NPMNPMNPMNPM NPM 4 6 8 10 6 8 10 12 6 7 8 9 2 4 6 8 2 4 6 8 log ● 2 ● ● ● (expression) ● ● ● ● ● ● ● ● ● ● ● p<0.001 ns p<0.001 ns p<0.001 ns p<0.001 p<0.001 p=0.001 p<0.001 p<0.001 ns p<0.001 ID1 AHR KLF5 PPARG HMGB2 H NBC NBC NBCNBCNBC NBC p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 2 4 6 8 10 6 8 10 log 6 8 10 12 2 4 6 8 2 4 6 8 1 3 5 ● ● 2 ● ● (expression) ● ● ● ● ● ● ● ● ● ● ● ● ● ● p<0.001 p<0.001 p=0.01 p<0.001 p<0.001 p<0.001 PPARG HMGB2 AHR KLF5 ID1 RBPJL G PC1 (49%) PC1 JUNB ST18 NFIC EPAS1 JUN SREBF2 NFE2L1 ELF3 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ISL1 RUNX1 MYC ELF1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ZBTB38 ID3 RBPJL MLXIPL EHF ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ZBTB16 ID2 RBPJ MLXIP EGR1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● XBP1 ID1 PRRX1 MEIS2 DRAP1 ● ● TSHZ3 HSF4 PPARG MEIS1 DMTF1 ● ● ● ● ● TCGA_PDA ● TSC22D3 HMGB3 PEG3 MEF2A CREB3L1 ● ● ● ● ICGC_PDA ● ● TSC22D1 HMGB2 PAX6 MAZ BPTF ● )%31( 2CP ● )%31( ● ● ● PNET ● ● ● ● ● TFDP1 HMGB1 NR5A2 MAFB BHLHE40 ● ● ● ● ● Normal ● ● STAT6 HMGA1 NR4A1 KLF6 BHLHA15 ● Top5_TFs STAT3 HIF1A NPAS2 KLF5 BCL6 F STAT2 FOSB NKX6-3 KLF13 ATF4 PC1 (36%) PC1 STAT1 FOS NKX2-2 JUND AHR D Kras pSMAD2 IHC G12D Normal ;Cdkn2a PNET 2CP )%11( PDA PCA of all TFs in each case each in TFs all of PCA Smad4 Smad4 ICGC & GTEx RNAseq GTEx & ICGC -/- E C -/- +/+ GAPDH GAPDH SMAD2/3 Non-specific Loading pSMAD2/3 pSMAD2/3 SMAD4 SMAD4 - + - + - + - + - - + - + + - + - - + - + + - + - + - - + β: TGF T1H4 CTLR3 HT47 HT11 HT28 HT33 LMCB3 HT30 HT25 HT42 HT24 HT22 NL5 Organoid: B Deep Deletion Deep Missense Mutation (likely driver) (likely Mutation Missense Amplification Truncating Mutation Truncating Missense Mutation (likely passenger) (likely Mutation Missense Genetic Alteration: Genetic (43%) SMAD4 (38%) SMAD4 (6%) SMAD3 (1.3%) SMAD3 (20%) SMAD2 (5%) SMAD2 (7%) TGFBR2 (8%) TGFBR2 (5%) TGFBR1 (6%) TGFBR1 β core pathway alterations in PD in alterations pathway core β TGF- A (UTSW): A β core pathway alterations in PD in alterations pathway core β TGF- A (TCGA): A (38%) SMAD4 (43%) SMAD4 (70%) TP53 (54%) TP53 (46%) CDKN2A (41%) CDKN2A (91%) KRAS (93%) KRAS Most common genetic alterations in PDA (UTSW): PDA in alterations genetic common Most Most common genetic alterations in PDA (TCGA): PDA in alterations genetic common Most A Supplementary Fig. S1 Fig. Supplementary Supplementary Fig. S1: TGF-β signaling and transcriptional networks in PDA A) cBioportal oncoprints of common genetic alterations and TGF-β pathway alterations in PDA. Each column represents one case. B) Western immunoBlot analysis of pSMAD2 and SMAD4 in human PDA organoids treated with or without 100 pM TGF-β for 2h. C) pSMAD2 immunohistochemistry (IHC) of mouse KrasG12D;Cdkn2a-/- PDA in the pancreas (right) and KrasG12D; Cdkn2a-/-;Smad4-/- PDA metastasis in the lung (left). D) Genes represented within the top 5 expressed transcription factors of at least one sample in 225 samples of normal pancreas, PNET, and PDA. E) PCA of GTEx and ICGC RNA-seq datasets from using the complete list of transcription factors expressed in these samples. F) PCA of GTEx, ICGC, and TCGA RNA-seq datasets using the gene set in Supplementary Fig. S1D. G) GEO2R analysis of the expression of the top PDA-enriched PC1 genes (refer to Figure 1E) in the pancreatic microarray dataset GSE71729. N=Normal pancreas, B=Basal PDA, C=Classical PDA. p-values are from two-sided, unpaired t-tests of B versus N, and C versus N. H) GEO2R analysis of the expression of PDA-enriched PC1 genes in pancreatic microarray dataset GSE71729. N=Normal pancreas, P=Primary PDA tumor, M=Metastasis of PDA. P- values from two-sided, unpaired t-tests of P versus N, and M versus P. .