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 make the stock solution at a concentration of 10 mM for Adv and 5 mM for RCS. Doxorubicin (DOX) was purchased from Cayman Chemical Co. z-VAD-fmk was purchased from Peptide Institute (Japan).

The human oral squamous cell carcinoma cell line CAL27, the human pharyngeal squamous carcinoma cell line Detroit562, human tongue squamous carcinoma cell line UPCI-SCC154, the human breast mammary gland adenocarcinoma cell lines MCF7 and MDA-MB-231, and the human lung adenocarcinoma cell line 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 from the 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 treated with control medium containing less than 1% DMSO to match the amount of solvent brought in by the drug. All cell line experiments were conducted within 10 passages after thawing. Mycoplasma contamination was tested routinely using the e-Myco™ Mycoplasma PCR Detection kit ver.2.0 (iNtRON Biotechnology, Inc.).

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 gift from Dr Feng Zhang; plasmid cat. no. 48139; Addgene) with the following sequence (5′-CAC CGT CCA TTG CTT GGG ACG GCA A-3′), selected with puromycin for 2 days, and grown without puromycin to select single colonies. TP53-KO MCF7 cells were also established in the same way.

To establish CAL27 cells expressing H2B-mCherry and AcGFP-α-tubulin, CAL27 cells were infected with lentiviruses and positive clones were selected using puromycin 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).

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 UUA AGU GUU AAU AdT dT and antisense UAU UAA CAC UUA AAU AAG CdA dC; siHDAC6#3: sense GGU UUU UGC UUU UUC AAC UdT dT and antisense AGU UGA AAA AGC AAA AAC CdG dC; siHDAC6#4: sense GCA UAU GUA AUA AAG UAC AdT dT and antisense UGU ACU UUA UUA CAU AUG CdA dA; control siLuc: sense CUU ACG CUG AGU ACU UCG AdT dT and antisense UCG AAG UAC UCA GCG UAA GdT dT. siRNAs were diluted to 200 nM in Opti-MEM I (Thermo Fisher Scientific, Inc.). Transfection was performed using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Knockdown (KD) efficiency was assessed using western blotting.

The dead cell counts were assessed by staining with propidium iodide (PI) (FUJIFILM Wako Chemical Corp.), and the number of red fluorescent signals was counted using the IncuCyte ZOOM (Sartorius) automated live cell imaging system. The cells were treated with Adv with or without RCS in the presence of PI (2.5 µg/ml) for up to 48 h in 96-well plates in tetraplicate (30).

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 microscope (BZ-X800; KEYENCE Co.).

The cells were lysed using RIPA lysis buffer (Nacalai Tesque) added together with a protease and phosphatase 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, and then transferred onto Immobilon-P membranes (Millipore Corp.). These membranes were probed with primary antibodies, 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) were purchased 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), and anti-phospho-histone H2A.X (Ser139) Ab (#9718, 1/1,000) were purchased from Cell Signaling Technology, Inc.

CAL27/H2B-mCherry/AcGFP-α-tubulin cells were seeded on collagen-coated glass-bottom dishes (CELLview #627870, Greiner). The next day, 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 the cell fate.

All quantitative data are expressed as mean ± standard deviation (SD). Statistical analyses for cell death inhibition with z-VAD-fmk treatment, and cell death in 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-phase duration, the Kruskal-Wallis test followed by Dunn's multiple comparison test was used. Statistical significance was 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 number of cell deaths as the dependent variables and the concentration 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 to kill almost all cells, the analysis indicated an 'antagonistic effect', because the dead cell count in the combination treatment group was smaller than the dead cell count in each mono treatment group.

To assess the effect of the combined treatment of Adv and RCS on TP53-mutated HNSCC cells, namely CAL27, HSC-3, SAS, Detroit562, and OSC-19 cells, and HPV-positive UPCI-SCC-154 cells, which showed 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- and time-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 ubiquitous, breast cancer cell lines (MDA-MB-231 and MCF7) and a lung 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 was observed 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-mutated cells 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 as compared 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, we established TP53-KO MCF7 cells using the CRISPR-Cas9 system (Fig. S2A). Although wild-type MCF7 cells showed slight 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.

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 and observed their cell morphology. In response to drug treatment, CAL27 cells showed nuclear fragmentation and chromatin condensation (Fig. 3A), which are characteristic features of cells 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 the cleavage of caspase-3 in CAL 27 cells (Fig. 3B). Additionally, Adv-and RCS-induced cell death was abolished in the presence 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 catastrophe, which subsequently leads to apoptotic or non-apoptotic cell death (31). To investigate whether the co-administration of Adv and RCS enhanced mitotic catastrophe, we established CAL27 cells stably expressing AcGFP-α-tubulin and histone-H2B-mCherry to monitor mitosis. Time-lapse imaging showed that most cells treated with Adv could not complete mitosis and underwent cell death, which indicated mitotic 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 catastrophe. Of note, when CAL27 cells were simultaneously exposed to Adv and RCS, dead cell counts were increased and most 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.

Next, to address how RCS enhances Adv-induced mitotic catastrophe, we assessed the G2/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 phosphorylation of Chk1 (Ser345), which indirectly inactivates CDK1. This increased p-Chk1 presumably compensated for 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-administration of the two drugs resulted in further increase in γ-H2A.X expression, a marker of DNA double-strand breaks. These data suggest 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 the co-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 that observed 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 cells on 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). This suggests that some of the TP53-mutated cell lines may have gained a compensatory mechanism to p53 and are insensitive to co-administration of RCS with Adv.

It has been reported that although RCS selectively inhibits HDAC6 (IC₅₀=4.7 nM), it also inhibits HDAC1, 2, and 3 (IC₅₀=58, 48, and 51 nM, respectively) at higher concentrations (33). To clarify whether the enhanced cell death mediated by 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 HDAC6 (Fig. 5A). Although KD of HDAC6 by itself did not change 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 terms of cell death induction, treatment with Adv in HDAC6-KD cells significantly and synergistically increased cell death, as in the case of CAL27 cells concomitantly treated with RCS and Adv (Fig. 5C-E). This suggested that increased DNA 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.