Xin Xu1,2, Ting Wei3, Weijie Zhong4, Rosalind Ang5, Ye Lei5, Hui Zhang6, Qingshan Li7. 1. The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong, 510630, People's Republic of China. 2. Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, 511458, People's Republic of China. 3. Department of Hematology, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, 510180, People's Republic of China. 4. Department of Geriatrics, Hematology and Oncology Ward, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, 510180, People's Republic of China. 5. Precision Immunology Institute, Mount Sinai School of Medicine, New York, NY, 10029, USA. 6. Institute of Immunology and Molecular Medicine, Jining Medical University, Jinan, Shandong, 272067, People's Republic of China. 7. Department of Hematology, Guangzhou Red Cross Hospital, Jinan University, No. 396 Tongfuzhong Road, Haizhu District, 510220, Guangzhou, Guangdong, People's Republic of China. eyxuxin@scut.edu.cn.
Abstract
BACKGROUND: Non-germinal center B-cell-like diffuse large B-cell lymphoma (non-GCB-DLBCL) has worse clinical outcome than GCB-DLBCL, and some relapsed/refractory non-GCB-DLBCL (R/R non-GCB-DLBCL) are even resistant to CD20 monoclonal antibody (rituximab). Bruton's tyrosine kinase inhibitors (BTKis) are new drugs for B-cell lymphoma. BTKis can promote apoptosis of DLBCL by inactivating nuclear transcription factor κB (NFκB) signaling pathway. Cylindromatosis (CYLD) is a tumor suppressor and ubiquitinase. CYLD can inactivate NFκB signaling pathway through ubiquitination and regulate the apoptosis of hematological tumors. The ubiquitination of CYLD can be regulated by phosphorylation, suggesting that the regulation of CYLD phosphorylation can be a potential mechanism to promote the apoptosis of hematological tumors. Therefore, we hypothesized that BTKis could promote the apoptosis of non-GCB-DLBCL by regulating the phosphorylation of CYLD, especially in rituximab resistant cases, and we proved this hypothesis through both in vivo and in vitro experiments. METHODS: The baseline expression levels of CYLD phosphorylation in non-GCB-DLBCL patients and cell lines were detected by Western Blotting. The non-GCB-DLBCL cell lines were treated with BTKis, and apoptosis induced by BTKis treatment was detected by Western blotting, cell viability assay and Annexin V assay. To verify whether the effect of BTKis on apoptosis in non-GCN-DLBCL cells is CYLD dependent, the expression of CYLD was knocked down by lentiviral shRNAs. To verify the effect of BTKis on the phosphorylation of CYLD and the apoptosis in vivo and in rituximab resistant non-GCB-DLBCL, the xeograft model and rituximab resistant non-GCB-DLBCL cells were generated by tumor cell inoculation and escalation of drug concentrations, respectively. RESULTS: BTKis induced apoptosis by down-regulating CYLD phosphorylationin in non GCB-DLBCL, xenograft mouse model, and rituximab-resistant cells, and this effect could be enhanced by rituximab. Knocking-down CYLD reversed apoptosis which was induced by BTKis. BTKis induced CYLD-dependent apoptosis in non-GCB-DLBCL including in rituximab-resistant cells. CONCLUSIONS: The present results indicated that CYLD phosphorylation is a potential clinical therapeutic target for non-GCB-DLBCL, especially for rituximab-resistant relapsed/refractory cases.
BACKGROUND: Non-germinal center B-cell-like diffuse large B-cell lymphoma (non-GCB-DLBCL) has worse clinical outcome than GCB-DLBCL, and some relapsed/refractory non-GCB-DLBCL (R/R non-GCB-DLBCL) are even resistant to CD20 monoclonal antibody (rituximab). Bruton's tyrosine kinase inhibitors (BTKis) are new drugs for B-cell lymphoma. BTKis can promote apoptosis of DLBCL by inactivating nuclear transcription factor κB (NFκB) signaling pathway. Cylindromatosis (CYLD) is a tumor suppressor and ubiquitinase. CYLD can inactivate NFκB signaling pathway through ubiquitination and regulate the apoptosis of hematological tumors. The ubiquitination of CYLD can be regulated by phosphorylation, suggesting that the regulation of CYLD phosphorylation can be a potential mechanism to promote the apoptosis of hematological tumors. Therefore, we hypothesized that BTKis could promote the apoptosis of non-GCB-DLBCL by regulating the phosphorylation of CYLD, especially in rituximab resistant cases, and we proved this hypothesis through both in vivo and in vitro experiments. METHODS: The baseline expression levels of CYLD phosphorylation in non-GCB-DLBCL patients and cell lines were detected by Western Blotting. The non-GCB-DLBCL cell lines were treated with BTKis, and apoptosis induced by BTKis treatment was detected by Western blotting, cell viability assay and Annexin V assay. To verify whether the effect of BTKis on apoptosis in non-GCN-DLBCL cells is CYLD dependent, the expression of CYLD was knocked down by lentiviral shRNAs. To verify the effect of BTKis on the phosphorylation of CYLD and the apoptosis in vivo and in rituximab resistant non-GCB-DLBCL, the xeograft model and rituximab resistant non-GCB-DLBCL cells were generated by tumor cell inoculation and escalation of drug concentrations, respectively. RESULTS:BTKis induced apoptosis by down-regulating CYLD phosphorylationin in non GCB-DLBCL, xenograft mouse model, and rituximab-resistant cells, and this effect could be enhanced by rituximab. Knocking-down CYLD reversed apoptosis which was induced by BTKis. BTKis induced CYLD-dependent apoptosis in non-GCB-DLBCL including in rituximab-resistant cells. CONCLUSIONS: The present results indicated that CYLD phosphorylation is a potential clinical therapeutic target for non-GCB-DLBCL, especially for rituximab-resistant relapsed/refractory cases.
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