BACKGROUND CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) are approved for the treatment of ER+ advanced breast cancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. We previously reported a novel therapeutic agent, ERX-11, that binds to the estrogen receptor (ER) and modulates ER-coregulator interactions. Here, we tested if the combination of ERX-11 with agents approved for ER+ BCa would be more potent. METHODS We tested the effect of combination therapy using BCa cell line models, including those that have acquired resistance to tamoxifen, letrozole, or CDK4/6 inhibitors or have been engineered to express mutant forms of the ER. In vitro activity was tested using Cell Titer-Glo, MTT, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, RT-qPCR, and mass spectrometry approaches. Xenograft, patient-derived explants (PDEs), and xenograft-derived explants (XDE) were used for preclinical evaluation and toxicity. RESULTS ERX-11 inhibited the proliferation of therapy-resistant BCa cells in a dose-dependent manner, including ribociclib resistance. The combination of ERX-11 and CDK4/6 inhibitor was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BCa cells, in vitro, in xenograft models in vivo, xenograft-derived explants ex vivo, and in primary patient-derived explants ex vivo. Importantly, the combination caused xenograft tumor regression in vivo. Unbiased global mass spectrometry studies demonstrated profound decreases in proliferation markers with combination therapy and indicated global proteomic changes in E2F1, ER, and ER coregulators. Mechanistically, the combination of ERX-11 and CDK4/6 inhibitor decreased the interaction between ER and its coregulators, as evidenced by immunoprecipitation followed by mass spectrometry studies. Biochemical studies confirmed that the combination therapy significantly altered the expression of proteins involved in E2F1 and ER signaling, and this is primarily driven by a transcriptional shift, as noted in gene expression studies. CONCLUSIONS Our results suggest that ERX-11 inhibited the proliferation of BCa cells resistant to both endocrine therapy and CDK4/6 inhibitors in a dose-dependent manner and that the combination of ERX-11 with a CDK4/6 inhibitor may represent a viable therapeutic approach.

中文翻译：

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雌激素受体调节剂结合调节剂（ERX-11）增强了CDK4 / 6抑制剂对雌激素受体阳性乳腺癌的活性。

背景技术CDK4 / 6抑制剂与内分泌疗法（AE / AI / SERDs）的组合被批准用于治疗ER +晚期乳腺癌（BCa）。但是，并非所有患者都能从CDK4 / 6抑制剂治疗中受益。我们以前报道过一种新型治疗剂ERX-11，它与雌激素受体（ER）结合并调节ER-调节剂的相互作用。在这里，我们测试了ERX-11与批准用于ER + BCa的药物的组合是否更有效。方法我们测试了使用BCa细胞系模型进行联合治疗的效果，其中包括已获得他莫昔芬，来曲唑或CDK4 / 6抑制剂耐药性或经工程改造可表达ER突变型的模型。使用Cell Titer-Glo，MTT和凋亡测定法测试了体外活性。使用Western印迹，报告基因测定，RT-qPCR，和质谱方法。异种移植，患者衍生的外植体（PDE）和异种移植的外植体（XDE）用于临床前评估和毒性。结果ERX-11以剂量依赖性方式抑制治疗耐药性BCa细胞的增殖，包括核糖体耐药性。ERX-11和CDK4 / 6抑制剂的组合在体外，体内异种移植模型，体外异种移植衍生的外植体和原发性内分泌协同作用下均能降低对内分泌治疗敏感的Bca细胞和对内分泌治疗耐药的BCa细胞的增殖离体来自患者的外植体。重要的是，该组合在体内引起异种移植肿瘤消退。全球无偏质谱研究表明，联合治疗可显着降低增殖标志物，并表明E2F1，ER，和ER协调器。从机理上讲，ERX-11和CDK4 / 6抑制剂的组合降低了ER及其核心调节剂之间的相互作用，免疫沉淀和质谱研究证明了这一点。生化研究证实，联合治疗显着改变了参与E2F1和ER信号转导的蛋白质的表达，这主要是由转录移位驱动的，如基因表达研究中所述。结论我们的结果表明ERX-11以剂量依赖的方式抑制了对内分泌治疗和CDK4 / 6抑制剂耐药的BCa细胞的增殖，并且ERX-11与CDK4 / 6抑制剂的组合可能代表了一种可行的治疗方法。 。免疫沉淀和质谱研究证明，ERX-11和CDK4 / 6抑制剂的组合降低了ER及其调节剂之间的相互作用。生化研究证实，联合治疗显着改变了参与E2F1和ER信号转导的蛋白质的表达，这主要是由转录移位驱动的，如基因表达研究中所述。结论我们的结果表明ERX-11以剂量依赖的方式抑制了对内分泌治疗和CDK4 / 6抑制剂耐药的BCa细胞的增殖，并且ERX-11与CDK4 / 6抑制剂的组合可能代表了一种可行的治疗方法。 。免疫沉淀和质谱研究证明，ERX-11和CDK4 / 6抑制剂的组合降低了ER及其调节剂之间的相互作用。生化研究证实，联合治疗显着改变了参与E2F1和ER信号转导的蛋白质的表达，这主要是由转录移位驱动的，如基因表达研究中所述。结论我们的结果表明ERX-11以剂量依赖的方式抑制了对内分泌治疗和CDK4 / 6抑制剂耐药的BCa细胞的增殖，并且ERX-11与CDK4 / 6抑制剂的组合可能代表了一种可行的治疗方法。 。生化研究证实，联合治疗显着改变了参与E2F1和ER信号转导的蛋白质的表达，这主要是由转录移位驱动的，如基因表达研究中所述。结论我们的结果表明ERX-11以剂量依赖的方式抑制了对内分泌治疗和CDK4 / 6抑制剂耐药的BCa细胞的增殖，并且ERX-11与CDK4 / 6抑制剂的组合可能代表了一种可行的治疗方法。 。生化研究证实，联合治疗显着改变了参与E2F1和ER信号转导的蛋白质的表达，这主要是由转录移位驱动的，如基因表达研究中所述。结论我们的结果表明ERX-11以剂量依赖的方式抑制了对内分泌治疗和CDK4 / 6抑制剂耐药的BCa细胞的增殖，并且ERX-11与CDK4 / 6抑制剂的组合可能代表了一种可行的治疗方法。 。