Dynamics, Regulation and Function of p53 in Single Cells

项目摘要 信号系统的动力学对于控制基因表达程序和细胞结果至关重要。 肿瘤抑制蛋白p53是一种转录因子，协调对细胞应激的反应， 我们以前发现，DNA损伤后其动力学（蛋白质水平随时间的变化）取决于 并在决定细胞存活或死亡方面发挥作用。然而，许多问题 仍然是关于不同的细胞环境如何影响p53动力学和最终的细胞结果， 在相互冲突的细胞结果之间进行选择，以及如何最好地利用p53动力学进行治疗 目的 我们的工作目标是获得一个全面的定量了解如何p53动态调节 我们希望能够在单细胞中获得细胞结果，并将我们的发现应用于临床需求。细胞环境 可以影响p53的动态，因此，我们将首先研究如何p53动态调节的因素， 作为培养的肿瘤球体和体内肿瘤中的3D细胞结构。然后我们将调查 在培养和体内环境中癌症相关p53突变体的动力学和细胞结果。的影响 p53对基因表达的动力学模式将在单细胞中通过使用新的技术来确定， 支持整合p53动态的实时成像数据与单细胞RNA测序。我们还将 研究p53动力学如何影响RNA和蛋白质水平的基因表达，以及 大量群体中p53翻译后修饰的动力学。这些研究将揭示 p53动力学模式对靶基因的RNA和蛋白质的影响，以及这些基因的组合 动态模式指导细胞结果。我们还将使用实时成像系统来确定 可以优化临床相关的治疗方法以诱导所需的p53动力学和细胞凋亡 癌症的结果。我们将确定辐射剂量和时间如何影响p53的动态 和功能，并优化通过p53介导的诱导肿瘤细胞死亡的组分的时间表。 机制等许多癌症过度表达p53抑制剂Mdm 2或Mdmx，并且对它们的抑制剂敏感。 抑制作用通过定量和调节p53动力学，我们将确定如何微调其抑制 使Mdm2或Mdmx过表达细胞对DNA损伤敏感，同时保留健康细胞。肿瘤细胞可以 被免疫系统清除，这个过程受到肿瘤基因表达程序的影响。 因此，我们将研究p53动力学如何影响肿瘤细胞和免疫细胞之间的相互作用， 并致力于优化p53靶向治疗与免疫治疗的组合， 免疫系统杀死肿瘤细胞。总的来说，这些研究将提供新的机制见解， p53动力学和控制细胞命运的功能之间的联系，并将为新的组合 癌症治疗的治疗方法。

Project Summary The dynamics of signaling systems are critical for controlling gene expression programs and cellular outcomes. The tumor suppressor protein p53 is a transcription factor orchestrating the response to cellular stresses, and we previously found that its dynamics (changes in its protein levels over time) following DNA damage depend on the stimulus and play a role in determining whether a cell will survive or die. However, many questions remain about how different cellular contexts influence p53 dynamics and ultimate cellular outcomes, how p53 chooses between conflicting cellular outcomes, and how p53 dynamics can best be leveraged for therapeutic purposes. The goals of our work are to obtain a comprehensive quantitative understanding of how p53 dynamics regulate cellular outcomes in single cells and to apply our findings to address clinical needs. The cellular environment can influence p53 dynamics, therefore we will first investigate how p53 dynamics are regulated by factors such as 3D cellular architecture in cultured tumor spheroids and in in vivo tumors. We will then investigate the dynamics and cellular outcomes of cancer-associated p53 mutants in cultured and in vivo settings. The effects of p53 dynamical patterns on gene expression will be determined in single cells by using novel technology that supports integrating live imaging data of p53 dynamics with single-cell RNA sequencing. We will also investigate how p53 dynamics influence gene expression at the RNA and protein levels, as well as the dynamics of p53 post-translational modifications in bulk populations. These studies will reveal the impact that p53 dynamical patterns have on the RNA and protein of its target genes, and how the combinations of these dynamical patterns guide cellular outcomes. We will also use our live-imaging systems to determine how clinically-relevant therapeutic approaches can be optimized to induce the desired p53 dynamics and cellular outcomes in cancer. We will determine how the doses and timings of radiation fractions affect p53 dynamics and function, and optimize the schedule of fractions for inducing tumor cell death via p53-mediated mechanisms. Many cancers overexpress the p53 inhibitors Mdm2 or Mdmx and are susceptible to their inhibition. Through quantifying and modulating p53 dynamics we will determine how to fine-tune their inhibition to sensitize Mdm2 or Mdmx overexpressing cells to DNA damage while sparing healthy cells. Tumor cells can be cleared by the immune system, and this process is influenced by the tumors' gene expression programs. Therefore, we will investigate how p53 dynamics influence interactions between tumor cells and immune cells, and work towards optimizing combinations of p53-targeting therapeutics with immunotherapies to maximize tumor cell killing by the immune system. In total, these studies will provide new mechanistic insights into the links between p53 dynamics and function in controlling cell fates, and will inform novel combinatorial therapeutic approaches to cancer treatments.