Selective CDK7 inhibition suppresses cell cycle progression and MYC signaling while enhancing apoptosis in therapy-resistant estrogen receptor positive breast cancer. Selective CDK7 inhibition suppresses cell cycle progression and MYC signaling while enhancing apoptosis in therapy-resistant estrogen receptor positive breast cancer

Purpose: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER) positive (ER+) breast cancer (BC). Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine resistant ER+ BC models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ BC. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ BC remain elusive. Herein, we sought to unravel these mechanisms. Experimental Design: We conducted multi-omic analyses in ER+ BC models in vitro and in vivo including models with different genetic backgrounds. We also performed genome wide CRISPR knock-out library screens to identify potential therapeutic vulnerabilities in CDK4/6i resistance models. Results: We found that the on-target anti-tumor effects of CDK7 inhibition in ER+ BC are in part p53 dependent, involve cell-cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ETs and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118, however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Lastly, genome wide CRISPR/Cas9 screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i resistant models. Conclusions: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ BC. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors for sensitivity to CDK7 inhibitor-based treatments. Overall design: 1. Comparative gene expression profiling analysis of RNA-seq data for ER-WT MCF7 cells and its ER-mutant derivatives with and without CDK7 mutation and in two PDX models (WT:1415 and Mut:1526). 2. Comparative gene expression profiling analysis of RNA-seq data for Palbociclib-sensitive and -resistant T47D and MCF7 cells.

研究目的：内分泌治疗（endocrine therapy, ET）与CDK4/6抑制剂（CDK4/6i）耐药是雌激素受体阳性（estrogen receptor, ER）阳性（ER+）乳腺癌（breast cancer, BC）中的临床难题。细胞周期蛋白依赖性激酶7（cyclin-dependent kinase7, CDK7）是内分泌耐药ER+ BC模型中的候选治疗靶点，选择性CDK7抑制剂（CDK7i）目前正处于ER+ BC治疗的临床研发阶段。然而，CDK7i在ER+ BC中发挥抗肿瘤活性的具体分子机制仍未明确。本研究旨在阐明上述机制。

实验设计：本研究在体外及体内的ER+ BC模型（涵盖不同遗传背景的模型）中开展多组学分析。此外，本研究还通过全基因组CRISPR敲除文库筛选，鉴定CDK4/6i耐药模型中潜在的治疗易感靶点。

研究结果：本研究发现，ER+ BC中CDK7抑制的靶标抗肿瘤活性部分依赖于p53，其作用机制涉及细胞周期抑制与c-Myc表达抑制。此外，CDK7抑制可产生细胞毒性效应，这与内分泌治疗及CDK4/6抑制剂的细胞生长抑制特性截然不同。CDK7抑制可抑制ER蛋白S118位点的磷酸化，但长期CDK7抑制则会增强ER信号通路激活，这一结果支持将ET与CDK7i联合使用的治疗方案。最后，全基因组CRISPR/Cas9筛选鉴定出CDK7与MYC信号通路作为CDK4/6i耐药模型中的推定易感靶点，且CDK7抑制可有效抑制CDK4/6i耐药模型的生长。

研究结论：综上，本研究结果支持开展选择性CDK7抑制剂联合内分泌治疗的临床研究，以克服ER+ BC的治疗耐药性。此外，本研究还指出，c-Myc活性升高与p53功能完整可作为CDK7抑制剂治疗的敏感性预测标志物。

整体实验设计：1. 对ER野生型（ER-WT）MCF7细胞及其带有或不带有CDK7突变的ER突变衍生细胞系，以及两例PDX模型（野生型：1415、突变型：1526）的RNA测序数据进行比较基因表达谱分析。2. 对帕博西尼（Palbociclib）敏感型与耐药型T47D及MCF7细胞的RNA测序数据进行比较基因表达谱分析。