Integrative approaches to elucidate p53 transcriptional networks during carcinogenesis

项目总结/摘要 TP 53肿瘤抑制基因在超过一半的人类癌症中发生突变，但其机制 p53在体内抑制癌症仍然不完全清楚。值得注意的是， 基于p53通路的癌症治疗。在这个建议中，我们努力解构通过 p53抑制癌症，以阐明p53丢失后失调的途径， 有针对性的治疗。我们之前对p53进行了无偏倚的体内shRNA和CRISPR/Cas9筛选， 对肿瘤抑制重要的靶基因，并将Zmat 3鉴定为两个筛选中的最高命中。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起作用，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 假设在转录后水平上研究p53通路，例如通过剪接， 蛋白质组学分析，将产生新的见解p53介导的肿瘤抑制。在主题1中，我们建议 鉴定p53依赖性剪接和蛋白质组变化，包括Zmat 3依赖性和Zmat 3依赖性 独立的，这可以解释小鼠LUAD和HCC中的肿瘤抑制。我们将测试 使用定量体内肿瘤测定，在这些LUAD和HCC抑制分析中发现的基因 叫做Tuba-seq在主题2中，我们将继续我们的观察，即p53在肺中的作用 再生，其中它驱动肺损伤后的肺泡1型细胞分化，以抑制LUAD。通过 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 表达KrasG 12 D的肺泡2型细胞以及p53转录程序如何随细胞状态变化 在小鼠模型中的LUAD进化。我们还将询问肿瘤微环境（TME）中的细胞如何 影响野生型和p53缺陷型肿瘤中的癌细胞轨迹。为了确定基因的功能， 癌细胞状态转换和癌细胞与TME组分之间的串扰，我们将采用 scPerturb-seq.在这个多元化的补充，坦博博士将执行一个新的项目有关的主题2的 R35通过使用蛋白质组学鉴定p53来扩展我们对p53在LUAD抑制中的作用的理解 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 具有改变的肿瘤抑制特性的结构域突变体，以鉴定与肿瘤抑制最相关的那些p53-相互作用物。 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。 小鼠移植系统，我们将确定这些p53相互作用的p53靶基因调控的作用 和抑制扩散。最终，我们将确定那些与LUAD最相关的p53相互作用蛋白质 在scPerturb-seq体内实验中的抑制。总之，这些研究将解构p53介导的 在前所未有的分子深度抑制体内肿瘤，这将提供关键的新见解如何 在癌症治疗策略中调节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.