HDAC-selective Inhibitor Cay10603 Has Single Anti-tumour Effect in
Burkitt’s Lymphoma Cells by Impeding the Cell Cycle*
Xiu-juan MA1
, Gang XU2
, Zhi-jie LI1
, Fang CHEN3
, Di WU1
, Jia-ning MIAO1
, Yue ZHAN1
, Yang FAN1#
Medical Research Center, Shengjing Hospital, China Medical University, Shenyang 110004, China
Department of Pediatrics, Shengjing Hospital, China Medical University, Shenyang 110004, China
Department of Hematology Laboratory, Shengjing Hospital, China Medical University, Shenyang 110004, China
Huazhong University of Science and Technology 2019
Summary: Histone deacetylases (HDACs) inhibitors are novel in cancer therapy nowadays.
HDAC6-selective inhibitors exert advantageous effects due to higher selectivity and less toxicity.
We explored the anti-tumor effect and the molecular mechanism of cay10603, a potent HDAC6
inhibitor in Burkitt’s lymphoma cells. Our study revealed cay10603 inhibited the proliferation
of Burkitt’s lymphoma cell lines, and induced caspase-dependent apoptosis. Cay10603 inhibited
the expression of CDKs and cyclins to impede cell cycle progression in both Burkitt’s lymphoma
cell lines. Cay10603 also showed the additive effect with vp16 notably. Our data presented the
promising anti-tumor effect of cay10603 in the Burkitt’s lymphoma therapy.
Key words: Burkitt’s lymphoma; HDAC6; cell cycle; apoptosis
Burkitt’s lymphoma (BL) is a highly-invasive
non-Hodgkin lymphoma which is the fastest growing
human tumour, and manifested as acute leukaemia.
BL can be categorized into Epstein-Barr-virus
(EBV)-associated endemic subtype, sporadic subtype
and immunodeficiency related subtype. Currently
multidrug chemotherapy is the principal means for
BL treatment, which is composed of doxorubicin,
alkylators, vincristine, and vp-16, etc.[1]. The high
toxicity of chemotherapy drugs in these regimens
limited the application to the older patients, leading
to the relapse and poor prognosis[2, 3]. Furthermore,
the immune suppression associated with the
intensive chemotherapeutic regimens also makes
the administration difficult for the endemic and
immunodeficiency related BL patients. Hence, less
toxic but effective therapeutic strategies are necessary.
In recent years, histone deacetylases (HDACs)
have been recognized as promising therapeutics
for human tumors[4, 5], especially in hematological
malignancy therapy[6–9]. HDACs regulate the dynamic
balance of acetylation and deacetylation of both histone
and non-histone to play critical role in the growth and
transformation of tumours. Eighteen HDACs have
been identified in humans, and are divided into four
classes: class I (HDAC1, 2, 3 and 8), class II (HDAC4,
5, 6, 7, 9 and 10), class III (SIRT1-7) and class IV
(HDAC11)[10]. In spite of two HDAC inhibitors
(HDACi), Romidepsin (FK228) and SAHA (vorinostat,
class I & II HDACi) being approved by the US Food
and Drug Administration (FDA) for cutaneous T-cell
lymphoma treatment, the cytotoxicity caused by the
pan-HDACi off-target effects limits its application in
research[11]. Inhibitors against specific HADC isomer
offer an advantage due to less toxicity. HDAC6 is
class IIb HDAC, which is located predominantly in
cytoplasm. The over-expression of HDAC6 suggests
poor prognosis and more aggressive course in B
and T cell lymphomas[12]. HDAC6 modulates the
acetylation of multiple non-histone substrates such as
tubulin, HSP90, p53, which were involved in cancer
development and progression[13, 14]. HDAC6 inhibitors
influence the biological behaviour of tumour cells by
regulating such key regulator. In many solid tumours,
HDAC6 was reported to be highly expressed, and
the anti-tumour effect of HDAC6 inhibitors were
also discussed[15, 16]. Cay10603 is a novel potent and
selective inhibitor of HDAC6, but not further explored
to date. Cay10603 was reported to be of high selectivity
and exert about 10-fold stronger effect than SAHA to
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Current Medical Science 39(2):2019 229
block pancreatic cancer cell growth[17]. It also showed
promising effect on inhibiting the proliferation in lung
adenocarcinoma cells[18]. However, the anti-tumour
effect of cay10603 in BL was not mentioned and the
related molecular mechanism remained unclear.
Our present study investigated the anti-tumour
effect of cay10603 on BL cells and the underlying
mechanism by using CA46 (no EBV infection)
and Raji cells (EBV infection). We demonstrated
cay10603 inhibited the proliferation of both BL cell
lines and induced apoptosis by activating caspase￾dependent apoptotic pathways. Cay10603 inhibited
the expression of CDK1/CDK2 and cyclins to impede
cell cycle progression. Furthermore, we also observed
cay10603 enhanced the chemo-sensitivity of vp16. Our
data reported for the first time the anti-tumour effect of
HDAC6-selective inhibitor cay10603 on BL cells, and
it provided the encouraging evidence for cay10603 as a
potential option for BL therapy.
1 MATERIALS AND METHODS
1.1 Cell Lines and Cell Culture
BL cell lines, CA46 and Raji cells were purchased
from the Cell Culture Center of Institute of Basic
Medical Sciences, Chinese Academy of Medical
Sciences (Beijing, China). CA46 cells were cultured
in RPMI-1640 (Gibco, China) containing 20% fetal
bovine serum (Corning, USA) and Raji cells were
cultured in RPMI-1640 (Gibco, China) containing
10% fetal bovine serum (Corning, USA). Other culture
conditions were the same for both cell lines, containing
1% penicillin and streptomycin (Hyclone, USA) at
37°C in 5% CO2 incubator.
1.2 Reagents and Antibodies
Cay10603 was provided by Selleck (USA).
Reagents were dissolved in DMSO (Sigma, USA),
and stored at –20°C until use. The vp16 was provided
by Hengrui Medicine Co., Ltd (China). Cell Titer
96® AQueous Non-Radioactive Cell Proliferation
Assay (MTS) was purchased from Promega (USA).
Apoptosis was quantified using the Annexin V-FITC
and Propidium Iodide (PI) binding assay, purchased
from Dojindo (Japan). Cell Cycle Detection Kit
was purchased from Key GEN BioTECH (China).
Antibodies against human Cyclin D1, Cyclin E, Cyclin
B, CDK1, CDK2 were provided by Wenleibio Co., Ltd
(China), and antibodies against human β-actin, cleaved
caspase 3, PARP, caspase 8 were obtained from Cell
Signaling Technology (USA). Antibodies against
human α-tubulin, acetyl-α-tubulin, HDAC6 were
obtained from ImmunoWay Biotechnology Company
(USA).
1.3 MTS Assay
The MTS assay was performed according to the
manufacturer’s specification, with slight modifications
on protocol described. BL cells were seeded in 96-well
plates in normal medium (2.5×104 cells/well in 100 μL/
well), then cay10603-containing medium was added to
the wells (50 μL/well up to a total volume 150 μL/well)
at concentrations ranging from 0.01 to 5 μmol/L for
24, 48 and 72 h. Subsequently, 20 μL of MTS reagents
were added to each well, after 4 h of incubation,
absorbance at 490 nm was measured. The percentage
of cell proliferation was calculated by dividing the
absorbance of treated BL cells by absorbance of
control cells within each group. All experiments were
conducted at 3 to 5 times.
1.4 Cell Cycle Assay
Cell cycle was quantified according to the
manufacturer’s specification with slight modifications,
using the Cell Cycle Detection Kit. Cells were seeded
in 24-well plates (1×106
cells/well in 3 mL/well). After
incubated with 2 μmol/L cay10603 for 12, 24 and 48
h, cells were harvested, washed with PBS and added to
500 μL of 70% ethanol at –20°C overnight. Then, the
cells were washed with PBS three times, and stained
with 50 μL RNase A for 30 min at 37°C, subsequently
stained with 450 μL PI for 30 min at 4°C. The stained
cells were analyzed for DNA content by flow cytometry
(FACS Calibur) and the percentage of cells in different
stages of the cell cycle was quantified.
1.5 Apoptosis Assay
Apoptosis was quantified using the Annexin
V-fluorescein isothiocyanate (FITC) and propidium
iodide (PI) binding assay, following the manufacturer’s
instructions (Dojindo, Japan), and analysed by flow
cytometry (FACS Calibur). CA46 and Raji cells were
exposed to 2 μmol/L cay10603 at a density of 3.3×105
cells/mL (1×106
cells/well in 3 mL/well) for 48 h. Cells
were harvested, washed with PBS, and added with 300
μL 1×binding buffers, then incubated with Annexin
V-FITC and PI in the dark at room temperature for 15
min. Annexin V+ cells were considered as apoptotic
cells, in which Annexin V+/PI- and Annexin V+/PI+
indicated early and late apoptosis respectively.
1.6 Western Blotting Analysis
Proteins were detected by Western blotting with
indicated antibodies. Briefly, BL cells were exposed to
cay10603 (2 μmol/L) for 12, 24 and 48 h, respectively.
Cells were harvested, washed and lysed with the RIPA
lysis buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl,
1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS,
protease inhibitor cocktail (Sigma, USA), 1 mmol/L
phenylmethylsulfonyl fluoride (PMSF). Total cell
lysates were denatured with 5× sample buffer. Protein
lysates (30 μg) were separated by SDS-PAGE gel and
transferred to PVDF membranes (Millipore, USA).
Membranes were incubated with primary antibodies
diluted according to manufacture’s recommendations
and horseradish peroxidase-conjugated goat anti￾rabbit or anti-mouse IgG (1:2000 dilutions). Signals
230 Current Medical Science 39(2):2019
were observed using enhanced chemiluminescence
substrate reagents (Amersham Life Science, USA)
and visualized by chemiluminescence imaging system.
The density for each band was analyzed via Gel-Pro
Analyser (version 4.0) software (Media Cybernetics,
USA).
1.7 Statistical Analysis
Statistical analyses were performed using
SPSS software (version 22.0). Data are presented as
mean±standard deviation (SD). All the experiments
were performed in triplicates and repeated at least three
times. The unpaired Student’s t-test was used for the
comparison of two groups, and P<0.05 was considered
to be significant.
2 RESULTS
2.1 Cay10603 Significantly Inhibited Proliferation
of Both CA46 and Raji Cells
HDAC6 was endogenously expressed in two BL
cell lines as shown in fig. 1A. Cay10603 acted as a
potent deacetylase inhibitor demonstrated by high
acetylation of target protein α-tubulin (fig. 1B). The
anti-proliferative effect of cay10603 on BL cells
was evaluated with escalating concentrations 0.01–5
μmol/L for 24–72 h. Cay10603 inhibited the growth
of CA46 and Raji cells in a dose- and time-dependent
manner (fig. 1C and 1D). Much stronger cell growth
inhibition was observed after 48 and 72 h treatment in
both cell lines. CA46 cells seemed more sensitive than
Raji cells, as shown in fig. 1C and 1D at 48 h. IC50 for
CA46 was 0.13 μmol/L, while 1.47 μmol/L for Raji
cells.
Fig. 1 HDAC6-selective inhibitor cay10603 inhibited growth of CA46 and Raji cells in a time- and dose-dependent manner
A: HDAC6 expressed in CA46 and Raji cells endogenously. B: The activity of HDAC6 was detected by Western blotting.
C: Cay10603 treatment inhibited proliferation of CA46 cells. Different concentrations of cay10603 (0.01–5 μmol/L) induced
growth inhibition in CA46 cells at 24, 48, and 72 h. D: Cay10603 treatment inhibited proliferation of Raji cells. Different
concentrations of cay10603 (0.01–5 μmol/L) induced growth inhibition of Raji cell lines at 24, 48, and 72 h. Data showed that
the mean±SD were representative of at least three independent experiments. Cay indicated cay10603.
2.2 Cay10603 Induced Cell Cycle Arrest in BL Cell
Lines
Cay10603 (2 μmol/L) treatment impeded cell
cycle progression of both BL cells but in different
patterns. After cay10603 treatment, CA46 cells
were arrested at G2/M and cell cycle was blocked
at G0/G1 phase rapidly, especially at 24 h after
cay10603 treatment (fig. 2A). As shown in fig. 2B,
cell population in G2/M phase was increased by
12.2% [(41.1±3.2)% vs. (28.9±3.0)%, P<0.01] 12 h
after cay10603 treatment, and increased by 42.6%
[(69.7±5.0)% vs. (27.1±6.3)%], P<0.01) at 24 h. As
a consequence, cell accumulation in sub-G0/G1 phase
appeared in cay10603-treated CA46 cells, and the cells
accumulated dramatically at 48 h. We observed the
cell population in sub-G0/G1 phase reached 38.4 folds
of DMSO-treated cells [(30.3±11.1)% vs. (0.8±0.2)%,
P<0.01], indicating an increase of apoptosis.
While in Raji cells, the same dose of cay10603
exerted effects on cell cycle weakly and mildly. After
cay10603 treatment for 48 h, cell cycle arrested in
G0/G1 and a block in G2/M phase were observed (fig.
2A). Cell proportion of Raji cells in G0/G1 phase was
increased by 17.5% [(78.0±1.5)% vs. (60.5±4.92)%,
P<0.01] while that in G2/M phase was decreased by
10.7% [(11.0±1.2)% vs. (21.7±4.0)%, P<0.05] at 48
h after cay10603 exposure. At 48 h, percentage of
cay10603-treated Raji cells in sub-G0/G1 phase was
increased from (2.1±0.8)% to (6.6±1.2)% (P<0.01).
2.3 Cay10603 Decreased Protein Expression of
Regulatory CDKs and Cyclins, Impairing Cell
Cycle Progression in BL Cells
Western blotting results revealed that cay10603
Cay Control Cay
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altered the expression of various CDKs and cyclins
in both cell lines. In CA46 cells, cay10603 resulted
in down-regulation of CDK1 (decreased by about
34.0%) and cyclin B (decreased by about 33.0%) as
early as 12 h and maintained in a low expression level
with time (fig. 3A), which was responsible for the cell
cycle arrest in G2/M. Except the critical alterations in
CA46 cells, we also observed reduced CDK2, cyclin
E and cyclin D1 within various exposure time. As for
Raji cells, we observed the down-regulation of both
CDK2 (reduced by 27.0% ) and CDK1 (reduced by
43.0%) 12 h after cay10603 treatment, and decreased
gradually to the minimum level at 48 h (reduced by
60.0% and 81.0%, respectively). At the same time,
expression of cyclin D1 (reduced by 43.0%) and cyclin
E (13.0% reduced) also decreased, but expression
of cyclin B was not significantly changed (fig. 3A),
responsible for the cell cycle arrest in G0/G1.
Fig. 2 Cay10603 induced cell cycle arrest in BL cells
A: Cell cycle profile of CA46 and Raji cell exposed to cay10603 (2 μmol/L) for 12, 24 and 48 h detected by flow cytometry. The
bars of M1, M2, M3 and M4 indicate the sub-G0/G1, G0/G1, S and G2/M phase, respectively. B: Cay10603 blocked cells cycle
progression of CA46 cells in G2/M phase. Quantification of the cell cycle distribution (%) of CA46 cells in different phases after
cay10603 treatment for 12, 24 and 48 h. C: Cay10603 blocked cells cycle progression of Raji cells in G0/G1 phase. Quantification
of the cell cycle distribution (%) of Raji cells in different phases after cay10603 treatment for 12, 24 and 48 h. Values represent
the mean±SD from three independent experiments. *
P<0.05; **P<0.01, ***P<0.001 vs. control group
CA46
Control
232 Current Medical Science 39(2):2019
2.4 Cay10603 Induced Caspase-dependent Apoptosis
in BL Cells
From the cell cycle analysis above, we noticed
cay10603 exposure (48 h) induced cell apoptosis. In
order to confirm the effect of cay10603 on inducing
apoptosis, we treated two BL cell lines with cay10603
and DMSO as control. The cell apoptosis was analyzed
by Annexin V/PI staining. Cay10603 increased
apoptosis of both cell lines at 48 h (fig. 4A). As shown
in fig. 4B, the apoptosis rate of CA46 cells increased
by 23.9 folds [(66.9±7.6)% vs. (2.8±1.0)%, P<0.001]
after cay10603 treatment. They appeared to be more
sensitive to cay10603 than Raji cells, which only
increased by 2.1 folds [(22.2±2.7)% vs. (10.8±2.2)%,
P<0.01]. In both cell lines, caspase-8, caspase-3
and PARP were cleaved after cay10603 treatment,
indicating the caspase cascade activation (fig. 4C).
After the caspase cascade was blocked by Q-VD-Oph,
a pan-caspase inhibitor, the activation of caspase-3,
caspase-8 and PARP was also blocked although both
cell lines were exposed to cay10603 (fig. 4C).
Fig. 4 Cay10603 activated caspase-dependent apoptosis in CA46 and Raji cells
A: Cay10603 induced apoptosis of CA46 and Raji cells detected by flow cytometry; B: Quantification of the apoptosis rate of
both BL cells. ***P<0.001 vs. control group; C: The cleaved caspase-8, cleaved caspase-3, and cleaved PARP were activated
after exposure to cay10603 (2 μmol/L) for 24 and 48 h. The expression levels of caspase-8, cleaved caspase-3, and PARP were
detected with corresponding antibodies by Western blotting. Q-VD-Oph (10 μmol/L) acted as an inhibitor for pan-caspase. Cay
indicated cay10603.
Fig. 3 Cay10603 reduced the expression of cell cycle regulatory
CDKs, cyclins in CA46 and Raji cells
Expression of CDK1, CDK2, cyclin B, cyclin D1, cyclin
E was detected with corresponding antibodies by Western
blotting. Representative data are shown, and tubulin
(α-tubulin) served as a loading control.
CDK1
Control 12 h 24 h
Current Medical Science 39(2):2019 233
2.5 Cay10603 Enhanced the Chemo-sensitivity of
Vp16
In both cells, combination of cay10603 with vp16
increased the apoptosis induced by vp16 alone (fig.
5A). As shown in fig. 5B, apoptosis caused by vp16
increased to 13.2 folds (71.1±2.8% vs. 5.4±1.0%,
P<0.001) in CA46 cells, while 2.1 folds (54.3±9.0%
vs. 26.0±0.42%, P<0.05) in Raji cells after combined
treatment with cay10603. Furthermore, the apoptosis
rate was also enhanced as compared with cay10603
alone [CA46, (71.1±2.8)% vs. (53.5±7.0)%; Raji,
(54.25±9.0)% vs. (24.0±0.5)%]. We then investigated
whether the caspase cascade was activated after
treatment of cay10603 combined with vp16. Our
results showed cay10603 promoted the caspase
cascade activation reliably as compared with vp16
alone, especially at 48 h (fig. 5C). The pan-caspase
inhibitor blocked the caspase-3/8 and PARP activation,
which proved the apoptosis was induced in caspase￾dependent manner (fig. 5D).
Fig. 5 Cay10603 increased the chemo-sensitivity of vp16 treatment in CA46 and Raji cells
A: Combination of cay10603 with vp16 increased the cell apoptosis compared with cay10603 or vp16 alone. B: Quantification
of the apoptosis rate of both cell lines. *
P<0.05, **P<0.01, ***P<0.001. C: The cay10603 (2 μmol/L) combined with vp16 (500
nmol/L) treatment for 24 and 48 h induced apoptosis by activating the caspase-dependent pathway. A panel of proteins with
caspase-8, cleaved caspase-3 and PARP were analysed by Western blotting. D: The Q-VD-Oph blocked the activation of caspase
cascade proteins. The expression of caspase-8, cleaved caspase-3 and PARP was detected after treatment with Q-VD-Oph (10
μmol/L) for 24 and 48 h, respectively. Representative data were shown. Cay indicated cay10603.
Control Cay Vp16 Cay+vp16
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Propidium iodide
234 Current Medical Science 39(2):2019
3 DISCUSSION
Considering the limitations of traditional
chemotherapy drugs, the research of targeted drugs for
treating BL is imperative. Many new targeted drugs
under development are promising in the treatment of
hematological tumors, such as the proteasome inhibitor
Carfilzomib[19]. In addition, chemotherapy resistance
is reversed when targeted drugs are combined with
chemotherapy drugs. Carfilzomib combined with
panobinostat (pan-HDACi) has entered Phase I clinical
trials in relapsed and refractory multiple myeloma[20].
However, the pan-HDACi off-target effect produces
severe cytotoxicity, which limits its application in
tumor therapy. HDAC6 selective inhibitors become
research hotspots due to their high efficiency and low
toxicity.
HDACs played a critical role in carcinogenesis and
tumour progression. HDAC6 influences the acetylation
modification of many critical regulatory targets
involved in cell fate[21–23]. Nowadays, HDAC6 selective
inhibitors attract much attention in personalized cancer
therapy because of the advantage of high selectivity to
tumour cells rather than normal cells[24–26]. In this study,
we elucidated for the first time the biological effects
of HDAC6-selectivity inhibitor cay10603 on BL cell
lines. For EBV infection is critical for BL, we chose
EBV infection cells, Raji cells and EBV infection free
cell line, CA46 to conduct our study. Raji cell is resistant
to apoptosis[27, 28], CA46 cell is reported to be resistant
to multiple death stimuli, for lacking of pro-apoptotic
proteins like Bax, Bak and Bcl-Xs although free of
EBV[29]. Our data indicated that cay10603 inhibited
the growth of both BL cell lines. CA46 cell was more
sensitive than Raji cell, for 48-h IC50 for CA46 cells
was nearly 10% of that of Raji cells. Cay10603 induced
apoptosis of both BL cell lines, especially for CA46
cell. We speculated the different sensitivity might be
related to the EBV infection state of cells. The cell type
of origin differed between EBV-positive and negative
BL, where EBV-positive BL arises from memory cells,
but EBV-negative disease might originate from an
earlier germinal centre counterpart[30]. We considered
the different response to cay10603 might result from
the infection of EBV or not, but the specific mechanism
needs further study.
We verified cay10603 induced apoptosis by
activating caspase-cascade (figs. 4 and 5). Furthermore,
we also revealed that cay10603 markedly increased
apoptosis induced by vp16, a common agent in BL
chemotherapeutic regimen, indicating that cay10603
is superior for BL chemotherapy, for improving anti￾tumour effect as well as lessening the toxicity of
chemotherapeutic agent. BL is a highly invasive non￾Hodgkin lymphoma with malfunctional regulation on
cell cycle control, which leads to rapid cell growth[31].
Thus focusing on the adjustment of cell cycle
progression may be a breakthrough point in BL therapy.
In present study, we proved that cay10603 induced
cell cycle arrest in BL cells, although two cell lines
differed in the phase of cell cycles. Cyclins activation
and CDK-cyclin complexe formation run the cell cycle
successfully and initiate mitosis[32–34]. In our data,
cay10603 down-regulated the expression of CDK1 and
cyclin B in CA46 cells, which induced the cell cycle
block at G2/M and hindered the cell cycle progression.
While the reduced expression of CDK2 and cyclin E in
Raji cells also interpreted the cell cycle arrest in G0/G1
phase. For there are diverse compensatory regulation
among CDKs-cyclins[35], we also observed several
other alterations on CDKs-cyclins in both cell lines
(fig. 3). Our result is consistent with the study of Pai
et al, which reported HDAC6 inhibitor blocked the cell
cycle in G0/G1 phase in lung cancer cells by promoting
the degradation of CDK2[36]. The altered expression
of CDKs-cyclins induced the cell cycle arrest, which
might be the major mechanism underlying the anti￾proliferation effect of cay10603 on BL. The detail
mechanisms by which cay10603 acts on CDKs-cyclins
in hematological malignancies requires further study.
Among the diverse key proteins HADC6 target,
p53 is a critical molecule regulating cell cycle and
apoptosis[37]. At least 30% of BL patients have
mutant p53 detected by biopsy, as have also been
found in CA46 and Raji cells[38]. It’s well known p53
acetylation was associated with cell cycle progression
and apoptosis[39–41]. We observed p53 of both BL
cell lines acetylated at lysine 381 (data not shown).
Furthermore, in CA46 cells, cay10603 also inhibited
the total expression as well as phosphorylated p53 at
Ser15 (data not shown). Above observations suggested
p53 might exert the anti-tumour effect on cay10603 by
regulating the cell cycle and the detailed mechanism
demands further study.
In present study, we verified for the first time the
anti-tumour effect of cay10603 on BL cells by impeding
the cell cycle. Our data provided encouraging evidence
for HDAC6-selective inhibitor cay10603 as a potential
option for BL therapy.
Conflict of Interest Statement
We declare no financial and personal relationships with
other people or organizations that can influence our work
inappropriately. There is no professional or other personal
interest in any product, service and/or company that could
be construed as influencing the position presented in the
manuscript entitled. All other authors have no conflict of
interest.
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