HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer (2020)
Figure 3

Figure 3.

Human non-classical and KDM6A-deficient PDAC exhibit HNF1A-deficient phenotypes

A. Human orthologs of genes that were up- and down-regulated in Hnf1aaKO pancreas were also up- and down-regulated in human pancreas with low vs. high HNF1A expression (lowest vs. highest expression deciles, respectively). A random list of 717 genes controlled for similar expression levels was used for comparison. Violin plots include median and interquartile ranges. Dots are average values for each gene. Kruskall Wallis P<0.0001.

B. GSEA demonstrates that down- or upregulated genes in Hnf1aaKO mice (downward or upward arrows) showed down- or upregulation, respectively, in gene lists ranked by differential expression in non-classical vs. classical PDAC molecular subtypes from the TCGA-PAAD study (Cancer Genome Atlas Research Network. Electronic address & Cancer Genome Atlas Research, 2017). All enrichments had GSEA FDR q values < 0.01.

C. Analysis of HNF1A function in 121 high-purity cases of the ICGC-PACA-AU cohort identified tumors with most pronounced downregulation of direct HNF1A target genes. We performed GSEA with a gene set of 106 human orthologs of HNF1A direct targets showing downregulation in Hnf1aaKO pancreas. For each tumor sample, we performed differential expression against all other samples, and used GSEA to ascertain abnormal expression of the mouse HNF1A-dependent gene set in the tumor. Samples were ranked by the resulting normalized enrichment score (NES), and classified as either HNF1A LoF samples (purple, NES < 0; P < 0.05), or Control 1 (beige, NES < 0; P > 0.05) and Control 2 (grey, NES>0). HNF1A LoF samples were predominantly non-classical tumors (Collisson et al., 2011, Moffitt et al., 2015, Bailey et al., 2016). Putative loss of function KDM6A mutations (KDM6A LoF mutants) were found in 19% of HNF1A LoF tumors vs. 2% of all others (Fisher's P = 0.005). KDM6A mutations were considered functional if classified as ´high´ functional impact in ICGC (small <=200bp deletions/insertions, single base substitutions), or as likely loss of function structural variants in (Bailey et al., 2016), all of which were frame-shift mutations. Other KDM6A mutations were classified as unknown. Heatmaps show Z-score normalized expression of deregulated genes in Hnf1aaKO pancreas. We confirmed that 85% of 106 downregulated and 60% of genes of 146 upregulated human orthologs showed differential expression across the 3 HNF1A profiles (q<0.05, SAM multiclass analysis).

D. HNF1A mRNA levels differed in HNF1A LoF and control groups (Kruskal-Wallis, P<0.01), despite considerable variability and overlap between groups.

E. KDM6A mRNA levels were downregulated in HNF1A LoF tumors (Kruskal-Wallis, P<0.001).

Data information: Box plots in (D and E) show HNF1A and KDM6A expression in HNF1A LoF tumors (n=26) and Control 1 (n=39) and Control 2 (n=57) tumors. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x interquartile range outside box limits.