Fig 2: A Unique Enhancer Landscape Linked to ∆Np63 Occupancy in PDA(A) Heatmap representation of unsupervised hierarchical clustering of nine human cell lines or organoids representing PDA or normal pancreatic ducts based on H3K27ac occupancy at total H3K27ac ChIP-seq peaks. Scale bar indicates Pearson correlation coefficient. Normal organoids: hN34, hN35; PDA organoids: hF3, hT85; PATU: PATU8988S.(B) ChIP-seq density plots of TP63 and H3K27ac enrichments at squamous elements (top) or a set of 1,336 control H3K27ac regions (bottom) in the indicated cell lines. Each row represents a 10-kb interval centered on the midpoint of each H3K27ac peak.(C) ChIP-seq profiles of TP63 (top track) and H3K27ac at representative squamous elements close to KRT5 and KRT6A (left), TRIM29 (middle), and PTHLH (right).(D) GSEA plots evaluating the relative expression of genes proximal to squamous elements in squamous or progenitor patient samples from the study by Bailey et al. (2016). See also Table S5.(E) Pie chart showing the genomic distribution of squamous elements according to annotation of H3K27ac peaks by HOMER. TTS, transcription termination site; TSS, transcription start site.(F) Ontology analysis of genes located nearest to squamous elements versus control elements using GREAT. Gene Ontology (GO) terms related to biological process (left) and disease ontology (right) are shown.(G) Representation of motifs enriched at squamous elements versus control elements using TRAP. Human promoters were used as the comparison library. Left: fold change in p value. The TP53 position weight matrix is represented as TP53 family motif. Right: enrichment of the TP53 motif at squamous elements and control elements as determined by p value.See also Figure S2.

Fig 3: ∆Np63 Is Necessary and Sufficient to Drive Progenitor-to-Squamous Transcriptional Reprogramming in Human PDA Cells(A and B) Transcription factor expression in squamous and progenitor subtypes of PDA. Scatterplots show expressed transcription factors ranked by their mean log2 fold change in squamous versus progenitor patient samples from (A) Bailey et al. (2016) and (B) The Cancer Genome Atlas Research Network (2017).(C) TP63 expression in human organoids derived from normal pancreatic tissue or PDA tumor samples at the indicated disease stage. Data are from the study by Tiriac et al. (2018). *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired Student’s t test. See also Table S1.(D) Survival curve of patients from the indicated study stratified according to high or low TP63 expression and for which survival data were available (Moffitt et al., 2015). Samples were designated as TP63high or TP63low based on Z score expression values >0.35 or <0, respectively. p value was calculated using the log rank (Mantel-Cox) test.(E) Squamous-PDA and progenitor-PDA identity signatures defined from patient samples. The top 500 protein coding genes in squamous (n = 25) or progenitor (n = 30) patient samples from the study by Bailey et al. (2016) were used to define the respective signatures. Heatmap shows expression levels of signature genes. Scale bar indicates the standardized expression value. See also Table S2.(F) TP63 expression in human PDA cell lines. Heatmap (top) shows RNA-seq analysis of human PDA cell lines ranked according to fold change in the median expression level of genes corresponding to the squamous-PDA versus progenitor-PDA identity signatures; scale bar indicates log2 fold change. Bar chart (middle) and western blot analysis (bottom) show TP63 expression in the indicated human PDA cell lines.(G and H) TP63 knock out in BxPC3 cells. (G) GSEA plots evaluating the squamous-PDA and progenitor-PDA identity signatures upon TP63 knockout.(H) Representative western blot analysis for the indicated proteins. RNA was extracted and whole-cell lysates were prepared for the respective analyses on day 5 post-infection with sgRNAs, 3 days post-selection with G418.(I and J) ∆Np63 expression in SUIT2 cells. (I) GSEA plots evaluating the squamous-PDA and progenitor-PDA identity signatures upon ∆Np63 expression.(J) Representative western blot analysis in SUIT2 cells for the indicated protiens. RNA was extracted and whole-cell lysates were prepared for the respective analyses on day 7 post-infection, 5 days following G418 selection.(K and L) Acute ∆Np63 expression in progenitor-like PDA cells. (K) Representative western blot analysis in the indicated cell lines for the indicated proteins.(L) Table summarizing RNA-seq data evaluating the squamous-PDA identity signature upon induction of ∆Np63 expression in the indicated cell lines. RNA was extracted and whole-cell lysates were prepared for the respective analyses 48 hr following dox administration.See also Figure S1.

Fig 4: CRISPR generated loss of Foxp1 and Pten in the murine prostate. (A) AAV constructs for expression of sgRNAs to Pten or Pten and Foxp1 together with expression of Cre protein were generated. (B) Validation of the AAV particles were conducted in MEF isolated from Cas9-EGFPflox/flox mice. The expression of EGFP revealed viral transduction and Cre expression from the viral construct. Images were taken with 10× magnification (C) The efficiency of the sgRNA was determined with Sanger Sequencing and analysed with Synthego ICE software. (D) The AAV particles were delivered to the murine prostate with surgical injections and prostate tissues were examined 4 months post-treatment. White arrowheads mark transformed areas in the prostatic tissues (representative image, n = 5). (E) Paraffin sections from the prostates were stained for H & E (arrows mark PIN and arrowheads mark normal prostatic epithelial), p-Akt, Foxp1 and p63 (brown stain) to confirm PIN formation and disruption to the Pten pathway and loss of Foxp1 in the Cas9-EGFP transgene mice (n = 5). Arrows mark cells depleted for Foxp1 and arrowheads mark cells positive for Foxp1 or p63. (F) Indel frequency was measured on EGFP-positive biopics from the anterior prostate lobe. Sanger Sequencing data were analysed with Synthego ICE software (n = 4–5).

Fig 5: Ectopic expression of p63 in PDA cells promotes inflammation-associated transcriptional changes in the tumor microenvironment.(A–I) SUIT2-empty and SUIT2-p63 PDA cells harboring a luciferase transgene were transplanted into the pancreas of NSG mice. (A–B) Quantification of tumor volume (A) and images of tumors (B) on day 25 post-transplantation. (C) Experimental workflow for RNA-seq analysis of bulk tumor tissue. (D–E) Heatmap representations of differentially expressed genes that unambiguously map to (D) the human genome and therefore are derived from human cancer cells or (E) the mouse genome and therefore are derived from mouse stromal cells. Selected genes in each group are listed. Scale bar indicates standardized expression value. (F) Ontology analysis of the 500 significantly up-regulated mouse genes using Metascape. GO terms are ranked by their significance (p value) and the most significant terms (-log10 p value > 15) are highlighted. (G) GSEA plot evaluating the iCAF signature in the mouse stromal compartment of SUIT2-p63 versus SUIT2-empty tumors. (H) Quantification of flow cytometry analysis of CD45+CD11b+Ly6G+ neutrophils and CD45+CD11b+F4/80+ macrophages from bulk tumor tissues from SUIT2 xenografts. n = 5 mice per group. ***p<0.001 by Student’s t-test. (I) Representative flow cytometry plots from (H). (J–K) KPC mM1 organoids with a p63 cDNA or empty vector control and transplanted to the pancreas of C57BL6 mice. (J) Quantification of flow cytometry analysis of CD45+ cells in the tumors. n = 4–5 mice per group. ***p<0.001 by Student’s t-test. (K) Representative flow cytometry plots from (J). TCRß refers to T cell receptor ß.