INTERNATIONAL JOURNAL OF ONCOLOGY 60: 54, 2022

Ricolinostat enhances adavosertib‑induced mitotic catastrophe
in TP53‑mutated head and neck squamous cell carcinoma cells
KEITARO MIYAKE1, NAOHARU TAKANO2, HIROMI KAZAMA2, HIROYUKI KIKUCHI3,
MASAKI HIRAMOTO2, KIYOAKI TSUKAHARA1 and KEISUKE MIYAZAWA2
1

Department of Otorhinolaryngology, Head and Neck Surgery, Tokyo Medical University Hospital,
Shinjuku‑ku, Tokyo 160‑0023; Departments of 2Biochemistry, 3Preventive Medicine and Public Health,Tokyo Medical University, Shinjuku‑ku, Tokyo 160‑8402, Japan
Received August 19, 2021; Accepted March 10, 2022
DOI: 10.3892/ijo.2022.5344
Abstract. TP53 mutation is one of the most frequent gene muta‑
tions in head and neck squamous cell carcinoma (HNSCC) andcould be a potential therapeutic target. Recently, the WEE1
G2 checkpoint kinase (WEE1) inhibitor adavosertib (Adv) has
attracted attention because of its selective cytotoxicity against
TP53‑mutated cells and has shown promising activity in earlyphase clinical trials. In the present study, it was demonstrated
that combined treatment with Adv and a selective histone
deacetylase 6 (HDAC6) inhibitor, ricolinostat (RCS), syner‑gistically enhanced cell death induction in four out of five
HNSCC cell lines with TP53 mutation (CAL27, SAS, HSC‑3,
and OSC‑19), one HNSCC cell line with impaired TP53 func‑tion by HPV‑infection (UPCI‑SCC154), and TP53‑knockout
human lung cancer cell line (A549 TP53‑KO), but not in TP53
wild‑type A549 cells. Time‑lapse imaging showed that RCS
enhanced the Adv‑induced mitotic catastrophe. Consistent with
this, RCS treatment suppressed checkpoint kinase 1 (Chk1)(Ser345) phosphorylation and co‑administration of RCS with
Adv suppressed cyclin‑dependent kinase 1 (Tyr15) phosphory‑
lation along with increased expression of γ‑H2A.X, a marker
of DNA double‑strand breaks in CAL27 cells. These data
showed that RCS enhanced Adv‑induced premature mitotic
entry and cell death induction in the mitotic phase. However,
although HDAC6 knockdown enhanced Adv‑induced cell
death with γ‑H2A.X elevation, HDAC6 knockdown did not
repress Chk1 phosphorylation in CAL27 cells. Our data
demonstrated that the co‑administration of RCS with Adv
in HNSCC cells resulted in the suppression of Chk1 activity,
leading to synergistically enhanced apoptosis via mitotic
catastrophe in a p53‑dependent manner. This enhanced cell

Correspondence to: Dr Naoharu Takano, Department ofBiochemistry, Tokyo Medical University,
Shinjuku‑ku, Tokyo 160‑8402, JapanE‑mail: ntakano@tokyo‑med.ac.jp

6‑1‑1

Shinjuku,

Key words: mitotic catastrophe, p53, head and neck squamous cell
carcinoma, adavosertib, ricolinostat

death appeared to be partially mediated by the inhibition of
HDAC6 activity by RCS.
Introduction
Head and neck squamous cell carcinoma (HNSCC) is thesixth most common cancer in the world with approximately
800,000 cases being diagnosed every year (1). Advanced
HNSCC shows poor prognosis and resistance to standard
therapy consisting of cisplatin. Because 60‑80% of HNSCC
cases carry a TP53 mutation (2‑4) and human papillomavirus(HPV)‑positive HNSCC shows impaired p53 function (5),therapeutic targeting of the TP53 mutation has become afocus of research. Additionally, as p53 is functional in normal
somatic cells, targeted therapy against TP53 mutations canrelieve adverse effects. Thus, owing to its selective cytotoxicity
against TP53‑mutated carcinoma cells, adavosertib (Adv), a
WEE1 G2 checkpoint kinase (WEE1) inhibitor, has been the
recent focus of study (6,7).
WEE1 is a kinase that regulates replication stress and cell
cycle arrest at the G2/M checkpoint. DNA damage during
the S‑phase activates WEE1, which in turn phosphorylates
cyclin‑dependent kinase 1 (CDK1) at Tyr15 to inactivate its
kinase activity (8). Because p53 regulates the G1/S check‑point (9), TP53‑mutated cells rely on the G2/M checkpoint toarrest the cell cycle to repair their DNA damage. Therefore,
Adv treatment of TP53‑mutated cells leads to the cells entering
M‑phase without DNA repair, which in turn causes cell death
named mitotic catastrophe. Mitotic catastrophe is a type ofcell death characterized by DNA damage, abnormalities in the
mitotic apparatus, dysfunction of the mitotic checkpoint, andfailure in the occurrence of normal mitosis (10,11), but there
is no clear definition. As Adv abrogates cell cycle arrest, it
was suggested that the co‑administration of DNA‑damaging
drugs with Adv enhances cytotoxicity (7,12,13). In the present
study, we sought to identify drug combinations to enhance
Adv‑induced cytotoxicity without losing selective cell death in
TP53‑mutated cells.
It has been reported that histone deacetylase (HDAC)
inhibitors induce growth arrest, differentiation, and apoptosis
in vitro, and suppress tumor growth in xenograft mouse models
with various cancer cell lines including HNSCC (14‑16).

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MIYAKE et al: RICOLINOSTAT ENHANCES ADAVOSERTIB-INDUCED MITOTIC CATASTROPHE

Additionally, HDAC inhibitors have been shown to enhancecytotoxicity in combination with DNA‑damaging anticancer
drugs (17,18) or Adv (19‑21). We recently reported that rico‑linostat (RCS), a selective histone deacetylase 6 (HDAC6)
inhibitor, exhibits potent cell growth inhibition in HNSCC celllines in vitro in combination with the proteasome inhibitor
bortezomib (22). Of note, HDAC6 differs from other HDACs
by deacetylating cytoplasmic proteins, including α‑tubulin.
HDAC6 has also been reported to be involved in the cell
cycle via deacetylation of α‑tubulin (23). Additionally, inhibi‑tion of HDAC6 has been reported to sensitize cancer cells to
DNA‑damaging drugs (24‑26). As the disruption of the cell
cycle or DNA damage enhances mitotic catastrophe, in thepresent study, we assessed the effect of the combined treatmentof Adv and RCS on HNSCC cell lines with TP53 mutation(CAL27, SAS, HSC‑3, Detroit562, and OSC‑19) or impaired
p53 function by HPV‑infection (UPCI‑SCC154).
Materials and methods
Reagents. Adavosertib (AZD1775, MK‑1775, hereafter referred
to as Adv) was purchased from MedChemExpress. Ricolinostat(ACY‑1215, hereafter referred to as RCS) was purchased from
Selleck Chemicals. Adv and RCS were dissolved in dimethyl
sulfoxide (DMSO) (Wako Pure Chemical Industries) to makethe stock solution at a concentration of 10 mM for Adv and5 mM for RCS. Doxorubicin (DOX) was purchased from
Cayman Chemical Co. z‑VAD‑fmk was purchased from
Peptide Institute (Japan).
Cell lines and culture conditions. The human oral squamouscell carcinoma cell line CAL27, the human pharyngeal
squamous carcinoma cell line Detroit562, human tongue
squamous carcinoma cell line UPCI‑SCC154, the humanbreast mammary gland adenocarcinoma cell lines MCF7
and MDA‑MB‑231, and the human lung adenocarcinoma cellline A549 were purchased from the American Type Culture
Collection (ATCC). The human tongue squamous carcinoma
cell lines SAS, HSC‑3, and OSC‑19 cells were obtained fromthe JCRB Cell Bank (Osaka, Japan). A549 and MCF7 cells
possess wild‑type TP53. MDA‑MB‑231, CAL27, HSC‑3,
SAS, Detroit562, and OSC‑19 cells carry mutant TP53.
UPCI‑SCC‑154 cells are HPV‑positive and show impaired
p53 function. CAL27, Detroit562, MCF7, MDA‑MB‑231,
and A549 cells were cultured in Dulbecco's modified Eagle
medium (DMEM) supplemented with 10% fetal bovine
serum (FBS; Biosera) and 1% penicillin/streptomycin
solution (Wako Pure Chemical Industries). UPCI‑SCC154
cells were cultured in MEM supplemented with 10% FBS
and 1% penicillin/streptomycin solution. SAS and OSC‑19
cells were cultured in DMEM/ F12 medium supplemented
with 10% FBS and 1% penicillin/streptomycin solution.
HSC‑3 cells were cultured in Eagle's minimum essential
medium (EMEM) supplemented with 10% FBS and 1%
penicillin/streptomycin solution. All cell lines were cultured
at 37˚C in a humidified incubator containing 5% CO2 and
95% air. In all experiments, as a control, cells were treatedwith control medium containing less than 1% DMSO tomatch the amount of solvent brought in by the drug. All cellline experiments were conducted within 10 passages afterthawing. Mycoplasma contamination was tested routinely
using the e‑Myco™ Mycoplasma PCR Detection kit ver.2.0(iNtRON Biotechnology, Inc.).
Establishment of TP53‑KO A549 and TP53‑KO MCF7
cells. TP53‑KO A549 cells were established as previously
described (27). Briefly, A549 cells were transfected with
pSpCas9 (BB)‑2A‑Puro (PX459) V2.0 plasmid vector (a giftfrom Dr Feng Zhang; plasmid cat. no. 48139; Addgene) withthe following sequence (5'‑CAC​CGT​CCA​TTG​CTT​GGG​ACG​
GCA​A‑3'), selected with puromycin for 2 days, and grownwithout puromycin to select single colonies. TP53‑KO MCF7
cells were also established in the same way.
Establishment of CAL27/H2B‑mCherry/AcGFP‑ α‑tubulin
cells. To establish CAL27 cells expressing H2B‑mCherryand AcGFP‑ α ‑tubulin, CAL27 cells were infected withlentiviruses and positive clones were selected using puro‑
mycin and blasticidin. Lentiviruses were produced in 293T
cells (ATCC) by transfection of the following plasmids:
pMD2.G (gift from Dr Didier Trono: Addgene #12259),
psPAX2 (gift from Dr Didier Trono: Addgene #12260),
pLenti6‑H2B‑mCherry (gift from Dr Torsten Wittmann:
Addgene plasmid #89766) (28), and pLenti‑AcGFP‑α‑tubulin.
For pLenti‑AcGFP‑α‑tubulin construction, TUBA1A cDNA
was cloned into pAcGFP1‑Hyg‑C1 vector (Takara Bio Inc.),
and then, the AcGFP‑α‑tubulin fused gene was inserted into
the pLentiN vector (gift from Dr Karl Munger: Addgene#37444) (29).
RNA interference. For the gene silencing of HDAC6 in CAL
27 cells, HDAC6 siRNA and control siRNA were synthesized
as follows (Japan Bio Services Co., Ltd.): siHDAC6#2: sense
GCU​UAU​U UA​AGU​GUU​A AU​AdT​dT and antisense UAUUAA​CAC​U UA​A AU​A AG​CdA​dC; siHDAC6#3: sense GGU​UUU​UGC​U UU​U UC​A AC​UdT​dT and antisense AGU​UGA​AAA​AGC​A AA​A AC​CdG​dC; siHDAC6#4: sense GCA​UAU​
GUA​AUA​A AG​UAC​AdT​dT and antisense UGU​ACU​U UA​UUA​ CAU​ AUG​ C dA​ d A; control siLuc: sense CUU​ACG​
CUG​AGU​ACU​UCG​AdT​dT and antisense UCG​A AG​UAC​
UCA​G CG​UAA​GdT​dT. siRNAs were diluted to 200 nM in
Opti‑MEM I (Thermo Fisher Scientific, Inc.). Transfection
was performed using Lipofectamine RNAiMAX transfec‑
tion reagent (Thermo Fisher Scientific, Inc.) according to the
manufacturer's instructions. Knockdown (KD) efficiency wasassessed using western blotting.
Assessment of cell death. The dead cell counts were assessedby staining with propidium iodide (PI) (FUJIFILM Wako
Chemical Corp.), and the number of red fluorescent signals
was counted using the IncuCyte ZOOM (Sartorius) automatedlive cell imaging system. The cells were treated with Adv withor without RCS in the presence of PI (2.5 µg/ml) for up to 48 h
in 96‑well plates in tetraplicate (30).
Morphological assessment. The cells were spread on
glass slides using a Cytospin 4 Centrifuge (Thermo
Fisher Scientific, Inc.) to prepare glass slides, stained with
May‑Grünwald‑Giemsa and examined under a digital micro‑
scope (BZ‑X800; Keyence Co.).

INTERNATIONAL JOURNAL OF ONCOLOGY 60: 54, 2022

Western blotting. The cells were lysed using RIPA lysis
buffer (Nacalai Tesque) added together with a protease andphosphatase inhibitor cocktail (Nacalai Tesque). Equal
amounts of proteins (25 µg) were loaded onto the gels (7.5,
10 and 15% gels were used), separated by SDS‑PAGE, andthen transferred onto Immobilon‑P membranes (Millipore
Corp.). These membranes were probed with primary anti‑
bodies, such as anti‑p53 antibody (Ab) (sc‑126, 1/1,000),anti‑ β ‑actin Ab (sc‑47778, 1/1,000), anti‑HDAC6 Ab(sc‑11420, 1/1,000), anti‑acetylated α‑tubulin Ab (sc‑23950,
1/1,000), and anti‑ α‑tubulin Ab (sc‑5286, 1/1,000) werepurchased from Santa Cruz Biotechnology, Inc. Anti‑PARP
Ab (#9542S, 1/1,000), anti‑caspase3 Ab (#9665S, 1/1,000),anti‑phospho‑p53 Ab (#9286, 1/1,000), anti‑p21 Ab (#2947S,
1/1,000), anti‑phospho‑ATR Ab (#9947, 1/1,000), anti‑ATR
Ab (#2790, 1/1,000), anti‑phospho‑Chk1 (Ser345) Ab (#2348,
1/1,000), anti‑Chk1 Ab (#2360, 1/1,000), anti‑phospho‑Chk2(Thr68) Ab (#2197, 1/1,000), anti‑Chk2 Ab (#3440, 1/1,000),anti‑phospho‑Cdc2 (Tyr15) Ab (#4539, 1/1,000), anti‑Cdc2
Ab (#9116, 1/1,000), anti‑H2A.X Ab (#7631, 1/1,000), andanti‑phospho‑histone H2A.X (Ser139) Ab (#9718, 1/1,000)
were purchased from Cell Signaling Technology, Inc.
Assessment of mitotic catastrophes. CAL27/H2B-mCherry/
AcGFP‑ α‑tubulin cells were seeded on collagen‑coatedglass‑bottom dishes (CELLview #627870, Greiner). The nextday, the cells were treated with control medium, Adv, RCS,
or Adv + RCS, and time‑lapse images were obtained every
10 min using a confocal microscope LSM 700 equipped with
CO2, temperature, and humidity controller (Carl Zeiss) or
fluorescent microscope BZ‑X800 equipped with a time‑lapse
module (BZ‑H4XT) (KEYENCE). The cells were maintained
at 37˚C and 5% CO2, under humidified conditions. Time‑lapse
images from fluorescent microscopy were used to analyze thecell fate.
Statistical analysis. All quantitative data are expressed asmean ± standard deviation (SD). Statistical analyses for cell
death inhibition with z‑VAD‑fmk treatment, and cell deathin combination with Adv treatment and HDAC6 KD were
performed using two‑way ANOVA followed by Bonferroni's
multiple comparison test. For the analysis of the M‑phaseduration, the Kruskal‑Wallis test followed by Dunn's
multiple comparison test was used. Statistical significancewas set at P<0.05. All analyses were performed using
GraphPad Prism 5 software (GraphPad Software, Inc.). The
synergistic effect was assessed as follows. Based on nesting
all elapsed hours for each experiment, mixed‑effect linear
regression analyses were performed by setting the numberof cell deaths as the dependent variables and the concentra‑tion of the two anticancer drugs as independent variables.
To assess the possible synergistic effects of the two drugs,
the interaction term was added as one of the independent
variables in the model to calculate p‑for‑interaction. All
statistical analyses were performed using Stata version 17.0
(Stata Corp.). When the mono treatment was enough tokill almost all cells, the analysis indicated an ‘antagonistic
effect’, because the dead cell count in the combination treat‑ment group was smaller than the dead cell count in eachmono treatment group.

3

Results
Combined treatment of Adv and RCS synergistically inducescell death in TP53‑mutated HNSCC cells. To assess the effectof the combined treatment of Adv and RCS on TP53‑mutated
HNSCC cells, namely CAL27, HSC‑3, SAS, Detroit562, andOSC‑19 cells, and HPV‑positive UPCI‑SCC‑154 cells, whichshowed impaired p53, these cells were treated with Adv and
RCS for 48 h and PI‑positive dead cell number was measured
using a live cell imaging system. In HNSCC cells, treatment
with Adv and/or RCS induced cell death in a dose‑ andtime‑dependent manner. Notably, co‑administration of Adv
and RCS synergistically enhanced cell death in four out of five
TP53‑mutated HNSCC cell lines (except Detroit562 cells) as
well as in the HPV‑positive UPCI‑SCC‑154 cell line (Figs. 1
and S1). To address whether this synergistic effect was ubiqui‑
tous, breast cancer cell lines (MDA‑MB‑231 and MCF7) and alung cancer cell line (A549) were also treated with Adv and/or
RCS for 48 h (Figs. 1 and S1). Although co‑administration of
RCS with Adv led to enhanced cell death in MDA‑MB‑231
cells with TP53 mutation, little or no enhanced cell death wasobserved in MCF7 and A549 cells, both carrying wild‑type
TP53. Because Adv has been reported to induce mitotic
catastrophe in TP53‑mutated cells, we hypothesized that RCS
enhanced Adv‑induced mitotic catastrophe in TP53‑mutatedcells but not in wild‑type TP53 cells. To address this issue,
we next compared the induction of cell death between TP53
wild‑type and TP53 knockout (KO) A549 cells, which were
established using the CRISPR‑Cas9 system (Fig. 2A). Although
TP53‑KO A549 cells did not show altered sensitivity to Adv ascompared to wild‑type A549 cells, co‑administration of RCS
significantly enhanced cell death in TP53‑KO but not TP53‑WT
A549 cells (Fig. 2B). To further confirm this observation, weestablished TP53‑KO MCF7 cells using the CRISPR‑Cas9
system (Fig. S2A). Although wild‑type MCF7 cells showedslight synergistic cell death in the combined treatment of Adv
and RCS, TP53‑KO MCF7 cells showed increased synergistic
cell death by the combined treatment as shown in A549 cells(Fig. S2B). This indicated that the combination treatment of
Adv and RCS can synergistically induce cell death in cells
lacking p53 function.
Combined treatment of Adv and RCS in cancer cells leads tomitotic catastrophe. To identify the mechanism by which the
combined treatment of Adv and RCS leads to enhanced cell
death, we treated CAL27 cells with Adv and RCS for 24 h andobserved their cell morphology. In response to drug treatment,
CAL27 cells showed nuclear fragmentation and chromatincondensation (Fig. 3A), which are characteristic features ofcells undergoing apoptosis. Western blotting revealed that
Adv or RCS treatment induced the cleavage of caspase‑3,and co‑administration of the two drugs further increased thecleavage of caspase‑3 in CAL 27 cells (Fig. 3B). Additionally,
Adv‑and RCS‑induced cell death was abolished in the pres‑ence of a pan‑caspase inhibitor, z‑VAD‑fmk (Fig. 3C). These
data demonstrated that co‑administration of Adv and RCS
induced apoptosis. Adv is known to induce mitotic catas‑
trophe, which subsequently leads to apoptotic or non‑apoptotic
cell death (31). To investigate whether the co‑administrationof Adv and RCS enhanced mitotic catastrophe, we established4

MIYAKE et al: RICOLINOSTAT ENHANCES ADAVOSERTIB-INDUCED MITOTIC CATASTROPHE

Figure 1. Ricolinostat enhances adavosertib‑induced cytotoxicity in HNSCC cell lines. CAL27, UPCI‑SCC‑154, HSC‑3, SAS, MDA‑MB‑231, and A549 cells
were treated with Adv in combination with RCS for up to 48 h. Cells were monitored using IncuCyte live cell imaging system, and dead cell number wasassessed using PI staining. Time‑dependent and dose‑dependent cell death numbers are shown in the left and the middle panels, respectively. Representative
data of three independent experiments are shown. n=3, bar, mean ± SD. Synergistically enhanced cell death in the combination treatment was analyzed as
described in Materials and methods and summarized in the right panels. A significant synergistic effect with both Adv and RCS treatment with P<0.05 isshown in red. An antagonistic effect with P<0.05 is shown in blue. HNSCC, head and neck squamous cell carcinoma; Adv, adavosertib; RCS, ricolinostat.

CAL27 cells stably expressing AcGFP‑ α ‑tubulin andhistone‑H2B‑mCherry to monitor mitosis. Time‑lapse
imaging showed that most cells treated with Adv could notcomplete mitosis and underwent cell death, which indicatedmitotic catastrophe (Fig. 3D‑F). Additionally, Adv treatment

resulted in a prolonged duration of mitosis (Fig. 3G). On
the contrary, although RCS treatment induced cell death,most of the cell death occurred during interphase, and most
RCS‑treated cells did not enter metaphase (Fig. 3E and F).
This indicated that, unlike Adv, RCS hardly induced mitotic

INTERNATIONAL JOURNAL OF ONCOLOGY 60: 54, 2022

5

Figure 2. Combined treatment of adavosertib and ricolinostat enhances cell death only in TP53‑KO A549 cells. (A) TP53 KO in A549 cells was confirmed
by western blotting. Expression of p53, phospho‑p53, and p21 was assessed after doxorubicin (DOX) (1 µM) treatment for 24 h. β‑actin was used as a loading
control. Relative band intensity of p‑p53/p53 was calculated and is summarized in the right panel. (B) TP53‑WT and TP53‑KO A549 cells were treatedwith Adv in combination with RCS for up to 48 h. Cells were monitored using IncuCyte live cell imaging system, and dead cell number was assessed by PI
staining. Dose‑dependent and time‑dependent cell death numbers are shown. The synergistic effect on cell death in combination treatment was assessed and issummarized at the right. Representative data of three independent experiments are shown. n=3, bar, mean ± SD. Adv, adavosertib; RCS, ricolinostat.

catastrophe. Of note, when CAL27 cells were simultaneously
exposed to Adv and RCS, dead cell counts were increased andmost of the cell death occurred during mitosis (Fig. 3D‑F).
However, the duration of the mitotic phase until cell death was
not further extended as compared to the cells treated with Adv
alone (Fig. 3G). These data showed that RCS enhanced the
Adv‑induced mitotic catastrophe.
RCS promotes Adv‑induced mitotic entry along with theincrement of γ‑H2A.X expression. Next, to address how RCS
enhances Adv‑induced mitotic catastrophe, we assessed theG2/M checkpoint and DNA damage response (DDR)‑related
proteins by western blotting in CAL27 cells (Fig. 4A). WEE1, a
Ser/Thr protein kinase family member, phosphorylates CDK1
at Tyr15, inhibits its activity, and acts as a negative regulator
for entry into mitosis (G2 to M transition) (32). Thus, Adv
treatment decreased CDK1 (Tyr15) phosphorylation (Fig. 4A).
This indicated that CDK1 was activated, leading to premature
mitotic entry. At the same time, we observed increased phos‑
phorylation of Chk1 (Ser345), which indirectly inactivates
CDK1. This increased p‑Chk1 presumably compensatedfor the increased CDK1 activity. In contrast, RCS treatment
suppressed Chk1 phosphorylation. Thus, co‑administration of
RCS with Adv suppressed p‑Chk1 and p‑CDK1. This appeared

to further promote forced mitotic entry to induce mitotic
catastrophe. In support of these findings, co‑administrationof the two drugs resulted in further increase in γ‑H2A.X
expression, a marker of DNA double‑strand breaks. These datasuggest that RCS enhanced Adv‑induced mitotic catastrophe
by suppressing p‑Chk1 and p‑CDK1 (Fig. 4A).
As shown in Fig. 1, the pronounced cell death induction
by combined treatment of Adv and RCS appeared to be
dependent on TP53 mutations. We examined whether theco‑administration of RCS suppressed p‑Chk1 in both WT and
TP53‑KO A549 cells (Fig. 4B). In both cells, Adv treatment
increased p‑Chk1 and suppressed p‑CDK1, similar to thatobserved in CAL27 cells. Although co‑administration of RCS
with Adv suppressed p‑CDK1 and p‑Chk1 in both cell lines,
γ‑H2A.X expression was increased only in TP53‑KO A549
cells. This may reflect the dependency of TP53‑mutated cellson the G2/M checkpoint. These data also suggest that RCS
enhances premature mitotic entry by suppressing p‑Chk1 in
A549 cells. Additionally, we assessed the DDR‑related protein
expression in Detroit562 cells which carry mutated‑TP53 but
did not show pronounced cell death by combined treatment of
Adv and RCS. Detroit562 cells showed almost the same result
as WT A549 cells and failed to further upregulate γ‑H2A.X
expression as compared to treatment with Adv alone (Fig. S3).

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MIYAKE et al: RICOLINOSTAT ENHANCES ADAVOSERTIB-INDUCED MITOTIC CATASTROPHE

Figure 3. Co‑administration of ricolinostat enhances adavosertib‑induced mitotic catastrophe in CAL27 cells. (A) CAL27 cells were treated with control, Adv(0.5 µM), RCS (5 µM), or Adv+RCS for 24 h and then stained with May‑Grünwald‑Giemsa stain. Scale bar, 25 µm. Arrowheads indicate nuclear chromatincondensation. (B) CAL27 cells were treated with control, Adv (0.5 µM), RCS (5 µM), and Adv + RCS for 24 h, and then, cleavage of PARP and caspase‑3
were assessed by western blotting. (C) CAL27 cells were treated with control, Adv (0.5 µM), RCS (5 µM), or Adv + RCS in the presence of z‑VAD‑fmk(0, 25, and 50 µM) for 24 h, and dead cell number was monitored using IncuCyte live cell imaging system by PI staining. n=7, bar, mean ± SD; *P<0.05 vs. 0 µM
z‑VAD‑fmk. (D) Live cell imaging of CAL27 cells expressing AcGFP‑α‑tubulin and Histone H2B‑mCherry. Cells were treated with control, Adv (0.5 µM),
or Adv + RCS (5 µM) and monitored using confocal microscopy. Representative images of cells after the nuclear envelope breakdown (NEBD) are shown.
Time after the NEBD is shown in the upper right corner of each image. Scale bar, 20 µm. (E) Five representative cell fates in each treated cell with control,
Adv (0.5 µM), RCS (5 µM), or Adv + RCS are shown in a tree diagram. (F) The ratio of cell death in mitosis or interphase in each treated cell is summarized
in the pie chart. n for each condition is shown at the bottom. Data from three independent experiments are summarized. (G) Time from NEBD to telophaseor cell death was assessed and summarized. n=75, 59, 29, 46. Data from three independent experiments are summarized. *P<0.05 vs. the control. PARP,
poly(ADP‑ribose) polymerase; Adv, adavosertib; RCS, ricolinostat.

This suggests that some of the TP53‑mutated cell lines may
have gained a compensatory mechanism to p53 and are insen‑
sitive to co‑administration of RCS with Adv.

RCS enhanced cell death induction via inhibition of HDAC6.
It has been reported that although RCS selectively inhibits
HDAC6 (IC50 =4.7 nM), it also inhibits HDAC1, 2, and 3

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Figure 4. Ricolinostat suppresses phosphorylation of Chk1 and further suppresses p‑CDK1 when co‑administered with adavosertib. (A) CAL27 cells and(B) TP53‑WT and TP53‑KO A549 cells were treated with Adv (0.5 µM), RCS (5 µM), and Adv+RCS for 24 h (CAL27 cells) or 48 h (A549 cells), and thenthe expression of DNA damage response‑related proteins (p‑Chk2, Chk2, p‑ATR, ATR, p‑Chk1, Chk1, p‑CDK1, CDK1, and γ‑H2A.X) was assessed bywestern blotting. To assess the inhibitory effect of HDAC6 by RCS, the level of acetylated (ac)‑ α‑tubulin was monitored. Expression of β‑actin was assessedas the loading control. The relative band intensity of each phosphorylated protein was calculated and summarized at the right. Representative data of three
independent experiments are shown. Adv, adavosertib; RCS, ricolinostat; WT, wild‑type; Chk, checkpoint kinase; ATR, ATR serine/threonine kinase; CDK1,
cyclin‑dependent kinase 1.

(IC50 =58, 48, and 51 nM, respectively) at higher concentra‑
tions (33). To clarify whether the enhanced cell death mediatedby RCS was due to the inhibition of HDAC6, we knocked

down HDAC6 using three different siRNAs (siHDAC6 #2, #3,
and #4). All three siRNAs efficiently knocked down HDAC6
and increased acetylated α‑tubulin, which is a substrate for8

MIYAKE et al: RICOLINOSTAT ENHANCES ADAVOSERTIB-INDUCED MITOTIC CATASTROPHE

Figure 5. HDAC6 knockdown (KD) enhances adavosertib‑induced cell death in CAL27 cells. (A) Knockdown efficiency of HDAC6 in CAL27 cells with threedifferent HDAC6 siRNAs was assessed by western blotting. (B) CAL27 cells were transfected with siLuc or three different siHDAC6 siRNAs (#2‑4) and thentreated with or without Adv (1 µM) for 24 h. Expression of DDR‑related proteins was detected by western blotting. Expression of HDAC6 and acetylated(ac)‑α‑tubulin were monitored to assess the KD efficiency. Representative data of three independent experiments are shown. (C and D) CAL27 cells weretransfected with siLuc or three different siHDAC6 siRNAs (#2-4) and then treated with various concentrations of Adv up to 24 h. Cells were monitored using
IncuCyte live cell imaging system, and dead cell number was assessed using PI staining. Dose‑dependent (C) and time‑dependent (D) cell death numbers areshown. Representative data of three independent experiments are shown. n=3, bar, mean ± SD. *P<0.05 vs. siLuc. (E) The synergistic effect of cell death in the
combination of Adv treatment and HDAC6 KD shown in (C) and (D) was assessed and is summarized. Adv, adavosertib; RCS, ricolinostat; HDAC6, histone
deacetylase 6; DDR, DNA damage response.

HDAC6 (Fig. 5A). Although KD of HDAC6 by itself did notchange p‑Chk1 levels or γ‑H2A.X expression levels, Adv
treatment in HDAC6‑KD cells resulted in increased γ‑H2A.X
expression, as in the case of CAL27 cells treated with RCS and
Adv (Fig. 5B). However, we did not observe the suppression of

p‑Chk1 or p‑CDK1 in Adv‑treated HDAC6‑KD cells. In termsof cell death induction, treatment with Adv in HDAC6‑KD
cells significantly and synergistically increased cell death, asin the case of CAL27 cells concomitantly treated with RCS
and Adv (Fig. 5C‑E). This suggested that increased DNA

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9

Figure 6. Schema of the effect of adavosertib and ricolinostat on the G2/M checkpoint. In TP53‑mutated cells, DNA repair is dependent on the G2/M
checkpoint regulated by CDK1. CDK1 is a key regulator inducing cell cycle progression through the G2/M checkpoint. WEE1 phosphorylates and inhibits
CDK1. Adavosertib inhibits WEE1 and then CDK1 is dephosphorylated and activated. Chk1 inhibits Cdc25, which dephosphorylates and activates CDK1.
Ricolinostat decreases the phosphorylation of Chk1, indicating inhibition of Chk1. Chk, checkpoint kinase; ATR, ATR serine/threonine kinase; ATM, ATM
serine/threonine kinase; CDK1, cyclin‑dependent kinase 1; HDAC6, histone deacetylase 6; WEE1, WEE1 G2 checkpoint kinase.

damage and enhanced cell death by co‑administration of RCS
with Adv appeared to have been caused by HDAC6 inhibition,
but the enhanced mitotic catastrophe caused by RCS addition
to Adv treatment may be independent of HDAC6 inhibition.
Discussion
In the present study, the combined WEE1 G2 checkpoint
kinase (WEE1) inhibitor adavosertib (Adv) and the histone
deacetylase 6 (HDAC6)‑selective inhibitor ricolinostat (RCS)
treatment exhibited synergistic cytotoxicity in TP53‑mutated,
impaired p53 function by HPV‑infection, or TP53‑KO
HNSCC, lung cancer, and breast cancer cell lines likely via
enhanced induction of Adv‑induced mitotic catastrophe(Fig. 6). WEE1 phosphorylates the amino acids Tyr15 andThr14 of CDK1, which keeps the kinase activity of CDK1 low
and prevents entry into mitosis (32). As p53 regulates the G1/S
checkpoint, TP53‑mutated cells rely on the G2/M checkpoint
to arrest the cell cycle to repair their DNA damage. Thus,
TP53‑mutated cells with increased replication stress appeared
to exhibit greater sensitivity to WEE1 inhibitors (34,35). The
synergistic effect of the two‑drug combination shown in Fig. 1
appeared to be mediated through forced entry of the cell into
M phase by CDK1 activation. Indeed, the two‑drug combina‑
tion resulted in enhanced dephosphorylation of CDK1, which
is an active state of CDK1, as compared to that in the treat‑ment of Adv alone. This was accompanied by pronounced
γ‑H2A.X expression, which indicated an increase in DNA
double‑strand breaks in TP53‑mutated cells (Fig. 4A and B).

In Adv‑treated cells, a compensatory mechanism appeared
to be induced, leading to an increased expression of p‑Chk1.
However, combination treatment with RCS suppressed p‑Chk1
upregulation and further activated CDK1 to promote prema‑ture mitotic entry (Figs. 4 and 6).
Adv is an orally available, first‑in‑class, reversible WEE1
inhibitor (36). Most clinical trials using WEE1 inhibitors
have been conducted as a combination with chemotherapy orradiotherapy (37‑40). In the case of WEE1 inhibitor mono‑
therapy against recurrent uterine serous carcinoma, which
frequently possesses TP53 mutation, the treatment showedsignificant elongation of progression‑free survival (PFS) anda high response rate (41). Consistent with our results (Figs. 1
and 2), several previous studies have reported that sensitivity
to Adv is dependent on TP53 mutation status (6,7,42), whereasa few other reports have shown that TP53 status does not alter
sensitivity to Adv (43). This may suggest that TP53 single genemutation by itself is not sufficient to determine sensitivity to
WEE1 inhibitor monotherapy.
HDAC6 is a unique member of the HDAC subfamily
possessing two catalytic domains, namely DAC1 with
E3‑ligase activity and DAC2 with deacetylase activity (44).
HDAC6 deacetylates histones; however, cytoplasmic proteins,
namely α‑tubulin, cortactin, and heat shock protein 90(HSP90), are also deacetylated by HDAC6 (45‑47). The rolesof HDAC6 in tumorigenesis and cell‑cycle progression havebeen reported (23,48,49). The overexpression of HDAC6 has
been reported in acute myeloid leukemia, oral squamous cell
carcinoma, and ovarian cancer (50‑52), and higher HDAC610MIYAKE et al: RICOLINOSTAT ENHANCES ADAVOSERTIB-INDUCED MITOTIC CATASTROPHE

expression appears to correlate with tumor progression andmalignancy in hepatocellular carcinoma and esophageal
squamous cell carcinoma (53,54). In our study, HDAC6 inhibi‑tion by RCS suppressed cell cycle progression (Fig. 3), which
was consistent with previous reports showing that RCS orother HDAC inhibitors caused G2/M phase arrest (53,55). In
contrast, in the presence of Adv + RCS, the cells underwent
a pronounced mitotic catastrophe, indicating increased entry
into the M‑phase. The molecular mechanisms underlying thiscontradictory phenomenon remain unclear.
Our study demonstrated that HDAC6 inhibition by RCS
suppressed p‑Chk1 expression (Fig. 4A and B), whereas
HDAC6 KD slightly increased Chk1 (Fig. 5B). Thus, suppres‑sion of p‑Chk1 by RCS seemed to be HDAC6‑independent.
However, as described above, in addition to deacetylase
activity, HDAC6 possesses E3‑ligase activity, which ubiquiti‑nates and degrades Chk1 and functions in DDR. Therefore, it
was reported that HDAC6 deletion resulted in an increase in
Chk1, which enhanced radiation‑induced cell cycle arrest innon‑small cell lung cancer cells (44). In our system, although
HDAC6 KD suppressed both activities, RCS interacted onlywith DAC2 and E3‑ligase activity localized in DAC1 appeared
to be intact. If the increase in Chk1 due to DAC1 inhibition
overcomes the inhibition of p‑Chk1 caused by DAC2 inhibi‑tion, the HDAC6 KD experiment alone is not sufficient todetermine whether RCS suppresses p‑Chk1 via HDAC6. To
address this, we need to further evaluate the specific disrup‑tion/deletion of the DAC2 domain in HDAC6.
In terms of the Chk1‑CDK1 axis, Adv treatment of
HDAC6‑KD cells resulted in increased γ‑H2A.X and cell
death despite no decrease in p‑Chk1 or p‑CDK1 (Fig. 5). This
indicates that the enhanced cell death by siHDAC6 or RCS
is not limited to be mediated by Chk1‑CDK1. As previously
reported, we also showed that RCS treatment and HDAC6
KD resulted in α‑tubulin acetylation (Figs. 4 and 5) (45). It is
well‑known that post‑translational modifications of tubulins
contribute to microtubule dynamics, which are crucial for
proper spindle organization and cell cycle progression (56).
CYLD, a tumor suppressor gene product, has been reported tobind and inactivate HDAC6 along with increased acetylationof tubulin, which negatively regulates cell‑cycle progres‑sion (23). In addition, tubastatin A, an HDAC6 inhibitor, hasbeen reported to disrupt maturational progression and meiotic
apparatus assembly in mouse oocytes (57). Therefore, it is still
possible that HDAC6 inhibitors including RCS cause abnormal
mitosis by constitutively enhancing the acetylation of tubulins,
leading to disruption of microtubule dynamics. This may also
play a role in promoting the Adv‑induced mitotic catastrophe.
To the best of our knowledge, this is the first report showingthat RCS with a higher selectivity against HDAC6 suppresses
p‑Chk1. Although suppression of p‑Chk1 by RCS may be
independent of HDAC6, enhanced DNA damage and cell
death were observed in both RCS‑treated and HDAC6‑KD
cells in combination with Adv treatment. As prominentcell death in Adv + RCS‑treated cells was only observed in
TP53‑mutated and TP53‑KO cells, this drug combination
appears to be much less toxic to normal cells with intact TP53
than in TP53‑mutated cancer cells. Applying this drug combi‑
nation in the experiment with the tumor xenograft model
appears to have potential for future research. Although in vivo

studies remain to be carried out, this drug combination withhigh specificity for cells lacking p53 function could be a goodcandidate for the treatment of HNSCC patients carrying TP53
mutations as well as human papillomavirus (HPV)‑positive
HNSCC patients lacking p53 function.
Acknowledgements
Not applicable.
Funding
This study was supported by JSPS KAKENHI Grant
Number 20K07298 (to NT) and the Cancer Research Grant
afforded by the Tokyo Medical University Cancer Research
Foundation (to KM).
Availability of data and materials
The datasets used during the present study are available fromthe corresponding authors upon reasonable request.
Authors' contributions
KM (Miyazawa) and NT conceived and designed the present
study. KM (Miyake), NT and HK (Kazama) performed theexperiments. KM (Miyake), NT and HK (Kikuchi) analyzed,
interpretated the data. KM (Miyake), NT and KM (Miyazawa)
confirmed the integrity of the data. KM (Miyazawa), NT,
MH, KT and KM (Miyake) wrote, reviewed and/or revised
the manuscript. All authors read and approved the manuscript
and agree to be accountable for all aspects of the research inensuring that the accuracy or integrity of any part of the work
are appropriately investigated and resolved.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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