Co-targeting CDK4/6 and BRD4 promotes senescence and ferroptosis sensitivity in cancer

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are approved for breast cancer treatment and show activity against other malignancies, including KRAS-mutant non-small cell lung cancer (NSCLC). However, the clinical efficacy of CDK4/6 inhibitors is limited due to frequent drug resistance and their largely cytostatic effects. Through a genome-wide cDNA screen, we identified that bromodomain-containing protein 4 (BRD4) overexpression conferred resistance to the CDK4/6 inhibitor palbociclib in KRAS-mutant NSCLC cells. Inhibition of BRD4, either by RNA interference or small-molecule inhibitors, synergized with palbociclib to induce senescence in NSCLC cells and tumors, and the combination prolonged survival in a KRAS-mutant NSCLC mouse model. Mechanistically, BRD4-inhibition enhanced cell cycle arrest and reactive oxygen species (ROS) accumulation, both of which are necessary for senescence induction; this in turn elevated GPX4, a peroxidase that suppresses ROS-triggered ferroptosis. Consequently, GPX4 inhibitor treatment selectively induced ferroptotic cell death in the senescent cancer cells, resulting in tumor regression. Co-targeting CDK4/6 and BRD4 also promoted senescence and ferroptosis vulnerability in pancreatic and breast cancer cells. Together, these findings reveal therapeutic vulnerabilities and effective combinations to enhance the clinical utility of CDK4/6 inhibitors. Overall design: To invertigate the gene expression program that can be affected by inhibition of CDK4/6 or BRD4, we treated KRAS mutant cell line H358 with CDK4/6 inhibitior (palbociclib) or BRD4 inhibitor (IBET726), or both for 9 days. We then performed gene expression profiling analysis using the RNA-Seq data generated in these cells.

细胞周期蛋白依赖性激酶4/6（CDK4/6）抑制剂已获批用于乳腺癌治疗，且对包括KRAS突变型非小细胞肺癌（NSCLC）在内的多种恶性肿瘤均展现出抗肿瘤活性。然而，由于频繁出现的耐药性以及其主要为细胞生长抑制的作用效果，CDK4/6抑制剂的临床疗效受到限制。本研究通过全基因组cDNA筛选，发现在KRAS突变型NSCLC细胞中，含溴结构域蛋白4（BRD4）过表达会赋予细胞对CDK4/6抑制剂帕博西尼的耐药性。通过RNA干扰或小分子抑制剂抑制BRD4，可与帕博西尼协同诱导NSCLC细胞及肿瘤发生细胞衰老，且该联合治疗方案可延长KRAS突变型NSCLC小鼠模型的生存期。从机制上来说，BRD4抑制可增强细胞周期阻滞与活性氧（ROS）积累，这二者均是诱导细胞衰老所必需的过程；这一过程继而会上调GPX4——一种可抑制活性氧诱导铁死亡的过氧化物酶。因此，GPX4抑制剂治疗可选择性诱导衰老癌细胞发生铁死亡性细胞死亡，最终实现肿瘤消退。同时靶向CDK4/6与BRD4还可在胰腺癌细胞和乳腺癌细胞中增强细胞衰老与铁死亡易感性。综上，本研究揭示了可提升CDK4/6抑制剂临床应用价值的治疗靶点与有效联合治疗方案。实验整体设计：为探究CDK4/6或BRD4抑制所影响的基因表达程序，我们将KRAS突变细胞系H358分别用CDK4/6抑制剂帕博西尼、BRD4抑制剂IBET726，或二者联合处理9天。随后我们利用这些细胞生成的RNA测序（RNA-Seq）数据开展基因表达谱分析。