Cell lines

Human cancer cell lines (HH [CTCL], ATCC_CRL-2105 and MOLT-4 [acute lymphoblastic leukemia], ATCC_CRL-1582) were purchased from the American Type Culture Collection (Manassas, VA, USA). DND-41 (T-cell acute lymphoid leukemia) was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). All cell lines were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2.

Reagents

BV was obtained from Takeda Pharmaceutical Company Limited (Tokyo, Japan). IgG was purchased from GeneTex (Irvine, CA, USA). Fourteen candidate drugs were assessed as potential synergistic partners of BV. Belinostat, romidepsin, and darinaparsin were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Pralatrexate, chidamide, deoxycoformycin, dexamethasone, lenalidomide, gemcitabine, doxorubicin, etoposide, and MK2206 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Selinexor was purchased from Ark Pharm (Arlington Heights, IL, USA). Nelarabine was purchased from Sigma-Aldrich (St Louis, MO, USA).

Cell viability assays

To assess cell viability, HH (4 × 103 cells/well), DND-41 (4 × 104 cells/well), and MOLT-4 (3 × 104 cells/well) cells were seeded and cultured for 24 h and then treated with BV (0.0068, 0.020, 0.068, 0.20, 0.68, 2.0, 6.8, 20 and 68 nM) for 72 h. Adenosine 5′-triphosphate content was detected using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) and luminescence was measured by ARVO X light (PerkinElmer, Waltham, MA, USA). Half-maximal inhibitory concentrations (IC50) were calculated using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).

Flow cytometry analysis

Cells were collected and incubated for 30 min at 4 °C with phycoerythrin (PE)-labeled mouse anti-human CD30 antibodies (#130-098-686, Miltenyi Biotec, Bergisch Gladbach, Germany), with isotype-matched control antibodies (#130-092-213, Miltenyi Biotec), or without antibodies. After washing with phosphate-buffered saline, cell-associated fluorescence was detected by the BD LSRFortessa Cell Analyzer (Becton Dickinson, Franklin Lakes, NJ, USA), and CD30 expression levels were analyzed using FlowJo software (Becton Dickinson).

Matrix concentration screening test

HH cells were seeded and treated with BV or one of the 14 other anticancer drugs, either as single agents or in combination, using a 7 × 10 concentration matrix for 72 h. Cell viabilities were detected and evaluated as described in the ‘Cell viability assays’ subsection. The concentration ranges were 0.012–4.1 nM for BV and 0.001–10 µM for each of the 14 other anticancer drugs and the reproducibility study for chidamide was conducted using proper concentration ranges (0.004–1.2 nM for BV and 0.001–10 µM for chidamide). Commercially obtained IgG (0.004–1.2 nM) was used as the negative control for BV. Optimal molar concentration ratios (BV:chidamide = 1:2500 and 1:25,000) were used for isobologram analysis. The anticancer effects of each drug pair were scored by the R package SynergyFinder 1.8.0. [16, 17], in which the difference between the actual effect and the expected effect was calculated based on the Bliss model and expressed as a Bliss score. The expected effect represented the additive effect estimated from the anticancer activity of each drug (A and B). For example, Bliss score C = A + B − A × B. A, B, and C were percentage fractional inhibitions; thus, they represent the magnitude of synergistic or antagonistic effects, corresponding to positive or negative values, respectively. Combination indices (CIs) were calculated using CalcuSyn Version 2.0 (BIOSOFT, Cambridge, UK) based on the Chou–Talalay method [18]. This provided quantitative definitions for an additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) of drug combinations. To plot an isobologram, fraction-affected levels were predicted using CalcuSyn Version 2.0.

DNA fragmentation assay

DNA fragmentation in cells was detected using the Cell Death Detection ELISAPLUS (Sigma-Aldrich) and a Viento XS plate reader (BioTek, Winooski, VT, USA), according to the manufacturer’s instructions, following 24-h incubation with BV, chidamide, or the combination of BV and chidamide (Sample size, n = 2).

Caspase 3/7 assay

Cells were seeded and treated with BV or chidamide as single agents or in combination for 24 h. Caspase 3/7 activities were measured using the Caspase-Glo 3/7 Assay System (Promega) according to the manufacturer’s instructions (Sample size, n = 2). Luminescence was detected by the ARVO X light.

Western blotting

Cells were lysed in buffer containing 62.5 mM Tris hydrochloride, 10% glycerin, 2% sodium dodecyl sulfate, protease inhibitor (Sigma-Aldrich), and phosphatase inhibitor (Sigma-Aldrich). Xenograft tumors were disrupted using TissueLyser II (Qiagen, Hilden, Germany) in the same buffer.

Protein concentrations in the whole cell or tissue lysate were measured using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (5 µg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with PVDF blocking buffer (TOYOBO, Osaka, Japan). Membranes were incubated with the following primary antibodies diluted in Can Get Signal™ solution 1 (TOYOBO) overnight at 4 °C: acetyl-histone H3 (#9649, Cell Signaling Technology [CST], Danvers, MA, USA), histone H3 (#9715, CST), poly (adenosine diphosphate-ribose) polymerase (PARP; #9532, CST), cleaved PARP (#5625, CST), cleaved caspase 3 (#ab214430, Abcam, Cambridge, UK), caspase 3 (#ab179517, Abcam), Bim (#2933, CST), Mcl-1 (#ab28147, Abcam), Bcl-2 (#15071, CST), survivin (#af886, R&D Systems, Minneapolis, MN, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#3683, CST). Membranes were then incubated with anti-rabbit or anti-mouse immunoglobulin G secondary antibodies (#7074 or #7076, CST) diluted in Can Get Signal Solution 2 (TOYOBO). Protein–antibody interactions were detected with ECL Select (Amersham, Buckinghamshire, UK) according to the manufacturer’s instructions. Signal intensity was measured using ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA) and analyzed with ImageQuant TL software (GE Healthcare).

In vivo mouse xenograft study

Five million HH cells were mixed with BD Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and inoculated in the right flank of 6-week-old female mice with severe combined immunodeficiency (CLEA, Tokyo, Japan). No sex difference in apoptosis was assumed. Mice were randomly assigned to four groups of five mice each and treated with either vehicle (0.2% carboxymethylcellulose saline and 0.1% Tween 80 for chidamide, and saline for BV), BV (0.1 mg/kg once a week, intravenously), chidamide (15 mg/kg once a day, oral gavage), or BV combined with chidamide (at the same dose and frequency as those used for single-agent treatment). For the negative control experiments shown in Fig. 4e and f, mice were administrated either combination of IgG (0.1 mg/kg once a week, intravenously) and vehicle, BV and vehicle, chidamide and IgG, or chidamide and BV. Drugs were administered when tumors reached an average volume of 100 mm3. Tumors were measured twice a week using digital calipers, and volumes were calculated as [L × (W × W)]/2, in which L is the longest diameter (in mm) and W is the shortest diameter (in mm). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shonan Health Innovation Park accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

Next-generation sequencing

Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Amplicon multiplex sequencing experiments were performed using the Ion AmpliSeq Transcriptome Human Gene Expression kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, target transcripts were amplified by polymerase chain reaction (PCR) from complementary DNA libraries synthesized from 1 ng of total RNA. Reactants were ligated to adapters and pooled at equal concentrations; multiplex sequencing at over 8 million reads per sample was performed using Ion Proton high-throughput sequencers (Thermo Fisher Scientific). Before identification of differentially expressed genes, genes were selected to satisfy the condition that they were expressed at a minimum of one read per million in the sample with the greatest expression levels among all the compared samples. Differentially expressed genes with a p value below 0.05 and an absolute log2-fold change above 0.5 were identified using the voom function in the limma package in R.

Gene pathway analysis

Gene pathway analysis was performed using the R packages ReactomePA 1.26.0, with an adjusted p value cut off below 0.05, and GO Function, with the false-discovery rate cut off below 0.5 [19]. The intersection of all assigned genes in AmpliSeq panel and human annotated genes in reference databases were selected as background. Principal component analysis was used to reduce the number of variables.

Quantitative reverse transcription PCR assays

In total, 4000 HH cells were harvested, and CDC45 messenger RNA (mRNA) expression was determined by quantitative reverse transcription (qRT) PCR using a FastLane Cell Probe Kit (Qiagen) according to the manufacturer’s instructions. Cycling parameters were 50 °C for 30 min and 95 °C for 15 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Data were normalized using GAPDH as an internal control, and relative mRNA expression levels were calculated using the 2−ΔΔCt method [20].

Statistical analysis

Data were expressed as mean values and standard errors. Statistical significance was calculated using Student’s t-test. p < 0.05 was considered statistically significant.