Elucidating the Mechanism of CDK4/6 Inhibitor-Mediated Radiosensitization of ER+ Breast Cancers

项目摘要/摘要 细胞周期蛋白依赖性激酶4和6(CDK4/6)抑制剂作为一线疗法用于治疗女性 转移性雌激素受体阳性(ER)乳腺癌有文献记载的改善 在该患者群体中的无进展和总体生存。此外，临床研究目前正在进行 确定它们在高危ER乳腺癌患者的早期、非转移性环境中的效用。尽管 这些有希望的研究表明，CDK4/6抑制剂尚未与放射治疗联合使用 患者接受的治疗是标准护理的一部分。我们实验室的初步数据表明，CDK4/6 抑制导致多个ER乳腺癌细胞系的放射增敏。这种放射增敏作用发生在 与临床批准的三种CDK4/6抑制剂Palbociclib、Riociclib和Abemaciclib的程度相似。 虽然我们已经证明了所有三种CDK4/6抑制剂都能导致ER乳腺的放射增敏 这种放射增敏的机制尚不清楚。在这份提案中，我们的目标是1)确定 CDK4/6抑制剂介导ER乳腺癌细胞放射增敏的机制及2)确定 CDK4/6抑制剂介导的放射增敏在ER乳腺癌体内模型中的作用 放射增敏通常通过细胞周期分布的改变或细胞效率的降低而发生。 DNA修复途径包括同源重组(HR)或非同源末端连接(NHEJ)。我们的实验室 有初步数据表明，短期抑制CDK4/6会导致DNA表达下降 修复蛋白，如CHK1和RAD51，在同源重组中发挥作用。CDK4/6抑制剂停用 通过G1/S细胞周期检查点，我们假设CDK4/6抑制剂介导的G1细胞 周期停滞可能限制细胞通过同源基因进行DNA双链断裂修复的能力 重组，导致体外和体内的细胞死亡。因此，这项工作的总体假设是 药物抑制CDK4/6导致抑制同源重组并导致 ER乳腺癌体内的临床相关放射增敏作用。在目标1中，我们将使用记者分析， 免疫荧光、Western blotts、qPCR和细胞周期分析以确定CDK4/6的作用 放疗对ER乳腺癌细胞周期进程及HR和NHEJ效率的抑制作用 细胞系。在目标2中，我们将使用ER乳腺癌的细胞系和PDX异种移植模型来评估其疗效 CDK4/6抑制剂在更多生理相关系统中介导的放射增敏作用的研究。建议数 研究将在科里·斯皮尔斯博士、詹姆斯·雷博士、泰德·劳伦斯博士、洛里·皮尔斯博士和 丹尼尔·海斯，将在密歇根大学演出。除了加深我们对 ER乳腺癌的有效治疗策略，这项建议推动了国家癌症研究所的 “了解癌症的机制”和“治疗癌症”的科学优先事项。

PROJECT SUMMARY/ABSTRACT Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are used as frontline therapy to treat women with metastatic estrogen receptor positive (ER+) breast cancers given their documented improvements in progression-free and overall survival in this patient population. Furthermore, clinical studies are currently defining their utility in the upfront, non-metastatic setting for women with high-risk ER+ breast cancers. Despite these promising studies, CDK4/6 inhibitors are not yet given in combination with the radiation therapy that patients receive as part of the standard of care. Preliminary data from our lab demonstrates that CDK4/6 inhibition leads to the radiosensitization of multiple ER+ breast cancer cell lines. This radiosensitization occurs to a similar degree with palbociclib, ribociclib, and abemaciclib, the three clinically approved CDK4/6 inhibitors. Although we have demonstrated that all three CDK4/6 inhibitors lead to the radiosensitization of ER+ breast cancer cells, the mechanism of this radiosensitization remains unclear. In this proposal, we aim to 1) determine the mechanism of CDK4/6 inhibitor-mediated radiosensitization of ER+ breast cancer cells and 2) determine the efficacy of CDK4/6 inhibitor-mediated radiosensitization in in vivo models of ER+ breast cancer. Radiosensitization typically occurs through changes in cell cycle distribution or decreases in the efficiency of DNA repair pathways like homologous recombination (HR) or non-homologous end joining (NHEJ). Our lab has preliminary data to suggest that short term CDK4/6 inhibition leads to a decrease in expression of DNA repair proteins like CHK1 and RAD51 that play a role in homologous recombination. CDK4/6 inhibitors halt progression through the G1/S cell cycle checkpoint, and we hypothesize that CDK4/6 inhibitor-mediated G1 cell cycle arrest may limit the ability of cells to undergo DNA double strand break repair through homologous recombination, leading to cell death both in vitro and in vivo. Thus, the overall hypothesis of this work is that pharmacologic CDK4/6 inhibition leads to inhibition of homologous recombination and leads to clinically relevant radiosensitization of ER+ breast cancers in vivo. In Aim 1, we will use reporter assays, immunofluorescence, western blots, qPCR, and cell cycle analysis to determine the effects of CDK4/6 inhibition and radiation on both cell cycle progression as well as HR and NHEJ efficiency in ER+ breast cancer cell lines. In Aim 2, we will use cell line and PDX xenograft models of ER+ breast cancer to assess the efficacy of CDK4/6 inhibitor-mediated radiosensitization in more physiologically relevant systems. The proposed studies will be conducted with guidance from Drs. Corey Speers, James Rae, Ted Lawrence, Lori Pierce, and Daniel Hayes, and will be performed at the University of Michigan. In addition to furthering our understanding of effective treatment strategies for ER+ breast cancer, this proposal advances the National Cancer Institute’s scientific priorities of ‘understanding mechanisms of cancer’ and ‘treating cancer’.