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。我们之前使用转录激活域突变小鼠所做的工作表明 P53急性DNA损伤反应背后的转录程序对肿瘤不是必需的 抑制，促使我们寻找P53介导的肿瘤抑制的新机制。因此，我们 对肿瘤重要的p53靶基因进行体内无偏shRNA和CRISPR/Cas9筛查 压制。我们确定了几个具有肿瘤抑制活性的p53靶基因，包括顶部的Zmat3 在两个屏幕上都很受欢迎。我们发现，Zmat3的表达在不同的环境中高度依赖于P53，并且Zmat3 抑制小鼠肺腺癌和肝细胞癌 模特们。Zmat3编码一种锌指RNA结合蛋白，我们发现该蛋白通过调节 剪接，揭示了P53介导的肿瘤抑制的一个新分支。由于最近的研究揭示了一个关键的 对于选择性剪接在癌症中的作用，我们假设在转录后研究p53通路 水平，如通过剪接和蛋白质组学分析，将产生对p53介导的肿瘤的新见解 压制。在主题1中，我们建议识别依赖于p53的剪接和蛋白质组变化，包括 Zmat3依赖和Zmat3非依赖的基因，可以解释小鼠LUAD的肿瘤抑制 还有肝癌。我们还将确定与Zmat3合作的基因，以抑制p53下游的癌症。我们 将测试在这些分析中发现的基因对LUAD和肝细胞癌抑制的重要性 体内肿瘤检测称为Tuba-Seq。在主题2中，我们将继续我们的观察，即P53重新用于 在肺再生中的作用，在其中它推动肺损伤时肺泡1型细胞的分化，以抑制 路德。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 表达KrasG12D的肺泡2型细胞的进化路径及P53转录程序的变化 在小鼠模型中，LUAD中的细胞状态发生了进化。我们还会问肿瘤中的细胞是如何 在野生型和p53缺陷型肿瘤中，微环境(TME)影响癌细胞的轨迹。为了定义基因 对癌细胞状态转换和癌细胞与TME之间的串扰具有重要功能 组件，我们将使用scPerturb-seq。LUAD进化的研究，其中我们表达了KrasG12D和 通过详细的动力学分析，分析癌细胞和TME水平上的后续事件，将 在体内以前所未有的分子深度解构P53介导的肿瘤抑制。总而言之，这些 研究将为如何在癌症治疗策略中调节P53通路提供至关重要的新见解。

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. Our previous work using transcriptional activation domain mutant mice suggested that the transcriptional programs underlying p53 acute DNA damage responses are not essential for tumor suppression, prompting us to search for new mechanisms of p53-mediated tumor suppression. We thus performed unbiased in vivo shRNA and CRISPR/Cas9 screens for p53 target genes important for tumor suppression. We identified several p53 target genes with tumor suppressor activity, including Zmat3 – the top hit in both screens. We found that Zmat3 expression is highly p53-dependent across contexts and that Zmat3 suppresses lung adenocarcinoma (LUAD) and hepatocellular carcinoma (HCC) in autochthonous mouse models. 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. As recent work has revealed a 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 also identify genes that cooperate with Zmat3 to suppress cancer downstream of p53. 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. Studies of LUAD evolution, in which we express KrasG12D and analyze subsequent events at both the cancer cell and TME levels through a detailed kinetic analysis, will deconstruct p53-mediated tumor suppression in vivo at an unprecedented molecular depth. Collectively, these studies will provide crucial new insight into how to modulate p53 pathways in therapeutic strategies for cancer.