Inhibitors of Bcl-2 and Bruton’s tyrosine kinase synergize to abrogate diffuse large B-cell lymphoma (DLBCL) growth. Inhibitors of Bcl-2 and Bruton’s tyrosine kinase synergize to abrogate diffuse large B-cell lymphoma (DLBCL) growth

Methods: DLBCL cell lines RIVA, U-2932 and OCI-LY3 were treated in vitro with ibrutinib (Ibru) or dimethyl sulfoxide (DMSO). Additionally, RIVA and U-2932 were orthotopically transplanted into MISTRG mice and mice were treated for two weeks with Ibru or vehicle. Tumor cells were isolated from the bone marrow at the study endpoint. The total RNA was isolated and the mRNA profiles were generated by next-generation sequencing using the Illumina TruSeq mRNA stranded protocol. Results: We have combined single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells. High Bcl-2 expression due to either BCL2 amplifications or translocations, in conjunction with chronic active BCR signalling accurately predict responses to dual Bcl-2/BTK inhibition.The combined data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a novel promising therapeutic strategy in DLBCL. Overall design: Examination of differential drug susceptibilities in DLBCL

方法：将弥漫大B细胞淋巴瘤（Diffuse Large B-Cell Lymphoma, DLBCL）细胞系RIVA、U-2932及OCI-LY3在体外分别用依鲁替尼（ibrutinib, Ibru）或二甲基亚砜（dimethyl sulfoxide, DMSO）处理。此外，将RIVA与U-2932细胞原位移植至MISTRG小鼠体内，并给予依鲁替尼或赋形剂处理两周。于实验终点从骨髓中分离肿瘤细胞，提取总RNA，采用Illumina TruSeq链特异性mRNA建库方案通过下一代测序生成mRNA表达谱。结果：本研究结合单药与联合药物应答谱分析，证实抗凋亡蛋白Bcl-2与酪氨酸激酶BTK的小分子抑制剂维奈克拉（venetoclax）及依鲁替尼联合使用，可高效杀伤DLBCL细胞。由BCL2基因扩增或易位导致的Bcl-2高表达，联合慢性活化B细胞受体信号通路，可精准预测双靶点Bcl-2/BTK抑制治疗的应答情况。联合分析数据显示，药物应答谱揭示的药物敏感性可归因于特定的突变特征与免疫组化生物标志物，提示Bcl-2/BTK联合抑制是DLBCL中一种极具潜力的新型治疗策略。整体实验设计：检测DLBCL细胞的差异药物敏感性。