Synergistic Antitumor Effect and Mechanism of Chidamide Combined with Gemcitabine, Oxaliplatin or Zanubrutinib in Diffuse Large B-Cell Lymphoma

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Aims: To observe the synergistic antitumor effect of chidamide (a novel orally benzamide class of histone deacetylase inhibitors, HDACi) combined with gemcitabine, oxaliplatin or zanubrutinib (a second generation Bruton's tyrosine kinase (BTK) inhibitor) in diffuse large B-cell lymphoma cell lines and explore the possible mechanisms. Methods: We used CCK-8 assay to test the inhibitory efficacy of single agent chidamide, gemcitabine, oxaliplatin, zanubrutinib and their combinations on DLBCL cell lines, respectively. And the combination index (CI) was calculated by the CalcuSyn software. The apoptosis and the cell cycle analysis of DLBCL cell lines under different treatments were assessed by Annexin V-APC/PI double-staining and PI staining using flow cytometry, respectively. The expression levels of acetylated histone and apoptosis-related proteins were detected by Western Blot. Results:Single-agent chidamide, gemcitabine and oxaliplatin caused growth arrest of the DLBCL cell lines (OCL-LY3、OCL-LY19、U2932、DOHH2) in a dose and time dependent manner, respectively(Figure1a,1b,1c). Single-agent zanubrutinib showed antitumor activity in two activated B-cell like DLBCL (U2932 and OCI-LY3) cell lines in a dose and time dependent manner while no antitumor activity in other two GCB-DLBCL (OCI-LY19 and DOHH2) cell lines (Figure1d). Chidamide combined with gemcitabine or oxaliplatin synergistically inhibited the proliferative viability of DLBCL cell lines. Also, chidamide could enhance the inhibitory effect of zanubrutinib in the two ABC-DLBCL cell lines. And the CI values of all groups were more than 1(Fig2-1~2-8). Chidamide could significantly increase the proportion of DLBCL cells in the G0/G1 phase in a dose-dependent manner(Figure 3). And chidamide combined with gemcitabine can synergistically enhance the cell cycle arrest in U2932 cells (Figure 4). Cell apoptosis rate increased along with the ascending dosage of chidamide in the four DLBCL cell lines. The effect of cell apoptosis became more obviously when combined with oxaliplatin or zanubrutinib (Figure 5.1~5.4). Chidamide significantly up-regulated the levels of acetylated H3 (Lys 9) and cleaved-caspase 3 while down-regulated Bcl-2 (Figure 6~7). Furthermore, PD-L1 protein was upregulated in chidamide-exposed DLBCL cell lines (Figure 8). Conclusion:Chidamide combined with gemcitabine, oxaliplatin or zanubrutinib synergistically inhibited the proliferative viability and cell apoptosis rate in DLBCL cell lines. Therefore, it is worthy to further explore the value of Chidamide combination therapy in DLBCL. Figure Disclosures No relevant conflicts of interest to declare.

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