Targeting Dependency on a Paralog Pair of CBP/p300 against De-repression of KREMEN2 in SMARCB1-Deficient Cancers

SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the “simultaneous inhibition of a paralog pair” concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers. Overall design: NT indicates that non-treated and non-transfected cells were harvested. A485 indicates that cells treated with 2 µM A-485 for 24 h were harvested. CP-C27 indicates that cells treated with 0.2 µM CP-C27 for 24 h were harvested. siRNA (siNT, siCBPsip300, siKREMEN2) indicates that cells transfeted with siRNA for 48 h or 96 h were harvested.

SMARCB1是SWI/SNF染色质重塑复合物（SWI/SNF chromatin remodeling complex）的一个亚基，同时也是横纹肌样瘤（rhabdoid tumors）与上皮样肉瘤（epithelioid sarcomas）的致病基因。本研究基于"同时抑制旁系同源基因对（paralog pair）"的理念，采用双siRNA筛选方法，鉴定出CREB结合蛋白（CBP）与p300蛋白（p300）的旁系同源基因对可作为SMARCB1缺陷型癌症的合成致死靶点。使用CBP/p300双重抑制剂处理，可抑制SMARCB1缺陷细胞来源的细胞系及肿瘤异种移植物的生长，而对SMARCB1正常型细胞来源的样本无此抑制效果。
含有SMARCB1的SWI/SNF复合物会与组蛋白H3第27位赖氨酸三甲基化（H3K27me3）及其甲基转移酶增强子zeste同源物2（EZH2）共同定位在KREMEN2基因座的启动子区域，从而导致KREMEN2的转录下调。与之相反，SMARCB1缺陷会使组蛋白H3第27位赖氨酸乙酰化（H3K27ac）及其乙酰转移酶CBP和p300被招募至KREMEN2基因座，伴随H3K27ac的定位，最终引发KREMEN2转录上调，且该过程与SMARCA1染色质重塑复合物协同发挥作用。同时抑制CBP/p300会导致KREMEN2转录下调，随后因KREMEN1无法与KREMEN2结合而发生单体化，进而诱导细胞凋亡，最终抑制抗凋亡信号通路。
综上，本研究结果表明，CBP/p300双重抑制剂有望成为SMARCB1缺陷型癌症的潜在治疗药物。
实验整体设计如下：NT组指收集未处理且未转染的细胞；A485组指收集经2 μM A-485处理24小时的细胞；CP-C27组指收集经0.2 μM CP-C27处理24小时的细胞；siRNA组（siNT、siCBPsip300、siKREMEN2）指收集经siRNA转染48小时或96小时的细胞。