In vitro, inhibitory effects of compound 23 were measured by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. Briefly, cells were placed in 96-well plates at a density of 2 × 10⁴ cells/well. After equilibration overnight at 37°C, the cells were treated with compounds at various concentrations. After 72 h of culture, the MTS reagent was added to each well, and the plates were incubated for another 4 h at 37°C. The samples were measured using a micro-plate reader at a wavelength of 490 nm (Biotek Instruments, Winooski, VT, United States). Half-maximal inhibitory concentration (IC₅₀) values were calculated with SigmaPlot 8.0 software (Systat Software, San Jose, CA, United States).

The cell cycle and apoptosis were analyzed by flow cytometry. EL4, NB4, and A20 cells (2×10⁶ cells/well) were placed in 24-well plates. After equilibration overnight, the cells were treated with various concentrations of compounds for 24 h. The effects of the compounds on cell cycle progression and apoptosis were determined by flow cytometry analysis. Procedures were conducted in accordance with the manufacturer’s recommendations. FxCycle™ PI/RNase Staining Solution for cell cycle analysis was purchased from Thermo Fisher Scientific (Waltham, MA, United States). An Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Beijing Kang Run Cheng Ye Biotech (Beijing, China).

EL4, NB4, and A20 cells were collected and washed twice with cold phosphate-buffered saline (PBS) before being lysed with radioimmunoprecipitation assay buffer for protein isolation. Protein samples (40 μg) were separated by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to PVDF membranes by wet electrophoresis. The membranes were blocked with 5% milk in Tris-buffered saline containing Tween 20 (TBST) for 30 min, followed by incubation overnight with primary antibodies (1:1,000 dilution) at 4°C. After washing three times with TBST, the membranes were incubated with secondary antibodies (1:1,000) for 1 h. The resulting bands were visualized using an enhanced chemiluminescence detection kit and Image Quant LAS 4000 (GE Healthcare, Piscataway, NJ, United States).

After culture and harvesting, the cells were suspended in saline at a density of 2 × 10⁷ cells/mL. The animal protocol followed guidelines and was approved by the Experimental Animal Management and Welfare Committee at the Institute of Materia Medica, Peking Union Medical College (Beijing, China). Male mice (BALB/c C57 and BALB/c) aged 6–8 weeks were subcutaneously injected in their right flanks with 0.2 ml of EL4 or A20 cell solution. The EL4- and A20-injected mice were then randomly divided into five groups: a control group that received saline intraperitoneally (i.p.) every day; three groups that received compound 23 dissolved in saline at doses of 3.75, 7.5, and 15 mg/kg intraperitoneally; and a positive control group that received 100 mg/kg of suberoylanilide hydroxamic (SAHA) orally. Body weight of the mice was measured twice each week. After sacrificing the mice, tumors were isolated and weighed. The percentage of tumor weight inhibition (TWI%) was calculated as follows: TWI %= (1—Tumor Weight treatment/Tumor Weight vehicle) × 100%. Statistical analysis was performed in EXCEL GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, United States), and the significance level was evaluated with a two-way Analysis of Variance (ANOVA) model.

The expression levels of PI3Kα (ENG00000121879), Akt (ENG00000142208), S6 (ENG00000108443), HDAC1 (ENG00000116478), TuBulin1α (ENG00000167552), ERBB2 (ENSG00000141736), and ERBB3 (ENSG00000065361) in different cell lines were analyzed by Cancer Cell Line Encyclopedia (CCLE, https://portals. broadinstitute. org/ccle), a database offering the expression level sorting of 84,434 genes in 1,457 cancer cell lines.

A series of dual HDAC and PI3K inhibitors were synthesized to examine their antitumor efficacy. Among those compounds, compound 23 showed more potent antiproliferative activity against solid tumors. The fact that HDAC and PI3K inhibitors had been approved by the FDA for treatment of hematologic cancers encouraged us to test the antiproliferative activity of compound 23 against hematologic tumor cells. In vitro antiproliferative activity of compound 23 was evaluated by the MTS assay in hematologic tumor cells including T lymphoma (EL4), B lymphoma (A20), and leukemia (NB4). As shown in Table 1, compound 23 displayed potent antiproliferative activity compared with reference compounds (SAHA and BKM120). Notably, compound 23 showed more remarkable antiproliferative activity in hematologic cell lines (IC₅₀ = 0.016–0.34 μM) compared with solid cancer cell lines (IC₅₀ = 0.11–6.8 μM) (Zhang et al., 2019).

In our previous study, compound 23 displayed selective inhibition of HDAC and PI3 kinase (Zhang et al., 2019). Therefore, western blot was applied to study the effects of compound 23 on PI3K and HDAC pathways. According to the literature, the inhibition of HDAC induced hyperacetylation of histone and its other substrates such as α-tubulin, which upregulated acetylation of H3 and α-tubulin; the inhibition of the PI3K/AKT/mTOR pathway downregulated phosphorylation levels of proteins downstream of AKT and mTOR, such as P70S6K, P85S6K, and S6. After treatment with compound 23, BKM120, or SAHA for 24 h, the three hematologic tumor cell lines were collected and fully lysed, and then the cell lysates were analyzed by western blot. As shown in Figure 2, compound 23 dose-dependently and simultaneously downregulated the levels of phosphor-AKT (p-AKT), phospho-p70 S6 kinase (p-P70 S6K), and phosphor-S6 ribosomal protein (p-S6), and upregulated acetyl histone H3 (Lys9) (Ac-H3) and acetyl-α-tubulin (Ac-α-tubulin) in the three cell lines. Moreover, compound 23 was more potent than BKM120 and SAHA in terms of downregulation of phosphorylation and upregulation of acetylation, respectively.

Recent studies have shown that HDAC and PI3K inhibitors can induce cell apoptosis (Chen et al., 2018). Since our previous results had shown that compound 23 significantly induced apoptosis in solid tumors (Zhang et al., 2019), we studied the proapoptotic effect of compound 23 in hematologic tumor cell lines, including EL4, NB4, and A20 cells. Annexin-V and propidium iodide (PI) staining were performed to investigate the effect on apoptosis. As shown in Figure 3, compound 23 significantly increased the proportions of early and late apoptotic cells of all three tumor cell lines in a dose-dependent manner and more potently induced apoptosis compared with BKM120 and SAHA at the same dose. Furthermore, it was remarkable that the apoptosis rates of EL4, NB4, and A20 cells treated with compound 23 at 500 μM were 52.37%, 62.65%, and more than 36.48%, respectively, which were both superior to the apoptosis rates (less than 30%) of HCT116 treated by compound 23 at the same dose (Zhang et al., 2019). Compared to the previous data, compound 23 has a more potent proapoptotic effect on hematologic tumors.

Since both HDAC and PI3K pathways are involved in cell processes such as proliferation, differentiation, and migration, here we evaluated cell cycle distribution to investigate the influence of compound 23 on hematologic tumor cells. As shown in Figure 4, histograms were prepared to demonstrate the influence on the cell cycle. Compound 23 arrested the cell cycle in G1 phase in all three cell lines. We observed that compound 23 and SAHA caused a significant cell cycle arrest at G1 phase in all three cell types. The proportions of cells in G1 phase in the control groups of EL4, NB4, and A20 cell lines were 36.7, 45.5, and 36.3%, respectively; in contrast, the proportions of G1 phase-arrested cells of EL4, NB4, and A20 cell lines significantly increased to 63, 66.5, and 65.8% in the compound 23 high-dose group. Conversely, BKM120 had no significant effect on the cell cycle. According to our results, compound 23 was able to arrest the cell cycle in G1 in different hematologic tumor cell lines.

Compound 23 inhibited the variation of hematologic cells, induced apoptosis, and promoted cell arrest in vitro. Therefore, we further assessed the antitumor potency of compound 23 against T lymphoma and B lymphoma in vivo. After 12 days of administration, the tumor tissues were isolated and weighed. The TWI% of compound 23 at 3.75 mg/kg, 7.5 mg/kg, and 15 mg/kg was 27.8, 42.9, and 78.1%, respectively (Figure 5A). As shown in Figure 5B, although compound 23 induced weight loss in animals during administration, it still significantly inhibited tumor growth in the high-dose group compared with both the vehicle and positive control groups. The tumor tissues from three tumor-bearing mice in each group of vehicle and compound 23 at 15 mg/kg group were randomly selected, and their supernatants were analyzed by western blot after full lysis. Compound 23 exhibited more potent antitumor activity compared with SAHA by simultaneously regulating PI3K and HADAC signaling pathways in vivo as shown in Figures 5C,D.

After confirming that compound 23 significantly inhibited the proliferation of mouse T lymphoma in vivo, we also explored whether it had inhibitory activity against B lymphoma. The TWI% of compound 23 at 7.5 mg/kg and 15 mg/kg was 24.9 and 60%, respectively (Figure 6A). Although compound 23 showed some toxicity by reducing body weight in animals at 15 mg/kg, it still significantly inhibited the proliferation of B lymphoma. In addition, it showed stronger antitumor activity than SAHA at 15 mg/kg in vivo (Figure 6B).

Comparing with the results of previous studies, we found that compound 23 showed a more potent antitumor activity against hematologic tumors compared with solid tumors. Many cellular processes in cancer are attributed to kinase signaling networks (Song et al., 2019). To understand the reasons for the discrepancy of the effects of compound 23 between solid tumors and hematologic tumors, we identified the gene expression levels of kinases in the PI3K/Akt/mTOR axis and HDAC pathway, such as PI3Kα, Akt, S6, HDAC1, and TuBulin1α, through the CCLE database (https://portals.broadinstitute.org). As shown in Figure 7A, we observed an analogous pattern of expression of the aforementioned kinases in both signaling pathways between solid tumors and hematologic tumors at the gene level. However, as shown in Figure 7B, the expression levels of ERBB2 and ERBB3 decreased in hematologic tumors compared with it in solid tumors. The overexpression of Erb2B in numerous types of primary human tumors and alterations in microRNA (miRNA) have been associated with tumor suppression or tumorigenesis in human cancer (Chen et al., 2009). Expression of certain oncogenes can result in activation of the PI3K/AKT signaling pathway. ErbB2 and ErbB3 form heterodimers that could activate the downstream targets of the PI3K/Akt signaling pathway (Hong et al., 2021). Thus, the low expression of ErbB2 and ErbB3 stabilized the PI3K/Akt signaling pathway in hematologic tumors (Table 1), thereby enabling compound 23 to exhibit more potent antitumor efficacy than against solid tumor cells. Based on these data, the expression levels of ErbB2 and ErbB3 at the gene level might be related to the effect of compound 23 and even the therapeutic effect of this type of compound, which may provide some reference value for the response rate of compound 23 in clinical treatment.

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