尽管CDK4/6在治疗HR+、HER2-晚期乳腺癌中具有生物学和治疗相关性，但CDK4/6抑制剂的详细作用方式尚未完全了解。特别是，CDK4在T172（pT172）的磷酸化对于产生活性构象至关重要，然而迄今为止尚未报告此类晶体结构。我们在此描述了活性CDK4-环D3与CDK4/6抑制剂abemaciclib结合的X射线结构，并讨论了催化能力复合物的关键方面。此外，尽管CDK4 T172磷酸化的作用作为CDK4/6抑制剂反应的潜在生物标记尚未被探究。我们在机制上表明，CDK4/6i稳定提前准备（pT172）的CDK4-环D复合物，并选择性地替换响应性肿瘤细胞中的p21。稳定活性CDK4-环D1复合物可以导致通过替代剂量方案随后的通路重新激活。因此，abemaciclib与CDK4的持续结合在乳腺癌细胞系中导致强大的细胞周期抑制并防止下游信号的反弹激活。总的来说，我们的研究提供了关键见解，表明CDK4/6抑制剂的长期治疗和CDK4/6-环D复合物的组成都是abemaciclib功效的关键决定因素，对于这类抗癌疗法有着重要的启示。 ©2022. 作者(们)。 Note: We kept the sentence structure as close to the original text as possible to maintain clarity and accuracy in translation.

Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.© 2022. The Author(s).