Investigating the Role of Cell Plasticity in Malignant Transformation

项目总结 随着肿瘤的发展，癌细胞获得了使它们能够适应各种压力的特征。事实上, 预测患者预后的最好指标之一是诊断时的疾病阶段，就像晚期肿瘤一样。 更具攻击性，更难治疗。然而，增强细胞的潜在机制增加了细胞 癌症进展过程中的可塑性仍然知之甚少。癌细胞的适应能力具有 对靶向治疗的使用提出了一个特别的问题，而靶向治疗经常被 后天抵抗力的出现。这项工作的目标是阐明调控的分子机制 细胞周期和细胞命运决定影响癌症的进展和对靶向治疗的耐药性。在 F99期，我的目标是识别调控视网膜母细胞瘤(RB)途径的新因素，并影响 细胞对细胞周期蛋白依赖性激酶4和6抑制剂(CDK4/6)的反应。CDK4/6，与细胞周期蛋白的复合体 D，使肿瘤抑制因子Rb磷酸化并失活，以推动细胞周期进展。最近开发的 CDK4/6抑制剂在临床上显示出一些希望，但每个患者最终都会服用这些抑制剂。 取得了进展，迫切需要确定抵抗机制。使用全基因组的体外基因组 CRISPR/Cas9筛选，我最近发现E3连接酶适配器AMBRA1的丢失是一种潜在的机制 对CDK4/6抑制的抗性。此外，AMBRA1缺失增加了Cyclin D蛋白的稳定性。我假设 AMBRA1及其伴随的E3连接酶复合体针对Cyclin D的降解和AMBRA1的丢失 可能是体内对CDK4/6抑制剂产生耐药的机制之一。我会用分子和生化分析 为了确定与AMBRA1协同作用的E3连接酶，以靶向Cyclin D。此外，我将结合肿瘤 多重CRISPR/Cas9基因打靶条码在小鼠非小细胞肺模型中的应用 以确定AMBRA1的缺失是否会在体内导致CDK4/6抑制剂耐药。在K00阶段，我 目的探讨肺腺癌(LUAD)细胞特性的分子调控机制。治疗 靶向突变受体酪氨酸激酶的LUAD小分子抑制剂可导致肿瘤复发 已经转分化为小细胞肺癌，这是一种侵袭性的神经内分泌癌， 治疗方案。然而，转分化的机制在很大程度上是未知的。我建议发展细胞 为了确定调节LUAD细胞的因素，建立了这种转分化过程的系和小鼠模型 确定并最终确定防止或逆转转分化的手段。共同努力，这一机构将 阐明肺癌获得性耐药和疾病进展的基本原理，这可能 也适用于其他癌症类型。

PROJECT SUMMARY As tumors progress, cancer cells acquire characteristics that allow them to adapt to various stresses. In fact, one of the best predictors of patient outcome is disease stage at the time of diagnosis, as advanced tumors are more aggressive and difficult to treat. However, the underlying mechanisms that potentiate increased cell plasticity throughout cancer progression remain poorly understood. The ability of cancer cells to adapt has posed a particular problem for the use of targeted therapies, which are frequently rendered ineffective by the emergence of acquired resistance. The goal of this work is to elucidate molecular mechanisms that regulate the cell cycle and cell fate decisions to influence cancer progression and resistance to targeted therapy. In the F99 phase, I aim to identify novel factors that regulate the retinoblastoma (RB) pathway and influence the cellular response to inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6). CDK4/6, in complex with Cyclin D, phosphorylate and inactivate the tumor suppressor RB to drive cell cycle progression. Recently developed CDK4/6 inhibitors have shown some promise in the clinic, but every patient given these inhibitors eventually progresses, creating an urgent need to identify mechanisms of resistance. Using an in vitro genome-wide CRISPR/Cas9 screen, I recently identified loss of the E3 ligase adaptor AMBRA1 as a potential mechanism of resistance to CDK4/6 inhibition. Further, AMBRA1 loss increased Cyclin D protein stability. I hypothesize that AMBRA1, with its accompanying E3 ligase complex, targets Cyclin D for degradation, and that AMBRA1 loss could be a mechanism of resistance to CDK4/6 inhibitors in vivo. I will use molecular and biochemical assays to identify the E3 ligase that cooperates with AMBRA1 to target Cyclin D. In addition, I will combine tumor barcoding with multiplexed CRISPR/Cas9-mediated gene targeting in mouse models of non-small cell lung cancer to determine whether loss of AMBRA1 leads to CDK4/6 inhibitor resistance in vivo. In the K00 phase, I aim to elucidate the molecular mechanisms regulating cell identity in lung adenocarcinoma (LUAD). Treatment of LUAD with small molecule inhibitors targeting mutant receptor tyrosine kinases can lead to relapsed tumors that have transdifferentiated into small cell lung cancer, an aggressive neuroendocrine cancer with limited treatment options. However, the mechanism of transdifferentiation is largely unknown. I propose to develop cell line and mouse models of this transdifferentiation process in order to identify factors that regulate LUAD cell identity and ultimately identify means to prevent or reverse transdifferentiation. Together, this body of work will elucidate fundamental principles of acquired resistance and disease progression in lung cancer, which may also be applicable to other cancer types.