Predicting effective pro-apoptotic anti-leukaemic drug combinations using co-operative dynamic BH3 profiling

The BH3-only apoptosis agonists BAD and NOXA target BCL-2 and MCL-1 respectively and co-operate to induce apoptosis. On this basis, therapeutic drugs targeting BCL-2 and MCL-1 might have enhanced activity if used in combination. We identified anti-leukaemic drugs sensitising to BCL-2 antagonism and drugs sensitising to MCL-1 antagonism using the technique of dynamic BH3 profiling, whereby cells were primed with drugs to discover whether this would elicit mitochondrial outer membrane permeabilisation in response to BCL-2-targeting BAD-BH3 peptide or MCL-1-targeting MS1-BH3 peptide. We found that a broad range of anti-leukaemic agents–notably MCL-1 inhibitors, DNA damaging agents and FLT3 inhibitors–sensitise leukaemia cells to BAD-BH3. We further analysed the BCL-2 inhibitors ABT-199 and JQ1, the MCL-1 inhibitors pladienolide B and torin1, the FLT3 inhibitor AC220 and the DNA double-strand break inducer etoposide to correlate priming responses with co-operative induction of apoptosis. ABT-199 in combination with pladienolide B, torin1, etoposide or AC220 strongly induced apoptosis within 4 hours, but the MCL-1 inhibitors did not co-operate with etoposide or AC220. In keeping with the long half-life of BCL-2, the BET domain inhibitor JQ1 was found to downregulate BCL-2 and to prime cells to respond to MS1-BH3 at 48, but not at 4 hours: prolonged priming with JQ1 was then shown to induce rapid cytochrome C release when pladienolide B, torin1, etoposide or AC220 were added. In conclusion, dynamic BH3 profiling is a useful mechanism-based tool for understanding and predicting co-operative lethality between drugs sensitising to BCL-2 antagonism and drugs sensitising to MCL-1 antagonism. A plethora of agents sensitised cells to BAD-BH3-mediated mitochondrial outer membrane permeabilisation in the dynamic BH3 profiling assay and this was associated with effective co-operation with the BCL-2 inhibitory compounds ABT-199 or JQ1.

仅含BH3结构域的凋亡激动剂BAD与NOXA分别靶向B细胞淋巴瘤因子2（BCL-2）与髓细胞白血病因子1（MCL-1），二者协同诱导细胞凋亡。基于该机制，靶向BCL-2与MCL-1的治疗药物联合使用时，其抗肿瘤活性或可得到增强。

我们借助动态BH3谱分析（dynamic BH3 profiling）技术，筛选出可使细胞对BCL-2拮抗作用致敏的抗白血病药物，以及可使细胞对MCL-1拮抗作用致敏的抗白血病药物：该技术通过药物预处理细胞，探究细胞在暴露于靶向BCL-2的BAD-BH3肽或靶向MCL-1的MS1-BH3肽时，是否会诱导线粒体外膜通透化。

研究发现，多种抗白血病药物——尤其是MCL-1抑制剂、DNA损伤剂与FLT3抑制剂——可使白血病细胞对BAD-BH3肽产生致敏性。

我们进一步针对BCL-2抑制剂ABT-199与JQ1、MCL-1抑制剂普拉迪诺利德B（pladienolide B）与托林1（torin1）、FLT3抑制剂AC220以及DNA双链断裂诱导剂依托泊苷（etoposide）开展分析，以将细胞致敏应答与药物协同诱导凋亡的效应相关联。

ABT-199分别与普拉迪诺利德B、托林1、依托泊苷或AC220联合使用时，可在4小时内强效诱导细胞凋亡，但MCL-1抑制剂与依托泊苷或AC220并未产生协同杀伤效应。

鉴于BCL-2的长半衰期特性，我们观察到BET结构域抑制剂JQ1可下调BCL-2的表达，并使细胞在48小时（而非4小时）时对MS1-BH3肽产生致敏性；后续实验证实，若在经JQ1长期预处理的细胞中加入普拉迪诺利德B、托林1、依托泊苷或AC220，可快速诱导细胞色素C释放。

综上，动态BH3谱分析是一种基于机制的实用工具，可用于理解并预测靶向BCL-2拮抗作用的药物与靶向MCL-1拮抗作用的药物之间的协同致死效应。在动态BH3谱分析实验中，大量药物可使细胞对BAD-BH3肽介导的线粒体外膜通透化产生致敏性，且该现象与ABT-199或JQ1这类BCL-2抑制类化合物的协同作用密切相关。