Integrative approaches to elucidate p53 transcriptional networks during carcinogenesis

项目摘要/摘要 TP53抑癌基因在超过一半的人类癌症中发生突变，但其机制是通过 P53在体内抑制癌症的作用尚不完全清楚。值得注意的是，没有标准的护理 基于P53途径的癌症治疗。在这项提案中，我们努力解构通过 哪种P53抑制癌症以阐明最终可能是 有针对性的治疗。我们之前在活体中进行了无偏向的shRNA和CRISPR/Cas9筛查P53 目标基因对肿瘤抑制很重要，并确定Zmat3在两个屏幕上都是最热门的。Zmat3 编码一种锌指RNA结合蛋白，我们发现它通过调节选择性剪接发挥作用，揭示了一种 P53介导的肿瘤抑制的新分支。鉴于选择性剪接在癌症中的关键作用，我们 假设在转录后水平研究P53通路，例如通过剪接和 蛋白质组学分析，将对P53介导的肿瘤抑制产生新的见解。在主题1中，我们建议 识别P53依赖的剪接和蛋白质组变化，包括Zmat3依赖和Zmat3- 独立基因，这可以解释小鼠LUAD和肝癌的肿瘤抑制。我们将测试它的重要性 使用体内肿瘤定量分析在这些分析中发现的LUAD和肝癌抑制基因的数量 被称为Tuba-seq。在主题2中，我们将继续我们的观察，即P53在肺中重新定位作用 再生，在此过程中，它推动肺损伤时肺泡1型细胞分化，以抑制LUAD。穿过 单细胞(Sc)rna-seq和scatac-seq分析，我们将询问p53状态如何决定进化路径。 KrasG12D表达的肺泡2型细胞及P53转录程序如何随细胞状态变化 在小鼠模型中LUAD的进化。我们还将询问肿瘤微环境(TME)中的细胞 影响野生型和p53缺陷型肿瘤的癌细胞轨迹。定义对基因功能具有重要意义的基因 癌细胞状态转换和癌细胞与TME组件之间的串扰，我们将使用 ScPerturb-序号在这份多元化补充资料中，Tambo博士将执行一个与主题2相关的新项目 R35通过蛋白质组学鉴定P53，以扩大我们对P53在LUAD抑制中作用的理解 互动伙伴。我们将进行串联亲和标签纯化野生型P53和P53反式激活 具有肿瘤抑制特性改变的结构域突变，以确定与P53最相关的相互作用因子 肿瘤抑制。然后，我们将测试这些P53相互作用蛋白在肿瘤抑制中的作用 我们将确定这些p53相互作用因子在p53靶基因调控中的作用 和抑制增殖。最终，我们将确定与LUAD最相关的P53相互作用蛋白 体内scPerturb-seq实验中的抑制作用。总而言之，这些研究将解构p53介导的 在体内以前所未有的分子深度抑制肿瘤，这将为我们提供至关重要的新见解 在癌症的治疗策略中调节P53通路。

PROJECT SUMMARY/ABSTRACT The TP53 tumor suppressor gene is mutated in over half of all human cancers, but the mechanisms through which p53 suppresses cancer in vivo remain incompletely understood. Notably, there are no standard-of-care cancer therapies based on the p53 pathway. In this proposal, we strive to deconstruct the pathways through which p53 suppresses cancer to illuminate pathways dysregulated upon p53 loss that could ultimately be targeted therapeutically. We previously performed unbiased in vivo shRNA and CRISPR/Cas9 screens for p53 target genes important for tumor suppression and identified Zmat3 as the top hit in both screens. Zmat3 encodes a zinc finger RNA-binding protein that we found acts by modulating alternative splicing, revealing a new branch of p53-mediated tumor suppression. Given the critical role for alternative splicing in cancer, we hypothesize that studying p53 pathways at the post-transcriptional level, such as through splicing and proteomics analyses, will yield novel insights into p53-mediated tumor suppression. In Theme 1, we propose to identify p53-dependent splicing and proteome changes, including both Zmat3-dependent and Zmat3- independent ones, that could explain tumor suppression in mouse LUAD and HCC. We will test the importance of genes found in these analyses for LUAD and HCC suppression using a quantitative in vivo tumor assay known as Tuba-seq. In Theme 2, we will pursue our observation that p53 repurposes a role in lung regeneration, in which it drives alveolar type 1 cell differentiation upon lung injury, to suppress LUAD. Through single cell (sc)RNA-seq and scATAC-seq analyses, we will ask how p53 status dictates the evolutionary path of KrasG12D-expressing alveolar type 2 cells and how p53 transcriptional programs change with cell state across LUAD evolution in mouse models. We will also ask how cells in the tumor microenvironment (TME) affect cancer cell trajectories in wild-type and p53-deficient tumors. To define genes functionally important for cancer cell state transitions and crosstalk between cancer cells and TME components, we will employ scPerturb-seq. In this diversity supplement, Dr. Tambo will perform a new project related to Theme 2 of the R35 to expand our understanding of p53 action in LUAD suppression by using proteomics to identify p53 interacting partners. We will perform tandem affinity tag purification of wild-type p53 and p53 transactivation domain mutants with altered tumor suppression properties to identify those p53-interactors most relevant for tumor suppression. We will then test the roles of these p53-interacting proteins in tumor suppression in a mouse transplant system and we will define the roles of these p53-interactors in p53 target gene regulation and proliferation suppression. Ultimately, we will identify those p53-interacting proteins most relevant for LUAD suppression in the scPerturb-seq experiments in vivo. Collectively, these studies will deconstruct p53-mediated tumor suppression in vivo at an unprecedented molecular depth, which will provide crucial new insight into how to modulate p53 pathways in therapeutic strategies for cancer.