METHODS: A panel of nine MCL cell lines were used for in vitro experiments. Four patient derived xenografts (PDX) obtained from patients with chemotherapy and ibrutinib-refractory MCL were used for in vivo proof-of-concept studies. Changes of the mitochondrial membrane potential, energy-metabolic pathways, AKT activity, and pro-apoptotic priming of MCL cells were evaluated by JC-1 staining, Seahorse XF analyser, genetically encoded fluorescent AKT reporter, and BH3 profiling, respectively. MCL clones with gene knockout or transgenic (over)expression of CDKN2A, MYC, CDK4, and RB1 were used to estimate impact of these aberrations on sensitivity to palbociclib, and venetoclax.
RESULTS: Co-targeting MCL cells with palbociclib and venetoclax induced cytotoxic synergy in vitro and in vivo. Molecular mechanisms responsible for the observed synthetic lethality comprised palbociclib-mediated downregulation of anti-apoptotic MCL1, increased levels of proapoptotic BIM bound on both BCL2, and BCL-XL and increased pro-apoptotic priming of MCL cells mediated by BCL2-independent mechanisms, predominantly palbociclib-triggered metabolic and mitochondrial stress. Loss of RB1 resulted in palbociclib resistance, while deletion of CDKN2A or overexpression of CDK4, and MYC genes did not change sensitivity to palbociclib.
CONCLUSIONS: Our data strongly support investigation of the chemotherapy-free palbociclib and venetoclax combination as an innovative treatment strategy for post-ibrutinib MCL patients without RB1 deletion.
方法:一组9个MCL细胞系用于体外实验。从化疗和依鲁替尼难治性MCL患者获得的四个患者来源的异种移植物(PDX)用于体内概念验证研究。线粒体膜电位的变化,能量代谢途径,AKT活动,通过JC-1染色评估MCL细胞的促凋亡引发,海马XF分析仪,基因编码的荧光AKT报告基因,和BH3配置文件,分别。基因敲除或转基因(过)表达CDKN2A的MCL克隆,MYC,CDK4和RB1用于评估这些像差对palbociclib敏感性的影响,和维尼托克.
结果:与palbociclib和venetoclax共同靶向的MCL细胞在体外和体内诱导了细胞毒性协同作用。观察到的合成致死性的分子机制包括palbociclib介导的抗凋亡MCL1下调,BCL2和BCL-XL上结合的促凋亡BIM水平增加以及由BCL2非依赖性机制介导的MCL细胞促凋亡引发增加,主要是palbociclib触发的代谢和线粒体应激。RB1丢失导致palbociclib耐药,而CDKN2A的缺失或CDK4和MYC基因的过表达并未改变对palbociclib的敏感性。
结论:我们的数据强烈支持对无RB1缺失的伊布替尼后MCL患者的无化疗帕博西尼和维奈托克联合治疗作为创新治疗策略的研究。