefit relative to
vehicle control animals.
Following this result, we evaluated the in vivo activity of AR-42
in an additional lymphoma model. The JeKo-1 MCL cell line
Figure 3. AR-42 exhibits class I and II DAC inhibitory activity. (A) CLL cells were incubated with or without AR-42 (0.90 mM) for the times
noted. Lysates from 697 cells incubated 16 hr without or with 0.04 mM romidepsin were included as controls. Extracts were analyzed by immunoblot
for acetylated tubulin and histone H3, plus GAPDH as a loading control. Data are representative of six patient samples. (B) CLL tumor cells were
incubated with or without DAC inhibitors romidepsin (0.04 mM), AR-42 (0.90 mM), or vorinostat (5.0 mM) as indicated. 697 cells incubated 24 hr with or
without 5.0 mM vorinostat were included as a control (left). Extracts were analyzed by immunoblot for acetylated tubulin and histone H3, plus GAPDH
as loading control. Data are representative of seven CLL samples. (C) CLL patient cells (n=4) were incubated without or with tubacin as indicated for
72 hr, and viability was analyzed by MTT assay. Data are shown relative to the time-matched untreated control. There were no significant changes in
viability across doses (p=0.265).
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[39,40] and its in vivo tumorigenicity has been previously described
[40,41]. Here, SCID mice engrafted with 40 million JeKo-1 cells
by tail vein injection were treated starting day 15 post-inoculation
either with AR-42 (20 mg/kg) or vehicle every third day by
intraperitoneal injection (n=5 per group). Mice receiving AR-42
showed a median survival of 20 days after the initiation of
treatment (95% CI 16–21; CV=11%), compared to 13 days (95%
CI 6–14; CV=13%) for the control group (p=0.003; figure 5B).
These studies in two aggressive models of human B-cell
lymphoma demonstrate the in vivo activity of AR-42 in B-cell
lymphoproliferative disorders. To explore the effects of AR-42 in a
more indolent leukemia, we utilized the Em-Tcl1 transgenic
leukemia mouse model previously described . These mice
develop disease very similar to that of CLL patients, including
chronic B-leukemic disease progression, elevated Igk+B cells,
splenomegaly, and infiltration of B-lymphocytes to the liver, lungs,
and kidney [42,43]. We employed a transplant model, in which
one million leukocytes from the enlarged spleen of a leukemic
Em-Tcl1 mouse were injected into a cohort of C.B-17 SCID mice
via tail vein, essentially as described by Wu et al. . Treatment
was initiated when leukemia was evident by a peripheral leukocyte
count of 20,000/mL averaged across the group (range 10,000–
60,000) and palpable spleens, which occurred at week 10 following
inoculation. At this point, mice were treated with vehicle or 75
mg/kg AR-42 (n=5 per group) Monday, Wednesday and Friday
for two weeks by oral gavage. AR-42 treatment resulted in a
significant reduction in peripheral blood lymphocytes, examined
two weeks after therapy initiation, relative to control mice
(p=0.0007; figure 5C). Leukemic mice treated with AR-42 also
had a significant survival advantage over vehicle-treated controls
(p=0.0251) with a median survival of 58 days (95% CI: 32–83;
CV=34%) after the initiation of therapy, compared to 37 days
(95% CI: 25–42; CV=25%) in the control group (figure 5D).
These studies utilizing three murine models of different types of B-
cell lymphoma collectively demonstrate in vivo activity of AR-42.
AR-42 is a novel class I and II DAC inhibitor that has shown
pre-clinical activity in a variety of solid tumor in vitro and in vivo
models . Here, we demonstrate that AR-42 has potent in vitro
and in vivo activity in multiple models of human B-cell malignancy
and provide data supporting its clinical development in this group
of diseases. Unlike other compounds whose efficacy is influenced
by human serum protein binding (e.g. flavopiridol), we found that
AR-42 has similar cytotoxic effect regardless of whether human or
bovine serum matrices are used. Importantly, we demonstrate that
AR-42 efficacy in CLL cells is not compromised by co-culture with
stromal cells, which have been widely shown to prevent
spontaneous apoptosis and mediate drug resistance in CLL tumor
cells . We validate the class I and class II DAC specificity of
AR-42 by demonstrating it promotes acetylation of histones and of
tubulin at concentrations that promote cytotoxicity in B-leukemia
cells, indicating its ability to inhibit both classes of DACs at
biologically relevant concentrations. AR-42 induces caspase-
dependent cell death, as cytotoxicity can be blocked by caspase
inhibition, although details of this mechanism remain to be
investigated. As shown with other DAC inhibitors, AR-42
augments the cytotoxic activity of TRAIL in CLL cells. This is
potentially due to reduction of c-FLIP protein, an effect we
previously reported in CLL cells using romidepsin . A study in
colon cancer cell lines  showed that the DAC inhibitor sodium
butyrate also caused substantial decrease in c-FLIP protein
concurrent with TRAIL sensitization, although similar studies in
several hematological cell lines using sodium butyrate and
vorinostat demonstrated TRAIL sensitization without reduction
of c-FLIP . The reason for differences in c-FLIP expression in
various cell types following DAC inhibitor treatment and the
importance of this in TRAIL sensitization remains unclear,
although antibody reagent differences must be considered as
reported here and also by Inoue et al. . Identifying biological
reasons for c-FLIP changes may shed light on the qualitative as
well as quantitative differences of the various DAC inhibitors, and
may guide future combination strategies. Regardless, these results
suggest the involvement of both the intrinsic and extrinsic
pathways of apoptosis in AR-42-mediated cytotoxicity in B-cells.
Importantly, AR-42 demonstrates in vivo activity in murine
models of Burkitt’s lymphoma, MCL, and CLL. With all three
models, increased survival with AR-42 is observed compared to
the vehicle control. Interestingly, in the Raji Burkitt’s lymphoma
Figure 4. AR-42 sensitizes CLL cells to TRAIL and mediates reduction of c-FLIP. (A) CLL patient samples (n=6) were incubated with or
without AR-42 (0.90 mM) and TRAIL (100 ng/mL). Romidepsin (0.04 mM) and F-ara A (2.0 mM) were included as positive and negative controls,
respectively. Cells were assessed by flow cytometry at 48 hr, and percent live (annexin and PI negative) cells were calculated relative to the time-
matched untreated samples. Co-incubation with AR-42 and TRAIL produced a significant reduction in cell viability (p,0.0001) compared to TRAIL or
AR-42 alone. (B) CLL cells were incubated 24 hr with or without AR-42, and lysates were analyzed for expression of c-FLIP. Romidepsin and F-ara A
were included as positive and negative controls, respectively. Example is representative of seven CLL samples.
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model, the class I/II DAC inhibitor vorinostat administered at its
maximum tolerated dose (MTD) lacked activity, whereas AR-42
showed statistically significant activity without discernable toxicity.
It should be noted that our selection of doses of each agent were
based on MTD in SCID mice as determined by weight loss greater
than 20%. We acknowledge that direct comparison of AR-42 and
vorinostat in vivo, even within the same model, is complicated by
potentially differing pharmacologic properties such as oral
absorption and half-life, as well as toxicities unrelated to weight
loss. Thus it remains to be determined whether this difference in
efficacy will be observed in leukemia patients. However, these data
collectively support future clinical development of AR-42 in the
treatment of lymphoid malignancies.
An important consideration with DAC inhibitors in the
treatment of hematologic malignancies is the development of
combination strategies with other targeted therapies. As has been
reported with other DAC inhibitors, AR-42 significantly sensitizes
CLL patient cells to TRAIL. This finding is important, as TRAIL
alone has little activity in CLL but also shows little or no toxicity
toward non-tumor cells. Thus, the combination of AR-42 and
TRAIL receptor agonists may provide improved clinical benefit
without substantial side effects. In particular, antibodies targeting
DR5 are quite attractive, as they have shown extended half-life.
The importance of dual inhibition of DAC classes I and II is
unclear. Most investigations of DAC inhibitors in B-cell malignancies
have used class I-specific inhibitors (e.g. romidepsin , entinostat
, and MGCD-0103 ). Clinical results in B-cell diseases using
each of these agents have been disappointing to date, although
vorinostat and romidepsin show significant activity in cutaneous T-
cell lymphoma and are FDA-approved for this purpose. Using
microarray analysis of CEM T-cell lymphoma cells treated with
vorinostat versus romidepsin , Peart et al. determined that the
In addition we observed no cytotoxic effect of DAC6 inhibition in
CLL patient cells, suggesting that acetylation of tubulin and/or
HSP90 is not required for DAC inhibitor-mediated cytotoxicity in
these cells. However AR-42 may influence other pathways controlled
by class II DACs, although these are not well defined. For example,
class II DACs can function as transcriptional co-repressors, and it is
possible that inhibition of these enzymes allows expression of genes
with pro-apoptotic effects . Based on the results presented here
Figure 5. AR-42 shows in vivo efficacy in multiple models of B-cell malignancy. (A) SCID mice were engrafted with two million Raji cells via
tail vein injection. Starting 3 days post-inoculation, mice were treated by oral gavage with vehicle only daily (n=11), vorinostat at the maximum
tolerated dose of 50 mg/kg daily (n=5), and AR-42 at 75 mg/kg (n=6) Monday, Wednesday and Friday. The median survival advantage in the AR-42
group relative to the vehicle control group was significant (p=0.001). (B) SCID mice were engrafted with forty million JeKo-1 cells via tail vein
injection. Starting on day 15 post-inoculation, mice were treated intraperitoneally with vehicle alone or 20 mg/kg AR-42 every three days (n=5 per
group). The improvement in median survival relative to control was significant (p=0.003). (C) SCID mice were engrafted with one million Em-Tcl1
leukemia cells via tail vein injection. When the circulating leukocyte count averaged 20,000 cells/mL across the group (dashed horizontal line; 69 days
post-inoculation), mice were randomized to two cohorts and treated Monday, Wednesday and Friday for two weeks with vehicle only (light bars) or
with AR-42 at 75 mg/kg via oral gavage (dark bars) (n=5, both groups). *The decrease in circulating leukocytes in AR-42 treated mice vs. controls at
week 12 relative to week 10 was significant (p=0.0007). (D) Animals in (C) were followed for survival (n=5 per group). The increase in median
survival in the AR-42 group relative to control was significant (p=0.025).
Deacetylase Inhibitor AR-42
PLoS ONE | www.plosone.org7 June 2010 | Volume 5 | Issue 6 | e10941
cell malignancies, we hypothesize that the more potent dual
inhibition of class I and II DACs allowed by AR-42 relative to other
available agents will produce clinical efficacy in B-cell leukemias
A major question arising from work with DAC inhibitors in
CLL and related B-cell lymphoid malignancies is whether there is
sufficient justification to pursue this class of drugs clinically. As
noted above, clinical investigations of DAC inhibitors in B-cell
malignancies have shown only modest activity. Romidepsin
produced a reduction in leukemic cell count in patients with
advanced CLL, but without partial or complete responses by NCI
criteria . Similarly, MGCD0103 was also studied in a phase II
trial including patients with relapsed CLL in which no clinical
responses were observed in 21 patients . In both studies,
significant fatigue and constitutional symptoms limited patient
willingness to continue therapy beyond 1–2 monthly treatments.
MGCD0103 has evidence of activity in other types of lymphoma,
as demonstrated by a preliminary phase II study of 38 patients in
which four responses were reported among follicular lymphoma
and large cell lymphoma subtypes . Also, in Hodgkin’s disease
a 40% response rate was observed among relapsed and refractory
patients . After a temporary hold to investigate pericarditis in a
subset of patients, clinical development of MGCD0103 continues.
In contrast to these class I DAC-specific agents, clinical
investigation of class I/II DAC inhibitors in B-cell malignancies
has been extremely limited. Vorinostat showed moderate activity
(four complete or partial responses out of 30 B-cell lymphomas) in
a study population that included multiple types of leukemia/
lymphoma . A phase II study of vorinostat in different types of
low-grade NHL demonstrated a 37% response rate in follicular
lymphoma and marginal zone lymphoma . In a preliminary
report, the class I/II DAC inhibitor panobinostat (LBH589)
produced a 38% objective response rate in Hodgkin’s disease .
To date, efficacy data of class I/II DAC inhibitors in CLL is nearly
non-existent, with just four CLL patients treated with vorinostat as
part of dose-escalation study in multiple types of leukemia .
Nonetheless, the encouraging in vitro and in vivo results reported
here and elsewhere with class I/II DAC inhibitors in B-cell
malignancies indicate that broader clinical exploration of these
agents is warranted.
Previous work by members of our group  and studies
described herein suggest that AR-42 has greater efficacy in vitro as
well as in vivo compared to vorinostat. These observations suggest
an improved potency and therapeutic index of AR-42 that will be
of key importance in the development of this agent, given the
widely observed constitutional symptoms observed with this class
of drugs. Also, AR-42 shares with vorinostat and panobinostat the
favorable property of oral availability, allowing far greater
feasibility and flexibility of administration. Pre-clinical pharma-
cology and toxicology also support clinical development (data not
shown), and an investigational new drug application has been
approved for a first-in-man study of AR-42. Based on these
collective findings, a phase I clinical trial of AR-42 in patients with
B-cell lymphoid malignancies including CLL is now underway.
Materials and Methods
Blood was obtained from CLL patients after obtaining written,
informed consent according to an Ohio State University
Institutional Review Board-approved protocol, in agreement with
the principles of the Declaration of Helsinki. All animal research
was reviewed and approved by The Ohio State University
Institutional Animal Care and Use Committee.
Patients, cell separation, and culture conditions
All patients previously received a diagnosis of CLL as defined by
National Cancer Institute (NCI) criteria, had elevated leukocyte
counts (.30,000 cells/mL), and were without treatment for at least
four weeks prior to blood collection. CD19-positive cells were
isolated from peripheral blood using Rosette-Sep reagents
(StemCell Technologies, Vancouver BC) and isolated by density
gradient centrifugation (Ficoll-Paque Plus; Pharmacia, Piscataway,
NJ). 697 cells were obtained from DSMZ (Braunschwieg,
Germany). Raji and HS-5  cell lines were obtained from
ATCC (Manassas, VA). The JeKo-1 MCL line [39,40] was the gift
of Dr. Raymond Lai (University of Alberta, Alberta, Canada). All
cells were cultured in RPMI 1640 supplemented with 10% heat-
inactivated fetal bovine serum (FBS), 100 U/ml penicillin and
100 mg/ml streptomycin (Sigma, St. Louis, MO), and 2 mM L-
glutamine (Life Technologies, Grand Island, NY), at 37uC and 5%
DAC inhibitors and other reagents
Romidepsin (depsipeptide) was obtained from the NCI.
Tubacin was the kind gift of Drs. Ralph Mazitschek and Stuart
Schreiber, The Broad Institute and Harvard University, Cam-
bridge MA. Vorinostat and AR-42 were synthesized in the
laboratory of Dr. Ching-Shih Chen, OSU College of Pharmacy.
Recombinant human TRAIL/Apo2L, used at 100 ng/mL, was
obtained from Cell Sciences, Inc. (Canton, MA).
bromide; Sigma) assays were performed as described . Cells
were incubated with or without drug for various times, and MTT
was added. Plates were incubated for an additional 24 hr before
processing and measuring by spectrophotometry. LC50and IC50
values were calculated using Prism software (GraphPad, San
Apoptosis and flow cytometric studies
After exposure to AR-42, cells were resuspended in buffer
containing annexin V-FITC and propidium iodide (PI) according
to the supplier’s instructions (BD Biosciences, San Diego, CA).
Annexin binding and PI positivity were assessed by flow cytometry
on a Coulter EPICS-XL. For caspase inhibition, 100 mM Z-VAD-
fmk (benzyloxycarbonyl valine-alanine-asparagine-fluoromethyl
ketone; MP Biomedicals, Aurora, OH) was added to cultures 15
minutes prior to drug addition.
Protein and mRNA quantification
Cell extracts were prepared as previously described . Total
protein in each sample was quantified using the BCA protein assay
(Pierce, Rockford, IL). Protein samples were separated along with
molecular weight markers (BioRad, Hercules, CA) by SDS-PAGE
and transferred onto nitrocellulose. Gel loading equivalence was
confirmed by Ponceau S staining (Sigma) of membranes and by
probing membraneswith a monoclonal
#MAB374, Millipore, Temecula CA). Blots were incubated with
chemiluminescent substrate (Pierce Super-Signal, Pierce) and
exposed to x-ray film or a ChemiDoc digital imaging system
(BioRad). Antibodies used were: acetylated histone H3 (#06-599,
Millipore), acetylated tubulin (#T7451, Sigma), Bcl-2 (#MO887,
Dako, Carpinteria, CA), polyADP-ribose polymerase (PARP)
(#AM30, EMD Biosciences, La Jolla, CA), and c-FLIP (#ALX-
804-428; Enzo Life Sciences, Plymouth Meeting PA). Real-time
Deacetylase Inhibitor AR-42
PLoS ONE | www.plosone.org8 June 2010 | Volume 5 | Issue 6 | e10941
RT-PCR was performed and analyzed as described  using
reagents, instruments and software from Applied Biosystems
(Foster City, CA).
In vivo studies
The use of C.B-17 SCID mice (Taconic Farm, Germantown,
NY) as a lymphoma model has been described . For cell line
engraftments, aliquots from the same culture of cells were
cryopreserved to ensure consistency of engraftments. Before
inoculation, cells were thawed and cultured for 10 days. Viability
was checked before engraftment to ensure greater than 90%
Raji Engraftment Model.
cells/ml in PBS at room temperature, and 26106cells were
inoculated via tail vein. Treatment began 3 days after engraftment.
AR-42and vorinostat were
methylcellulose w/v, 0.1% Tween 80 v/v, in sterile water). In
pilot studies, the maximum tolerated dose (MTD) of AR-42 and
vorinostat in these mice was determined to be 75 mg/kg and 50
mg/kg, respectively, when administered daily by oral gavage.
MTD was defined as the maximum dose resulting in weight loss of
less than 20% over the course of treatment. After engraftment,
mice were randomly placed into three groups that received the
following treatments: (a) vehicle alone, (b) AR-42 at 75 mg/kg
every other day, (c) vorinostat at 50 mg/kg daily. Mice received
treatment by oral gavage (10 ml/g body weight) for the duration of
the study. The mice were monitored daily and were sacrificed if
hind-limb paralysis, respiratory distress, or weight loss greater than
20% was observed. Survival (absence of the above criteria) was
used as an endpoint for this study.
Mantle cell lymphoma model.
models, 6–8 week old female C.B-17 SCID mice were used. Mice
were depleted of murine NK cells with intraperitoneal injections of
0.2 mg rat anti-mouse interleukin 2 (IL-2) receptor b monoclonal
antibodies (TMb1), one day before engraftment and then every
week, as described . Intravenous injection (in 200 ml of sterile
PBS) of 4.06107JeKo-1 cells results in a disseminated tumor after
3–4 weeks post injection and, without intervention, mice have a
mean survival of 28 days [40,41]. Starting 15 days post-injection
with JeKo-1 cells, a time when established tumor burden can be
documented in sentinel animals, mice (5 per group) received
vehicle alone (64% saline, 12% ethanol, 24% PEG-400) or AR-42
at 20 mg/kg every three days via intraperitoneal injection. The
end point of the study was survival as defined for the Raji SCID
Em-Tcl1 engraftment model.
of the Em-Tcl1 transgenic mouse as a CLL model has been
described [42,43]. An animal with a leukocyte count greater than
Cells were resuspended at 107
dissolvedin vehicle (0.5%
Similar to the previous
Development and validation
100,000/ml and with palpable splenomegaly was selected as a
donor for engraftment. Leukocytes were recovered from the spleen
of the donor, and one million cells were engrafted into C.B-17
SCID mice (female, age 6–8 weeks) via tail vein injection. Mice
were randomly placed into (a) vehicle alone, or (b) 75 mg/kg AR-
42 groups. Disease progression was monitored by peripheral
leukocyte count using blood smears in duplicate, read by workers
blinded to treatment group. Treatment began when both groups
reached an average of 20,000 cells/ml. AR-42 was administered
orally Monday, Wednesday, Friday for 2 weeks. Survival as noted
above was used as the endpoint for evaluation.
To test for differences between AR-42-treated cells in the
presence or absence of Z-VAD-fmk, a linear mixed effects model
was used to account for dependencies among samples from the
same patient. Main effects and differences were estimated from
this model. Linear mixed effect models were also used to test for
significant interactions between AR-42 and TRAIL. For assess-
ments of the effect of AR-42 pretreatment in CLL cells alone or
co-cultured with HS5 cells and differences in tumor load in Em-
TCL1 mice, outcomes were natural log-transformed to stabilize
variabilities among conditions and mixed effects models were then
applied to the data. From these models, relevant estimates with
95% confidence intervals were obtained. For survival assessments,
Kaplan-Meier estimates of the survival function for control and
AR-42-treated mice were generated. Median survival times with
95% confidence intervals were calculated, and the log-rank test
was used to compare the overall survival between the two groups.
P values of less than 0.05 were considered significant. All analyses
were performed using SAS/STAT software, Version 9.2 (SAS
Institute Inc., Cary, NC).
The authors are grateful to Drs. Ralph Mazitschek and Stuart Schreiber
(Howard Hughes Medical Institute Investigator) of Harvard University and
the Broad Institute, Cambridge MA for the gift of tubacin. We also thank
the members of our laboratory for helpful comments and the many patients
who donated blood for our studies.
Conceived and designed the experiments: DML LA DAW RAB JCB.
Performed the experiments: LA DAW MED RBE. Analyzed the data:
DML LA DAW MED RBE AJJ KAB CCH MAF MRP AL XZ DJ MRG
RAB JCB. Contributed reagents/materials/analysis tools: LA AJJ KAB
CCH MAF MRP DW SKK CC MRG CSC. Wrote the paper: DML LA
DAW RAB JCB.
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