Combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines

Abstract

Epstein-Barr virus (EBV) manipulates the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and survival of the host cells through the expression of viral proteins in distinct latency patterns. We postulate that the combination of bortezomib (proteasome inhibitor) and venetoclax (Bcl-2 inhibitor) [bort/venetoclax] will cause synergistic killing of post-transplant lymphoproliferative disorder (PTLD) through targeting the pro-survival function of latent viral proteins such as latent membrane protein-1 (LMP-1) and EBV nuclear antigen-3C (EBNA-3C). Bort/venetoclax could synergistically kill spontaneous lymphoblastoid cell lines (sLCLs) derived from patients with PTLD and EBV-associated hemophagocytic lymphohistiocytosis by inducing DNA damage response, apoptosis and G1-S cell cycle arrest in a ROS-dependent manner. Bortezomib potently induced the expression of Noxa, a pro-apoptotic initiator and when combined with venetoclax, inhibited Mcl-1 and Bcl-2 simultaneously. Bortezomib prevented LMP-1 induced proteasomal degradation of IκBα leading to the suppression of the NF-κB signaling pathway. Bortezomib also rescued Bcl-6 from EBNA-3C mediated proteasomal degradation thus maintaining the repression of cyclin D1 and Bcl-2 causing G1-S arrest and apoptosis. Concurrently, venetoclax inhibited Bcl-2 upregulated by either LMP-1 or EBNA-3C. Bort/venetoclax decreased the expression of phosphorylated p65 and Bcl-2 at serine 70 thereby suppressing the NF-κB signaling pathway and promoting apoptosis, respectively. These data corroborated the marked suppression of the growth of xenograft of sLCL in SCID mice (p<0.001). Taken together, the combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Fig 1. Bort/venetoclax promote synergistic cell death through inducing expression of apoptotic and DNA damage response markers as well as inhibitor of NF-κB but suppressed regulators for cell cycle progression in sLCLs.

sLCL 381 and sLCL 421 were treated with combination of bortezomib (0, 1, 2, 4, 8, 16, 32, and 64 nM) and venetoclax (0, 1.25, 2.5, 5, 10, 20 μM) for 24 hours and stained by MTT solution. OD570 nm and OD630 nm were measured, and cell viability was plotted. (a) Percentages of cell viability in bort/venetoclax treatments were determined. (b) Isobolograms and CI were applied for analysis of synergism of bort/venetoclax. (c-d) sLCL 381 and sLCL 421 cells were treated with conditions as described with or without 15 mM N-acetylcysteine (NAC). Proteins were extracted and subjected to western blot analysis with indicated antibodies. (e-f) sLCL 381 and sLCL 421 cells were treated with conditions as described. Proteins were extracted and subjected to co-immunoprecipitation with anti-Bak antibody. The expression of Mcl-1, Bcl-2 and Bak were detected by western blot analysis. (g) sLCL 381 and sLCL 421 cells were treated with conditions as described. The cells were fixed, incubated with anti-cleaved caspase-3 antibody overnight, and then probed by AF488-conjugated anti-rabbit secondary antibody for cytometric analysis. One representative set of flow cytometric analysis of the cell lines was presented. Percentage of cleaved caspase-3 in (h) sLCL 381 and (i) sLCL 421 cells upon the treatments was analyzed for statistical significance by using One-way ANOVA Bonferroni post-tests. *p < 0.05 was considered as statistically significant (*p < 0.05 and **p < 0.01). (j) sLCL 381 and sLCL 421 cells were either treated with DMSO or bort/venetoclax with or without 50 μM caspase-8 inhibitor (Z-IETD), 50 μM caspase-9 inhibitor (Z-LEHD), or 50 μM pan-caspase inhibitor (Z-VAD). Proteins were extracted and subjected to western blot analysis with indicated antibodies.

Fig 2. Lentivirus shRNA knockdown experiment and MTT analysis showing the effect of bortezomib/venetoclax on the cell viability of sLCL 381 and 421 panels.

Lentivirus shRNA knockdown on (a) BHRF-1, (b) EBNA-3C, (c) LMP-1 and (d) Noxa were performed in sLCL 381 and 421 cells. Scramble shRNA on wild-type Firefly Luciferase (shLuc) was performed as a control. The expression of three different clones of BHRF-1, EBNA-3C and LMP-1were detected by western blot analysis. sLCL 381 and 421 shLuc and shNOXA were treated with conditions as described to test the expression of Noxa which was detected by western blot analysis. The cells were treated with combination of bortezomib (0, 1, 2, 4, 8, 16, 32, and 64 nM) and venetoclax (0, 1.25, 2.5, 5, 10, 20 μM) for 24 hours and stained by MTT solution. OD570 nm and OD630 nm were measured, and cell viability was plotted. (e & g) Percentages of cell viability of sLCL 381 and 421 panels in bort/venetoclax treatments were determined. (f & h) Isobolograms were applied for analysis of synergism of bort/venetoclax.

Fig 3. Apoptosis induced by bortezomib/venetoclax was stronger in sLCL expressing BHRF-1, EBNA-3C, LMP-1 and Noxa.

(a) sLCL 381 and (b) sLCL 421 panels were were either treated with DMSO, 10 nM bortezomib (BTZ), 5 μM venetoclax (VTX) or bortezomib/venetoclax (BTZ/VTX) for 24 hours. The cells were stained by TUNEL as described and analyzed by flow cytometry. Fold change of percentage of BrdU in the cells upon treatments was calculated. The fold change was analyzed for statistical significance by using One-way ANOVA Bonferroni post-tests. *p < 0.05 was considered as statistically significant (***p < 0.001).

Fig 4. Synergistic apoptosis, DNA damage response and inhibition of NF-κB induced by bortezomib/venetoclax was related to the expression of BHRF-1, LMP-1, EBNA-3C and botezomib-induced Noxa in sLCL.

(a) shLuc, shLMP-1 and shE3C of sLCL 381and sLCL 421 were treated with conditions as described. Proteins were extracted and subjected to western blot analysis with indicated antibodies. (b) sLCL 381 and (c) sLCL 421 panels were treated with conditions as described. Proteins were extracted and subjected to western blot analysis with indicated antibodies.

Fig 5. Bortezomib/venetoclax suppressed G1-S transition in sLCLs expressing EBNA-3C, LMP-1 and bortezomib-induced Noxa.

sLCL 381 and sLCL 421 panels were treated with conditions as described. The treated cells were subjected to cell cycle analysis as described and analyzed by ModFit LT 3.0. (a-b) Cell cycle analysis showing the percentages of sLCL 381 and 421 cells in G1, S, G2/M phases. The results were analyzed for statistical significance using One-way ANOVA Dunnett’s Multiple Comparison Test. p-value less than 0.05 was considered statistically significant; *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Error bars represent the standard error of mean (SEM) of data obtained from three independent experiments.

Fig 6. Effects of bortezomib/venetoclax on the growth suppression of sLCL 381 xenografts in SCID mice.

SCID mice were engrafted with sLCL 381 xenograft and treated with conditions as described (n = 6 for each treatment group). (a) The average of tumor volumes on day 30 and (b) the average of tumor masses of mice of control and treated groups were shown. (c) The size of tumors during the period of experiment was measured twice weekly using a caliper. Data are presented as the mean tumor volumes of mice in both treatment and control groups on the day of treatment (day 1, 4, 8, 11, 15, 18, 22, 26 and 30). (d) The mice were weighed on day 1, 4, 8, 11, 15, 18, 22, 26 and 30 to assess the toxicity of the treatments. (e) pH2AX IHC staining of the xenografts were performed as described. The number of pH2AX positive cells were summarized in histograms and (f) a representative IHC image of each treatment group was shown. The results were analyzed for statistical significance using One-way ANOVA Dunnett’s Multiple Comparison Test. p-value less than 0.05 was considered statistically significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns = not significant. Error bars represent the standard error of mean (SEM) of data obtained from the SCID mice.

Fig 7. Schematic diagram illustrating the mechanism of action of bortezomib/venetoclax in sLCLs.