Trastuzumab-refractory, HER2 (human epidermal growth factor receptor 2)-positive breast cancer is commonly treated with trastuzumab emtansine (T-DM1), an antibody-drug conjugate of trastuzumab and the microtubule-targeting agent, DM1. However, drug response reduces greatly over time due to acquisition of resistance whose molecular mechanisms are mostly unknown. Here, we uncovered a novel mechanism of resistance against T-DM1 by combining whole transcriptome sequencing (RNA-Seq), proteomics and a targeted small interfering RNA (siRNA) sensitization screen for molecular level analysis of acquired and de novo T-DM1-resistant models of HER2-overexpressing breast cancer. We identified Polo-like kinase 1 (PLK1), a mitotic kinase, as a resistance mediator whose genomic as well as pharmacological inhibition restored drug sensitivity. Both acquired and de novo resistant models exhibited synergistic growth inhibition upon combination of T-DM1 with a selective PLK1 inhibitor, volasertib, at a wide concentration range of the two drugs. Mechanistically, T-DM1 sensitization upon PLK1 inhibition with volasertib was initiated by a spindle assembly checkpoint (SAC)-dependent mitotic arrest, leading to caspase activation, followed by DNA damage through CDK1-dependent phosphorylation and inactivation of Bcl-2/xL. Furthermore, we showed that Ser70 phosphorylation of Bcl-2 directly regulates apoptosis by disrupting the binding to and sequestration of the pro-apoptotic protein Bim. Importantly, T-DM1 resistance signature or PLK1 expression correlated with cell cycle progression and DNA repair, and predicted a lower sensitivity to taxane/trastuzumab combination in HER2-positive breast cancer patients. Finally, volasertib in combination with T-DM1 greatly synergized in models of T-DM1 resistance in terms of growth inhibition both in three dimensional (3D) cell culture and in vivo. Altogether, our results provide promising pre-clinical evidence for potential testing of T-DM1/volasertib combination in T-DM1 refractory HER2-positive breast cancer patients for whom there is currently no treatment available.

中文翻译：

---

在 HER2 阳性乳腺癌中，靶向 PLK1 通过 CDK1 依赖性磷酸化和 Bcl-2/xL 失活克服了 T-DM1 耐药性。


曲妥珠单抗难治性 HER2（人表皮生长因子受体 2）阳性乳腺癌通常采用曲妥珠单抗 emtansine (T-DM1) 治疗，曲妥珠单抗是曲妥珠单抗和微管靶向剂 DM1 的抗体药物缀合物。然而，由于耐药性的获得，药物反应随着时间的推移而大大降低，而耐药性的分子机制大多未知。在这里，我们通过结合全转录组测序（RNA-Seq）、蛋白质组学和靶向小干扰RNA（siRNA）敏化筛选，发现了一种新的T-DM1耐药机制，用于获得性和从头T-DM1耐药性的分子水平分析HER2 过度表达乳腺癌模型。我们确定了 Polo 样激酶 1 (PLK1)（一种有丝分裂激酶）作为耐药介质，其基因组和药理学抑制可恢复药物敏感性。 T-DM1 与选择性 PLK1 抑制剂 volasertib 联合使用时，两种药物在较宽的浓度范围内，获得性耐药模型和新发耐药模型均表现出协同生长抑制作用。从机制上讲，volasertib 抑制 PLK1 后的 T-DM1 致敏是由纺锤体组装检查点 (SAC) 依赖性有丝分裂停滞启动的，导致 caspase 激活，随后通过 CDK1 依赖性磷酸化和 Bcl-2/xL 失活造成 DNA 损伤。此外，我们发现 Bcl-2 的 Ser70 磷酸化通过破坏促凋亡蛋白 Bim 的结合和隔离来直接调节细胞凋亡。重要的是，T-DM1 耐药特征或 PLK1 表达与细胞周期进展和 DNA 修复相关，并预测 HER2 阳性乳腺癌患者对紫杉烷/曲妥珠单抗组合的敏感性较低。 最后，volasertib 与 T-DM1 组合在 T-DM1 耐药模型中在三维 (3D) 细胞培养和体内生长抑制方面具有极大的协同作用。总而言之，我们的结果为 T-DM1/volasertib 组合在目前尚无治疗方法的 T-DM1 难治性 HER2 阳性乳腺癌患者中进行潜在测试提供了有希望的临床前证据。