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...