MCL cell lines Jeko-1 and Granta-519 were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 units/ml and 100 µg/ml, respectively) (all from Thermo Fisher Scientific). The cells were cultured at 37°C in a humidified, 5% CO₂ atmosphere, and tested for mycoplasma contamination before experiments.

The commercial sources of antibodies used are listed in Supplementary Table S1. Ganetespib and ibrutinib were purchased from Selleck Chemicals, Inc. (Houston, TX). All chemicals were purchased from Sigma-Aldrich (Saint Louis, MO) unless otherwise specified. 10 mM stock solutions of ganetespib and ibrutinib were prepared in DMSO and stored at −20°C.

Jeko-1 and Granta-519 cells were plated in 24-well plates (5×10⁴ and 10×10⁴ cells/well, respectively), and treated with ganetespib or vehicle (DMSO, Sinopharm, Shanghai, China) for 72 h or briefly pretreated with ganetespib or vehicle for 12 h and ganetespib was then washed out. Then these cells were cultured in media with different concentrations of ibrutinib for various periods of time as indicated. Cell viability was determined using MTT (3- [4]-2, 5-diphenyltetrazolium bromide thiazolyl blue, Saiguo Biotech, Guangzhou, China). The cell viabilities were measured at least in three independent experiments. IC₅₀ values were calculated using CompuSyn (ComboSyn, Inc., Paramus, NJ).

Apoptosis and cell cycle assays were performed as previously described (Liu et al., 2015). Briefly, apoptosis was evaluated in drug-treated cells using Annexin V-FITC/propidium iodide (PI) staining kit (Yeasen, Shanghai, China). Jeko-1 and Granta-519 cells were trypsinized without EDTA and harvested. Cells were suspended in the binding buffer and incubated with Annexin V-FITC and PI staining reagents. For cell cycle assays, cells seeded in 6-well plates, harvested and stained with PI solution (KeyGen Biotech, Nanjing, China) with RNaseA (Biosharp, Hefei, China). Apoptosis and cell cycle samples were analyzed using a Beckman Coulter CytoFLEX Flow Cytometer (Brea, California). Data were analyzed using FlowJo software (FlowJo).

For EdU (5-Ethynyl-2′-deoxyuridine) staining, the harvested cells were stained with EdU (Ribobio, Guangzhou, Guangdong, China) after drug treatment, and then transferred to poly-lysine-treated adhesive slides, and then the slides were incubated and images of staining results were captured as previously described (Liu et al., 2021).

Manufacturer’s protocol was followed. Briefly, the cells were fixed with 4% paraformaldehyde for 30 min and treated with 0.5% TritonX-100 for 7 min at room temperature. After washed once with PBS, the cells were incubated with Biotin-11-dUTP (KeyGen) for 30 min at 37°C in dark. Then the cells were incubated with streptavidin-fluorescein (KeyGen) staining solution for 30 min at 37°C in dark and stained with DAPI for 5 min (Roche, Mannheim, Baden-Württemberg, Germany). Images were captured using Nikon Eclipse (Tokyo, Japan) microscope.

Immunoblot and immunofluorescence experiments were performed as previously described (Tu et al., 2011; Tu et al., 2013; Liu et al., 2015). In Immunoblot experiments, cells were lysed and protein concentrations were determined using BCA kits (Beyotime, Shanghai, China). Proteins were resolved on 8%-14% SDS-PAGE gels and transferred to PVDF membrane (Millipore, Billerica, MA). Membranes were blocked and then incubated at 4°C in primary antibody overnight, followed by incubation with HRP-conjugated secondary antibody and signal detection with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). In immunofluorescence experiments, Jeko-1 and Granta-519 cells were seeded on glass slides and treated as indicated. Then cells were fixed with freshly prepared 4% paraformaldehyde at R.T. for 10 min. Cells were incubated in 3% BSA/0.3% Triton X-100/PBS for 30 min, followed by 2 h incubation with primary antibodies in 3% BSA/0.3% Triton X-100/PBS at 22°C and incubation with appropriate secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific) as indicated. Images were captured using Nikon Eclipse (Tokyo, Japan) microscope. The commercial sources of antibodies used are listed in Supplementary Table S1.

All procedures involving mice were approved by the Institutional Animal Care and Use Committee of Jiangsu University. In vivo experiments were performed as previously described (Liu et al., 2021). All Nu/Nu nude mice were provided with sterilized food and water and housed in a barrier facility under a 12-h light/dark cycle. Six-week-old Nu/Nu nude mice were injected subcutaneously in the right flank with 2 × 10⁶ Jeko-1 cells. Tumors were measured every other day with vernier calipers, and tumor volumes were calculated using the equation: volume = 0.52 × W² × L, where L and W represent the length and width of tumors, respectively. When tumor size reached approximately 100 mm³ (25 days after initial transplantation), the mice were randomized into four groups (six mice/group, containing vehicle+vehicle, ganetespib+vehicle, vehicle+ibrutinib, and ganetespib+ibrutinib groups). Ganetespib (15 mg/kg formulated in 10/18 DRD [10% dimethyl sulfoxide, 18% Cremophor RH 40, 3.6% dextrose, and 68.4% water] or 10/18 DRD [vehicle]) was administered once a week by tail vein injection for 10 days. Ibrutinib (50 mg/kg formulated in 10/18 DRD or 10/18 DRD [vehicle]) was administered once a day by intraperitoneal injection for 10 days. At the end of the observation, mice were sacrificed and autopsied. Tumors were removed and measured. Differences between two groups were compared by the Mann–Whitney test, with p < 0.05 considered significant.

Tumors or tissues were paraffin embedded, sectioned (5 µm thick), and deparaffinized as previously described (Liu et al., 2021). Sections were either hematoxylin & eosin (H&E) stained or subjected to antigen retrieval for immunohistochemistry as previously described (Liu et al., 2021). Sections were exposed to appropriate species-specific secondary antibodies and exposed to 3,30-diaminobenzidine (KeyGen). Then sections were lightly counterstained with hematoxylin (KeyGen). The immunoreactivity scores were evaluated as previously described (Liu et al., 2015; Liu et al., 2021).

Data are presented as mean ± standard deviation from three independent experiments, unless otherwise stated. Statistically significant differences between different groups were determined by Student’s t tests (two-tailed) or one-way ANOVA followed by Bonferroni post-testing using GraphPad Prism version 5.00 (GraphPad, San Diego, CA) between different groups (^# p > 0.05; *p < 0.05; **p < 0.01; and ***p < 0.001).

At the beginning of our study, we tested the inhibitory effects of ibrutinib and ganetespib as single agents on Jeko-1 and Granta-519 cell lines. As shown in Figures 1A,B, the IC₅₀ values of ibrutinib on Jeko-1 and Granta-519 cells were 3.24 ± 0.36 and 5.72 ± 0.40 μM, respectively. At the same time, the IC₅₀ values of ganetespib on Jeko-1 and Granta-519 cells were 12.8 ± 1.57 and 45.7 ± 4.18 nM, respectively. These results demonstrated that both ibrutinib and ganetespib have potent inhibitory effects on cell proliferation of MCL cells.

In one of our recent studies, we found that transient exposure of ganetespib for only 12 h is sufficient to inhibit the growth of MCL cells (Liu et al., 2021). For the reasons previously mentioned, we hypothesized that pretreatment with ganetespib would probably increase the sensitivity of MCL cells to ibrutinib. In our experimental design, cells were pre-treated with ganetespib for 12 h, and then treated with different concentrations of ibrutinib for another 60 h, before the cell viabilities were measured (Figure 1C). As shown in Figure 1D,E, transient ganetespib treatment indeed increased the sensitivity of Jeko-1 and Granta-519 cells to ibrutinib as manifested by 2.50 and 2.44-fold decrease of IC₅₀ values, respectively.

In the following experiments, MCL cells were pre-treated with ganetespib for 12 h, and then treated with ibrutinib for 12, 24, 36, 48, and 60 h, respectively (Figure 1F). Compared with the vehicle group and the single-agent groups, the combined use of the two drugs significantly inhibited cell proliferation of both Jeko-1 and Granta-519 cells, and the inhibitory effects of the combination therapy were significantly better than those of the single-agent groups (Figure 1G). Since DNA replication is an essential event of cell proliferation, EdU staining assays were also used to evaluate the effects of the combinational use of ganetespib and ibrutinib on cell proliferation of MCL cells. The concentrations of ganetespib and ibrutinib were set to the IC₅₀ values determined earlier, and Jeko-1 and Granta-519 cells were pre-treated with ganetespib for 12 h, followed by ibrutinib treatment for another 24 h. Compared to the vehicle group, the combined use of the two drugs reduced the percentage of EdU positive cells from 50.09% to 16.42% in Jeko-1 cells, and also reduced the percentage of EdU positive cells from 45.39% to 27.36% in Granta-519 cells (Figures 1H–K). Additionally, the pre-treatment with ganetespib significantly reduced the percentage of EdU positive cells from 35.53% to 16.42% in Jeko-1 cells, and also reduced the percentage of EdU positive cells from 39.81% to 27.36% in Granta-519 cells when compared with ibrutinib single-agent group. Previous studies have found ibrutinib can inhibit NF-κB and AKT pathways, therefore the levels of these proteins were detected after treatment. Results demonstrated that ibrutinib reduced the protein levels of p-AKT in both cell lines, and the pretreatment with ganetespib led to larger reductions of the p-AKT levels (Figure 1L). Pretreatment with ganetespib also shows similar effects on NF-κB levels in nuclei in MCL cells (Figure 1M). These results supported that pretreatment with HSP90 inhibitor ganetespib sensitizes MCL cells to BTK inhibitor ibrutinib.

To gain insight into the mechanism by which ganetespib pretreatment sensitizes MCL cells to ibrutinib, Jeko-1 and Granta-519 cells with different treatments were subjected cell cycle analysis. Consistent with our previous study (Liu et al., 2021), transient ganetespib treatment induced cell cycle arrest at G0/G1 phase. When ibrutinib was used as a single agent, it also induced G0/G1 cell cycle arrest. As expected, when the cells were treated with ganetespib and ibrutinib sequentially, the portions of G0/G1 phase increased significantly when compared to the vehicle or the single-agent groups in both cell lines (Figures 2A–D). Although MCL is characterized by the aberrant expression of cyclin D1 (Swerdlow et al., 2016), neither ganetespib nor ibrutinib showed obvious effects on cyclin D1 expression in either cell lines and there was not a consistent trend in the expression levels of Cyclin D1 after drug treatments in this study (Figures 2E,F). Instead, sequential administration of ganetespib and ibrutinib induced the decreases of the protein levels of CDK2, CDK4, and CDK6 dramatically in these two cell lines (Figures 2E,F). Because Lee et al. (2018) proposed that the activation of Myc can contribute to ibrutinib resistance, we examined the levels of c-myc after drug treatments and found that c-myc did not change significantly under our experimental conditions (data not shown).

Next, we studied the effects of sequential administration of ganetespib and ibrutinib on cell apoptosis. The results showed both ganetespib and ibrutinib induced mild but significant cell apoptosis in Jeko-1 and Granta-519 cells (Figures 2G–J). More importantly, ganetespib pretreatment could dramatically increase the levels of apoptosis (Figures 2G–J). In order to further confirm the above results, TUNEL assays were carried out in the following experiments. Consistently, the numbers of TUNEL-positive cells were significantly increased when cells were pretreated with ganetespib compared with the single-agent treated groups in Jeko-1 cells (Figure 2K,L). Similar results were obtained when Granta-519 cell were treated (Figures 2M,N). In addition, Western blot results showed the upregulation of cleaved-caspase9 and the downregulation of BCL-2 in MCL cells in ganetespib-ibrutinib combination group, which explained changes of cell apoptosis after drug treatment at the molecular level (Figure 2O).

Our previous study found that transient ganetespib treatment resulted in increased DNA damage (Liu et al., 2021). Therefore, immunofluorescence (IF) analysis of γH2AX and 53BP1 foci were used to evaluate the effects on DNA damage by the drug treatment. Results showed that ganetespib treatment caused significant increase of numbers of positive 53BP1 foci in both cell lines as expected (Figures 3A–D). The combination use of ganetespib and ibrutinib also demonstrated similar effects on positive 53BP1 foci. Additionally, ganetespib treatment and the combination use of ganetespib and ibrutinib also increased the similar percentages of positive γH2AX cells (Figures 3E–H). These results demonstrated that the sequential administration of ganetespib and ibrutinib had similar effects on increasing DNA damage as the transient treatment with ganetespib.

Finally, we evaluated the anti-tumor activity of ganetespib and ibrutinib on Jeko-1 xenograft tumors. When the tumor volumes reached about 100 mm³, these tumor-bearing mice were randomly grouped and treated as indicated in Figure 4A. As a matter of fact, the tumors in mice with single-agent treatment grew significantly slower than those of the vehicle group (Figures 4B,C). As expected, the combination therapy of ganetespib and ibrutinib dramatically inhibited the growth of xenografts, reducing final volume by 74.49% (p < 0.001), when compared to the vehicle group. More importantly, the ganetespib-ibrutinib treatment showed better tumor inhibitory effects than either ganetespib or ibrutinib single-agent groups. Consistent results were obtained in tumor weight after mice were sacrificed (Figure 4D). IHC results indicated that the expression levels of Ki-67 (a major proliferation biomarker of MCL) and BCL-2 were greatly reduced in tumors from mice treated with ganetespib-ibrutinib, while the extent of reduction in either ganetespib or ibrutinib groups were much less (Figures 4E,F). In addition, the weight measurement results of major organs suggested that all these drug treatment showed no obvious toxicities to mice (Supplementary Figure S1). Taken together, these data demonstrated that the combination of ganetespib and ibrutinib dramatically inhibited the growth of xenograft tumors without obvious toxicities.