Mechanism of estrogen independent proliferation in ER+ breast cancer cells

摘要 联合细胞周期蛋白依赖性激酶（CDK）抑制剂与内分泌治疗可改善转移性乳腺癌的预后 雌激素受体阳性（ER+）、HER2阴性的乳腺癌患者。然而，这种组合的价值 在有可能治愈的早期患者中，我们的初步结果检查了进化 使用系列肿瘤活检的单细胞转录组学分析的早期乳腺癌肿瘤的轨迹 来自术前内分泌治疗单独（来曲唑）或与细胞周期联合的临床试验 抑制剂ribociclib。具有加速雌激素信号丢失的耐药肿瘤显示JNK上调 而那些在治疗过程中维持雌激素信号传导的人显示出CDK4/6活化的增强作用。 与ERBB4和ERK信号上调一致。细胞周期重建证实肿瘤细胞 可以在联合治疗期间重新激活，表明对增殖状态的更强选择。我们 假设在早期乳腺癌中对CDK4/6抑制抗性是由JNKMAPK驱动的 通过促进CDK6表达的途径刺激和细胞周期的再活化，或 降低细胞周期抑制剂功能。在目标1中，我们将使用CDK4/6调节的新机制模型 通过细胞周期抑制剂和促进剂（CIP）将雌激素和JNK信号传导与细胞周期进程偶联 在一系列敏感和耐药的等基因细胞系中测量驱动细胞周期活化的机制， CDK4/6和内分泌抑制剂以及患者肿瘤细胞中的表达。这项分析将揭示不同的信号 在雌激素、CDK4/6和JNK抑制治疗期间，细胞周期通路有助于细胞周期再活化， 不同细胞类型、不同时间和不同系统之间的每种耐药机制的特征。目标2利用我们的 从FELINE临床试验中收集患者肿瘤，以发现细胞内和肿瘤内耐药性 推动扩散的机制。基本的阻力机制将在超过300，000 使用已掌握的单细胞RNA测序数据从360个肿瘤样本中提取患者细胞， 细胞内信号状态单独或协同作用以驱动增殖。接下来， 将分析每个肿瘤，以量化肿瘤内异质性以及耐药群体在生长中的差异。 或缩小肿瘤。应用CIP来预测患者肿瘤细胞的增殖将 允许预测最有效地阻断细胞内和肿瘤内增殖的抑制剂策略。最后， 目标3将应用一系列具有临床潜力的JNK通路药物来设计和测试治疗策略， 维持对ER+癌细胞增殖的持久抑制。数学模型间的迭代反馈 和患者/实验数据有助于深入了解细胞周期调节和机制 失调导致抗药性。总之，这些实验将揭示雌激素 和替代介导的JNK信号传导，以及它们在抗性中的作用，并为治疗方案提供指导 更持久地控制癌细胞增殖。

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.