ABT-199 cooperates with Apatinib to reduce the viability of diverse DLBCL cells
In hematological malignancies, ABT-199 resistance is associated with a variety of gene mutations. On this basis, we enriched the mutated genes by GO. This requires us to use other chemotherapy drugs in combination when treating patients. In addition, compared with the normal group, patients with DLBCL had high expression of BCL-2 (Fig. 1A, S. 1A). To evaluate the potential cooperative effect of ABT-199 and Apatinib we assessed the effect on cell viability. Various DLBCL cell lines were treated with indicated concentrations of ABT-199 and Apatinib, under an electron microscope we found the cells shrinkage, and the fragmentation of membrane blebs (Fig. 1B, S. 1B), after which cell viability was tested using CCK-8 assay. Treatment with ABT-199 and Apatinib alone effectively inhibited cell growth in a dose-dependent manner. Notably, co-administration of ABT-199 and Apatinib led to markedly enhanced growth inhibition in all of these DLBCL cell lines (Fig. 1C, 1D, S. 1C, S. 1D, and S. 1E). The half-maximal inhibitory concentration (IC50) values (Table 1) of ABT-199 and Apatinib were lower at 24 h than at 12 h in all cell lines (P < 0.001). Then we examined whether the combination of ABT-199 and Apatinib affects the clonogenicity of DLBCL cells. The size of colonies was observed under an electron microscope and visualization. As shown in Fig. 1E, 1F, 1G, 1H, S. 2A, S. 2B, S. 2C, and S. 2D the colony-forming assay revealed that compared with ABT-199 and Apatinib monotherapy groups the combination group could significantly inhibit colony formation in all tested cell lines (P < 0.001).
Synergetic lethality of combined ABT-199 and Apatinib in DLBCL cells in vitro
To further verify the cytotoxicity effect of ABT-199 and Apatinib when used in combination, we treated DLBCL cells with ABT-199 at various concentrations (1, 2, 4 nM) in the presence or absence of different concentrations of Apatinib (10, 20, 40 μM) for 12 h and 24 h and then the percentage of apoptotic cells was detected by Annexin V/DAPI dual staining (Fig. 2A). The results showed that, even though exposure to different concentrations of ABT-199 and Apatinib single treatment can induce a certain degree of apoptosis, these events were significantly enhanced by combined treatment for 12 h and 24 h (Fig. 2B, 2C, 2D, 2E, 2F). The combination index (CI) calculation shows ABT-199 has a strong synergistic effect with Apatinib at different doses (Table 2).
The combination of ABT-199 and Apatinib altered cell cycle distribution, increased ROS generation, and mitochondrial injury
Subsequently, we analyzed the cell cycle status to further characterize the role of low-dose ABT-199 in enhancing Apatinib mediated cytotoxicity. In all tested DLBCL cell lines, compared with the ABT-199 or Apatinib alone, the percentage of S phase cells in the combined group decreased significantly after 24 h treatment, and the cell cycle was arrested in G0/G1 phase (Fig. 3A, 3B, 3C, 3D, 3E, and 3F). In addition, ABT-199 combined with Apatinib also regulated cycle-related proteins (Fig. 3L). Consistent with the results of apoptosis, ABT-199 and Apatinib also induced loss of mitochondrial membrane potential (MMP), reflected by markedly decreased fluorescence intensity ratio between JC-1 aggregate and monomer (Fig. 3G, 3H). To uncover the potential mechanism of apoptosis induced by these two agents, flow cytometry was used to detect intracellular ROS levels. After being co-treated with ABT-199 and Apatinib for 24 h, a notable increase in ROS generation was observed in OCI-Ly1 and MCA (Fig. 3I, 3K), compared to treatment with each single agent. In addition, our results showed that ABT-199 and Apatinib exerted an anti-proliferative and anti-apoptosis effect in DLBCL cells by inhibiting the MAPK/ERK and pro-survival pathways in OCI-Ly1 and MCA cell lines (Fig. 3J).
The regimen combining ABT-199 and Apatinib impairs tumor-forming capabilities in a xenograft model
To confirm the therapeutic effect of BCL-2 inhibitor combined with Apatinib on DLBCL in vivo, we tested the efficacy of ABT-199 plus Apatinib in the BALB/C nude mice (Fig. 4A). The DLBCL cell line OCL-Ly1 was subcutaneously injected for about 7 days, and the treatment was started when the tumor volume reached to ~75mm3, mice were randomly divided into the vehicle, ABT-199 (80 mg/kg), Apatinib (100 mg/kg), and the combination of ABT-199 plus Apatinib groups for 14 consecutive days, respectively. Interestingly, compared with the vehicle control as well as each agent, the combined group significantly reduced tumor burden, reflected by decreasing volume and weight of tumor (Fig. 4B, 4D, and 4E). In addition, the weight of mice did not decrease significantly and no other signs of notable toxicity were observed (Fig. 4C). Histological examination showed that the combination treatment inhibited the infiltration of DLBCL cells, while diseased mice treated with ABT-199 or Apatinib alone had more severe DLBCL infiltration (Fig. 4F). Western Blot revealed that the MAPK/ERK pathway was inhibited, which is consistent with the results in vitro (Fig. 4G). Together, these findings argue that ABT-199 and Apatinib might interact to cause robust apoptosis and inhibition of DLBCL cells by downregulation MAPK/ERK pathway and anti-apoptotic BCL-2 family proteins as well as upregulation of pro-apoptotic BAX in DLBCL cells.
Combined treatment with ABT-199 and Apatinib ameliorates DLBCL primary patient cells in vitro and in vivo
The anti-tumor effect of ABT-199 and Apatinib alone or in combination was further verified ex vivo in the primary blasts isolated from 12 DLBCL patients. We summarized the characteristics of the patients in Table 3. Following the results obtained from DLBCL cell lines, cotreatment of primary DLBCL blasts with ABT-199 at the concentrations ranging from 0.5 to 16 nmol/L and Apatinib from 5 to 160 μmol/L caused remarkable inhibited cells proliferation when compared with each agent alone (Fig. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, and 5K). We detected the apoptosis of primary DLBCL blasts and found that ABT-199 and Apatinib could significantly induce apoptosis, which was consistent with the results in cells lines (Fig. 5L). Similarly, Western Blot results showed that MAPK/ERK pathway and anti-apoptotic BCL-2, MCL-1 were significantly inhibited (Fig. 5M). Together, these results indicate that the combination of ABT-199 with Apatinib might be a preferable choice to targeted DLBCL blasts.
Co-treatment with ABT-199 and Apatinib alters genome-wide gene expression in DLBCL cells
To screen for the molecular target of co-treatment with ABT-199 and Apatinib, we performed RNA sequencing in the samples of DLBCL cells treated with ABT-199 and/or Apatinib. We found that 189 genes were differentially expressed after co-treatment ABT-199 and Apatinib compared with that in the single-treated samples (S. Table1). GO analysis indicated that multicellular organismal development was upregulated, while terms including oxidation-reduction process, inflammatory response, oxygen transport, and cell killing were downregulated (Fig. 6A, 6B). Pathway analysis revealed that Natural killer cell-mediated cytotoxicity, VEGF signaling pathway, and ECM-receptor interaction were significantly elevated. Meanwhile, the MAPK signaling pathway and ErbB signaling pathway were downregulated (Fig. 6C, D). Five key genes including HGF, EDNZ, SPP1, HMOX1, CCL5, ADM were significantly enriched according to the pathway activity network (Fig. 6E, 6F). Different K-cores were used to identify core regulatory genes involved in DLBCL treatment. As shown in Fig. S. 3C, we found that EDN1 was most possibly related to DLBCL development, which achieved a high ranking among the top 5 genes in terms of different K-cores after excluding genes with low abundance and matrix-related genes. Thus, we hypothesized that co-treatment exhibited its DLBCL attenuation effect by activating EDN1. GO analysis was used to analysis differentially expressed genes treated with ABT-199 and/or Apatinib for 3 days (all DEGs) and the percentage of GO term in the combination treatment group is shown in the pie chart from the DLBCL cells transcriptome (all DEGs) (Fig. S. 3A, S. 3B).
Endothelin-1(EDN1) gene plays an important role in apoptosis induced by ABT-199 and Apatinib
EDN1 is considered to be the most effective vasoconstrictor in the human cardiovascular system and plays a vital role in the development of tumors. In the previous study, we found the EDN1 gene through RNAseq assay, and it was also an anti- apoptotic protein that affects the overall survival and disease-free survival (Fig. 7A, 7B). Therefore, we verified its role in DLBCL by overexpression and knockdown of the EDN1 gene. Western Blot and flow cytometry showed the effect of overexpression of EDN1 gene in OCI-Ly1 and MCA cell lines (Fig. 7C, 7D, 7F, 7 G and S. 2E, S. 2F). Then we knocked out the EDN1 gene, and Western Blot was used to verify the knockout effect of EDN1 (Fig. 7H, 7I). Compared with the scramble, the expression of the EDN1 significantly decreased. The cell counts and the colony-forming also obviously decreased after shRNA interference (Fig. 7J, 7K, 7E, 7N, 7O, and S. 2G). The flow cytometry results also showed that the proportion of apoptosis of OCI-Ly1 and MCA cell lines after interference was remarkably increased as shown in Fig. 7L, 7M.
shRNA interference of EDN1 gene inhibits tumor burden in mice
To further explore the effect of EDN1 knockout in vivo, we selected BALB/C nude model mice and injected EDN1 knockout OCI-Ly1 and MCA cell lines into mice respectively. According to the results of cell lines, EDN1 gene knockout can effectively inhibit the growth of tumor cells and reduce tumor load, reflected by decreased volume and weight of tumor masses, compared to vehicle control and each agent (Fig. 8A, 8D, 8E, 8F, 8I, 8J). In addition, there was no significant difference in body weight between NC group and shEDN1 group (Fig. 8B, 8G). Histological examination found that the shEDN1 group remarkably inhibited the infiltration of DLBCL cells (Fig. 8C, 8H). After the mice were killed we ground the stripped tumor and extracted proteins, the results are consistent with those of cell lines. ABT-199 and Apatinib inhibited the MAPK/ERK and pro-survival pathways in the xenograft model (Fig. 8K, 8L). Thus, a mechanism involving ABT-199 and Apatinib apoptotic network is presented in hematologic malignancies (Fig. 8M).