Integrative approaches to elucidate p53 transcriptional networks during carcinogenesis

项目总结/摘要 TP53肿瘤抑制基因在超过一半的人类癌症中发生突变，但其机制通过 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。我们先前使用转录激活结构域突变小鼠的工作表明， 作为p53急性DNA损伤反应基础的转录程序对于肿瘤不是必需的， 抑制，促使我们寻找p53介导的肿瘤抑制的新机制。我们因此 进行了无偏倚的体内shRNA和CRISPR/Cas9筛选，以筛选对肿瘤重要的p53靶基因 镇压我们鉴定了几个具有肿瘤抑制活性的p53靶基因，包括Zmat3-最高的p53基因。 在两个屏幕上点击。我们发现Zmat3的表达在不同的环境中是高度依赖于p53的， 抑制肺腺癌（LUAD）和肝细胞癌（HCC）在本地小鼠 模型Zmat3编码一种锌指RNA结合蛋白，我们发现它通过调节替代性的 剪接，揭示了p53介导的肿瘤抑制的一个新的分支。最近的研究表明， 选择性剪接在癌症中的作用，我们假设在转录后水平研究p53通路， 水平，如通过剪接和蛋白质组学分析，将产生新的见解p53介导的肿瘤 镇压在主题1中，我们提出鉴定p53依赖的剪接和蛋白质组变化，包括 Zmat3依赖性和Zmat3非依赖性，这可以解释小鼠LUAD中的肿瘤抑制 和HCC。我们还将识别与Zmat 3合作抑制p53下游癌症的基因。我们 将使用定量方法测试这些分析中发现的基因对LUAD和HCC抑制的重要性 称为Tuba-seq的体内肿瘤测定。在主题2中，我们将继续观察p53重新利用了 在肺再生中的作用，其中它在肺损伤时驱动肺泡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状态如何决定细胞的增殖。 表达KrasG12D的肺泡2型细胞的进化路径以及p53转录程序如何改变 与小鼠模型中LUAD进化中的细胞状态。我们也会问肿瘤中的细胞 微环境（TME）影响野生型和p53缺陷型肿瘤中的癌细胞轨迹。来定义基因 对于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KrasG12D和 通过详细的动力学分析，在癌细胞和TME水平上分析随后的事件， 以前所未有的分子深度解构p53介导的体内肿瘤抑制。总的来说，这些 研究将为如何在癌症治疗策略中调节p53通路提供重要的新见解。

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.