PROJECT SUMMARY Lineage plasticity is a prominent feature of human pancreatic ductal adenocarcinoma (PDAC), which accompanies several stages of this disease ranging from tumor initiation, chemotherapy resistance, and the acquisition of metastatic traits. Our work in this area has relied heavily on the application of epigenomics and high throughput genetic screening in clinically relevant PDAC models. An important attribute of our work is to take deep-dives into causal molecular mechanisms, which naturally focuses on the lineage master regulator proteins that implement aberrant cell fate transitions. Our work seeks to challenge the view that transcription factors are undruggable by defining the molecular details of their cancer maintenance functions. Into the next funding cycle of this grant, our focus will continue to be on the basal (also known as adenosquamous) cell state, which is known to emerge aberrantly during PDAC progression. In our published work, we have defined mechanisms by which Np63 and ZBED2, two transcription factors that are expressed in the normal basal lineage but not in the normal human pancreas, drive basal identity in PDAC. The next funding cycle of this grant will pursue key unanswered questions related to these factors in PDAC biology. The first Aim will focus on MED12, a novel master regulator of basal identity in PDAC and transcriptional coactivator of Np63, which we identified by way of an unbiased genetic screen. We will perform CRISPR base-editor tiling screens coupled with molecular reporters to map MED12 mutations that disrupt Np63 function without altering other MED12 transcriptional functions. We will biochemically and epigenomically characterize these novel MED12 alleles to gain an atomic level understanding of its involvement in sustaining basal identity in PDAC. The second aim builds upon our prior work showing that a basal identity in PDAC reprograms the stroma via a unique secretory phenotype driven by Np63. We will establish novel in vivo basal-like PDAC models with inducible Np63 and MED12 knockdown to compare how each factor drives a secretory phenotype that reprograms the tumor stroma. The third aim focuses on ZBED2, which was unstudied prior to our recent publication, but we have shown that this factor can destabilize the classical-ductal identity in PDAC by antagonizing IRF1 function, which in turn leads to repression of the GATA6 gene. Since much of our prior work on ZBED2 occurred in tissue culture systems, the logical extension of this work will be to manipulate ZBED2, IRF1, and GATA6 in organoid models of PDAC in vivo, a system that fosters the lineage plasticity potential seen in human PDAC. We seek to test the hypothesis that acquisition of ZBED2 expression can ‘kickstart’ ductal-to-basal trans-differentiation by interfering with the IRF1-GATA6 axis. Collectively, this research will reveal several fundamental biological characteristics of basal identity in PDAC, which may have broader implic...