CD30 protects EBV‐positive diffuse large B‐cell lymphoma cells against mitochondrial dysfunction through increasing BNIP3 expression

Introduction: EBV-positive diffuse large B-cell lymphoma (EBV+DLBCL) predicts poor prognosis. Studies have found that CD30 expression was more frequent in EBV+DLBCL patients compared to EBV-negative (EBV-) DLBCL and high CD30 expression was associated with poor survivals in EBV+DLBCL. All these facts suggested a synergistic effect between CD30 expression and EBV infection. Methods: FARAGE and GM12878 are type II and III latency EBV-infected B cell lines while RAJI and DAUDI are type I. SUDHL-2, U2932 and RIVA are EBV-negative DLBCL cell lines. CRISPR/Cas9 editing was employed to engineer the loss-of-function models of genes involved in this study. Results: A total of 451 patients were included and the results showed patients had worse PFS (P = 0.007) and OS (P = 0.007) when CD30 was co-expressed with EBER in DLBCL (Figure 1A). Immunoblot analysis showed that CD30 expression in EBV latency II and III cell lines were higher than EBV latency I and EBV-negative cell lines (Figure 1B). Relevant experiments indicated that the tumor cells with high CD30 expression demonstrated rapid proliferation and were less susceptible to apoptosis (Figure 1C and 1D) and CD30-KO mice showed significantly smaller tumor size compared to CD30 wild type (WT) mice (Figure 1E). A LMP1 overexpression resulted in CD30 expression (Figure 1F). To elucidate how LMP1 regulated CD30, we found LMP1 overexpression increased p-Btk and p-p65 (Figure 1G). Immunoblot analysis detected a decrease in CD30 expression when latency II and III cells were treated with ibrutinib (a BTK inhibitor) and ibrutinib also abolished LMP1-induced p-p65 and p-IκBα (Figure 1H). We performed gene expression analysis following CD30 KO in FARAGE and the results indicated depression of CD30 inhibited BNIP3 expression (Figure 1I). Immunoblot analysis showed that BNIP3 expression in the KO group were significantly lower compared to those in the control group (Figure 1J). IHC staining results revealed a significant decrease of BNIP3 expression in CD30-KO mice (Figure 1K). We found that CD30 KO by CRISPR/Cas9 system led to a significant fall in the MMP (Figure 1L). Moreover, the accumulation of damaged mitochondria was observed in CD30-deficient tumor cells in mice by TEM (Figure 1M). Above results suggested that silencing of CD30 resulted in mitochondrial damage and the inhibition of mitochondrial activity. As BNIP3 was identified as a target gene for CD30, we investigated the effect of BNIP3 in EBV+DLBCL. Collectively, our results validated an important role of BNIP3 in promoting EBV+DLBCL survivals (Figure 1N and 1O). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Molecular Targeted Therapies, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.