Supplementary Fig. S1 A Most common genetic alterations in PDA (TCGA): Most common genetic alterations in PDA (UTSW): KRAS (91%) KRAS (93%) CDKN2A (46%) CDKN2A (41%) TP53 (70%) TP53 (54%) SMAD4 (38%) SMAD4 (43%) TGF-β core pathway alterations in PDA (TCGA): TGF-β core pathway alterations in PDA (UTSW): TGFBR1 (6%) TGFBR1 (5%) TGFBR2 (8%) TGFBR2 (7%) SMAD2 (5%) SMAD2 (20%) SMAD3 (1.3%) SMAD3 (6%) SMAD4 (38%) SMAD4 (43%) Genetic Alteration: Amplification Truncating Mutation Missense Mutation (likely passenger) Deep Deletion Missense Mutation (likely driver) B Organoid: NL5 HT22 HT24 HT42 HT25 HT30 LMCB3 HT33 HT28 HT11 HT47 CTLR3 TGFβ: - + - + - + - + - + - + - + - + - + - + - + - + SMAD4 pSMAD2/3 Loading Non-specific SMAD2/3 GAPDH C +/+ -/- E -/- Smad4 Smad4 ICGC & GTEx RNAseq PCA of all TFs in each case PDA
)%11( 2CP )%11( PNET ;Cdkn2a Normal G12D pSMAD2 IHC Kras
D AHR FOS JUND NKX2-2 STAT1 ATF4 FOSB KLF13 NKX6-3 STAT2 PC1 (36%) F BCL6 HIF1A KLF5 NPAS2 STAT3 Top5_TFs ● ● BHLHA15 HMGA1 KLF6 NR4A1 STAT6 ● ● ● Normal ● ● ● ● ● ● ● ● ● ● BHLHE40 HMGB1 MAFB NR5A2 TFDP1 ● ● ● ● ● PNET ● ● ● ● ● ● ● ●
BPTF HMGB2 MAZ PAX6 TSC22D12CP )%31( ● ● ● ● ● ● ● ICGC_PDA ● ● ● ● CREB3L1 HMGB3 MEF2A PEG3 TSC22D3 ● ● ● TCGA_PDA ● ● ● ● ● ● ● ● ● ● DMTF1 HSF4 MEIS1 PPARG TSHZ3 ● ●
● ● DRAP1 ID1 MEIS2 PRRX1 XBP1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● EGR1 ID2 MLXIP RBPJ ZBTB16 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● EHF ID3 MLXIPL RBPJL ZBTB38 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ELF1 ISL1 MYC RUNX1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ELF3 JUN NFE2L1 SREBF2 ● ● ● ● EPAS1 JUNB NFIC ST18 PC1 (49%)
G ID1 KLF5 AHR HMGB2 PPARG RBPJL p<0.001 p<0.001 p=0.01 p<0.001 p<0.001 p<0.001
● ● ● ● ● ● ● ● ● ● ● ● ● ●
(expression) ● ●
2 ● ● 1 3 5 2 4 6 8 2 4 6 8 6 8 10 12 log 6 8 10 2 4 6 8 10 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 NBCNBCNBC NBC NBC NBC H ID1 KLF5 AHR HMGB2 PPARG p<0.001 ns p<0.001 ns p<0.001 ns p<0.001 ns p<0.001 p=0.001
● ● ● ● ● ● ● ● ● ● ● (expression) ● ● ● 2 ● log 2 4 6 8 2 4 6 8 6 7 8 9 6 8 10 12 4 6 8 10 NPM NPMNPMNPMNPM 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.