Integrative approaches to elucidate p53 transcriptional networks during carcinogenesis

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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.