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

项目总结/摘要 细胞周期蛋白依赖性激酶4和6（CDK 4/6）抑制剂被用作一线治疗， 转移性雌激素受体阳性（ER+）乳腺癌，鉴于其在以下方面的记录改善： 无进展生存期和总生存期。目前，临床研究 确定其在高危ER+乳腺癌女性的前期非转移性环境中的效用。尽管 这些有希望的研究，CDK 4/6抑制剂尚未与放射治疗联合使用， 患者作为标准治疗的一部分。我们实验室的初步数据表明，CDK 4/6 抑制导致多种ER+乳腺癌细胞系的放射增敏。这种放射增敏作用发生在 与palbociclib、ribociclib和abemaciclib（三种临床批准的CDK 4/6抑制剂）相似。 尽管我们已经证明所有三种CDK 4/6抑制剂都导致ER+乳腺癌的放射增敏， 癌细胞，这种放射增敏的机制仍然不清楚。在本提案中，我们的目标是：1）确定 CDK 4/6介导ER+乳腺癌细胞放射增敏的机制和2）确定 CDK 4/6转运蛋白介导的放射增敏在ER+乳腺癌体内模型中的功效。 放射增敏通常通过细胞周期分布的变化或细胞周期效率的降低而发生。 DNA修复途径，如同源重组（HR）或非同源末端连接（NHEJ）。我们实验室 有初步数据表明，短期CDK 4/6抑制导致DNA表达减少， 修复蛋白如CHK 1和RAD 51在同源重组中起作用。CDK 4/6抑制剂停止 通过G1/S细胞周期检查点的进展，我们假设CDK 4/6通道介导的G1细胞 细胞周期停滞可能限制细胞通过同源的DNA双链断裂修复的能力， 重组，导致体外和体内细胞死亡。因此，这项工作的总体假设是 药理学CDK 4/6抑制导致同源重组的抑制， ER+乳腺癌在体内的临床相关放射增敏。在目标1中，我们将使用报告基因测定， 免疫荧光、蛋白质印迹、qPCR和细胞周期分析，以确定CDK 4/6的作用 抑制和放射对ER+乳腺癌细胞周期进程以及HR和NHEJ效率的影响 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疗效。 CDK 4/6转运蛋白介导的放射增敏作用在生理学上更相关的系统。拟议 研究将在Corey Speers、James Rae、Ted Lawrence、洛里皮尔斯和 丹尼尔海斯，并将在密歇根大学演出。除了进一步了解 ER+乳腺癌的有效治疗策略，该提案推进了国家癌症研究所的 “了解癌症机制”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’.