Mechanism of estrogen independent proliferation in ER+ breast cancer cells

摘要 细胞周期蛋白依赖性激酶(CDK)抑制剂联合内分泌治疗可改善转移性肿瘤的预后 雌激素受体阳性(ER+)，HER2阴性，乳腺癌患者。然而，这种组合的价值在于 在潜在可治愈的早期患者中，情况是不同的。我们的初步结果检验了进化 用连续肿瘤活检的单细胞转录图谱研究早期乳腺癌的生长轨迹 来自术前单独内分泌治疗(来曲唑)或结合细胞周期的临床试验 核糖核酸库。雌激素信号加速丢失的耐药肿瘤显示JNK上调 途径，而那些在治疗期间维持雌激素信号的人显示CDK4/6的激活增强 与ERBB4和ERK信号上调一致。细胞周期重建发现肿瘤细胞 在联合治疗期间可以重新激活，这表明对增殖状态的选择更强。我们 早期乳腺癌对CDK4/6抑制的抵抗是由JNK MAPK驱动的假说 通过促进CDK6的表达或通过促进细胞周期的重新激活来刺激细胞周期 细胞周期抑制功能减弱。在目标1中，我们将使用一种新的CDK4/6调节机制模型 通过细胞周期抑制物和促进剂(CIP)将雌激素和JNK信号与细胞周期进程相结合 检测一系列敏感和耐药等基因细胞系的细胞周期激活机制 对CDK4/6和内分泌抑制物以及患者肿瘤细胞的作用。这一分析将揭示出不同的信号 在雌激素、CDK4/6和JNK抑制治疗期间，这些通路有助于细胞周期的重新激活，并提供 不同细胞类型、不同时间和不同系统之间的每种抗性机制的特征。AIM 2利用我们的 从猫临床试验中收集患者肿瘤以发现细胞内和肿瘤内耐药 驱动扩散的机制。基本阻力机制的测量将超过300,000 使用已有的单细胞RNA测序数据从360个肿瘤样本中提取患者细胞以确定核心 细胞内信号状态单独或共同作用以驱动增殖。接下来，内含的细胞数量 将对每个肿瘤进行分析，以量化肿瘤内的异质性以及耐药人群在生长过程中的差异。 或在药物治疗期间肿瘤缩小。应用CIP计划跨患者肿瘤细胞的增殖将 允许预测最有效地阻止细胞内和肿瘤内增殖的抑制策略。最后， Aim 3将应用一系列具有临床潜力的JNK途径药物来设计和测试治疗策略 持久抑制ER+癌细胞的增殖。数学模型之间的迭代反馈 患者/实验数据有助于对细胞周期调节和机制的深入理解 失调会导致抵抗。总之，这些实验将揭示雌激素与雌激素之间的平衡 和替代介导的JNK信号及其在耐药性中的作用，并为治疗方案提供指导 更持久地控制癌细胞的增殖。

Abstract Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for metastatic estrogen receptor positive (ER+), HER2 negative, breast cancer patients. However, the value of this combination in potentially curable earlier stage patients is variable. Our preliminary results examined the evolutionary trajectories of early stage breast cancer tumors using single cell transcriptomic profiling of serial tumor biopsies from a clinical trial of preoperative endocrine therapy alone (letrozole) or in combination with the cell cycle inhibitor ribociclib. Resistant tumors with accelerated loss of estrogen signaling show up-regulation of the JNK pathway, while those that maintain estrogen signaling during therapy show potentiation of CDK4/6 activation consistent with ERBB4 and ERK signaling up-regulation. Cell cycle reconstruction identified that tumors cells can reactivate during combination treatment, indicating stronger selection for a proliferative state. We hypothesize that resistance to CDK4/6 inhibition in earlier stage breast cancer is driven by JNK MAPK pathway stimulation and reactivation of the cell cycle through promotion of CDK6 expression or decreased cell cycle inhibitor function. In Aim 1, we will use a new mechanistic model of CDK4/6 regulation by cell cycle Inhibitors and Promoters (CIP) that couples estrogen and JNK signaling with cell cycle progression to measure the mechanisms driving cell cycle activation in a series of isogenic cell lines sensitive and resistant to CDK4/6 and endocrine inhibitors and in patient tumor cells. This analysis will reveal how distinct signaling pathways contribute to cell cycle reactivation during estrogen, CDK4/6 and JNK inhibition treatments and provide signatures of each resistant mechanism across cell types, over time and between systems. Aim 2 leverages our collection of patient tumors from the FELINE clinical trial to discover the intracellular and intratumoral resistance mechanisms driving proliferation. Fundamental resistance mechanisms will be measured in over ~300,000 patient cells from 360 tumor samples using single cell RNA sequencing data already in hand to identify core intracellular signaling states that act alone or in concert to drive proliferation. Next, the population of cells within each tumor will be analyzed to quantify intratumoral heterogeneity and how resistant populations differ in growing or shrinking tumors during drug treatment. Applying CIP to project proliferation across patient tumor cells will allow prediction of inhibitor strategies that most effectively block intracellular and intratumoral proliferation. Lastly, Aim 3 will apply a series of JNK pathway drugs with clinical potential to design and test treatment strategies that maintain durable inhibition of proliferation in ER+ cancer cells. Iterative feedback between mathematical models and patient/experimental data serves to provide a deep understanding of cell cycle regulation and mechanisms of dysregulation leading to resistance. Together, these experiments will reveal the balance between estrogen and alternative mediated JNK signaling, and their roles in resistance and provide a guide for therapeutic regimes with more durable control of cancer cell proliferation.