Abstract
Kras-activating mutations display the highest incidence in pancreatic ductal adenocarcinoma. Pancreatic inflammation accelerates mutant Kras-driven tumorigenesis in mice, suggesting high selectivity in the cells that oncogenic Kras transforms, although the mechanisms dictating this specificity are poorly understood. Here we show that pancreatic inflammation is coupled to the emergence of a transient progenitor cell population that is readily transformed in the presence of mutant KrasG12D. These progenitors harbor a proto-oncogenic transcriptional program driven by a transient enhancer network. KrasG12D mutations lock this enhancer network in place, providing a sustained Kras-dependent oncogenic program that drives tumors throughout progression. Enhancer co-option occurs through functional interactions between the Kras-activated transcription factors Junb and Fosl1 and pancreatic lineage transcription factors, potentially accounting for inter-tissue specificity of oncogene transformation. The pancreatic ductal adenocarcinoma cell of origin thus provides an oncogenic transcriptional program that fuels tumor progression beyond initiation, accounting for the intra-tissue selectivity of Kras transformation.
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Data availability
The sequencing data generated by experiments, including bulk cells ATAC-seq, bulk cells RNA-seq, bulk cells ChIP-seq and single cells ATAC-seq are deposited under series GSE134236 in the GEO data repository. The super-series of GSE134236 is composed of the following subseries: GSE134230 (bulk ATAC-seq), GSE134235 (bulk RNA-seq), GSE134233 (bulk ChIP-seq) and GSE147726 (single-cell ATAC-seq). Flow cytometry sequential gating information, raw immunoblot and DNA gel images, as well as data used for all statistical analyses, are provided as source data. Source data are provided with this paper.
Code availability
The following open-source code and software was used in this study: trim-galore (v.0.6.0), HISAT2 (v.2.1.0), SAMtools (v.1.9), featureCounts (v.1.6.3), R (v.3.5.1), bowtie2 (v.2.3.4.3), Cell Ranger ATAC software (v.1.1.0), MACS2 (v.2.1.2), bamCoverage (v.3.2.0), MAnorm (v.1.1.4), Bedtools (v.2.28.0), STAR (version 2.7.0e), HOMER software (v.4.9.1) and GSEA (v.4.0.4). Default settings were used for all GSEA analyses. R package Seurat (v.3.0.2), R package Signac (v.0.2.4), R package DESeq2 (v.1.22.1), R package ChIPseeker (v.1.18.0), R package pheatmap (v.1.0.10) and R package VennDiagram (v.1.6.20) were also used.
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Acknowledgements
We thank R. Xu, S. Zhang and C. Tian for help with bioinformatic analysis. TO-KrasG12D cells were a gift from J. Massagué (Memorial Sloan Kettering Cancer Center). KC mice were a gift from F. Qian (Tsinghua University). We thank A. Bialkowska (SUNY Stony Brook) for technical advice. We thank R. Macdonald and S. Vanharanta for critical reading of the manuscript. C.J.D. was supported by start-up support from Tsinghua University and the Peking University-Tsinghua Center for Life Sciences along with grants from the the Beijing Natural Science Foundation (Z190022) and the Natural Science Foundation of China (no. 81871957). Wenming Wu was supported by the Beijing Natural Science Foundation (Z190022) and the National Science Foundation of China (no. 81773292). Yupei Zhao was supported by Chinese Academy of Medical Sciences (CAMS) Initiative for Innovative Medicine (CAMS-12M; 2019-12M-1-001, 2017-12M-1-001). Junya Peng was supported by the National Science Foundation of China (no. 31701171).
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Contributions
C.J.D. conceived and supervised the project. C.J.D. and Y.L. designed the experiments. Y.L. performed most experiments with help from Z.S. and D.Z. Y.H. performed most bioinformatic analysis. Xinde Liu performed ATAC-seq in PDA cell lines. J.X. provided the Ptf1a-CreERT2 mice. Xinhong Liu created dCas9-KRAB cell line. Z.S. performed lineage tracing and associated scRNA-seq. J.P., D.H. and M.X. performed single-cell RNA-seq in human tumors. X.L., Z.L. and Y.H. performed scATAC-seq analysis. Y.Z. and Wenming Wu supervised single cell RNA-seq experiments in human tumors. J.M., B.J. and W.X. performed initial ATAC-seq experiments and provided technical assistance. Wenze Wang performed pathological analysis of human and mouse tumor samples. J.W. provided technical guidance in small-scale RNA-seq experiments. H.D. performed proteomic analysis. M.Z. developed the AAV8 pancreatic infection system. M.C. performed some ChIP-seq experiments and supervised aspects of the project. C.J.D. wrote the paper with input from the other authors.
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Extended data
Extended Data Fig. 1 A Klf5 knock in mouse allows isolation of pancreatic ductal cells.
a, Expression of reported ductal TFs in TCGA PDA RNA-seq data. b, Ductal TF expression in PDA tumors (n=179) compared to normal pancreas (n=171) from GEPIA58. c, Immunofluorescence for mCherry, Klf5, and Krt19 in the healthy pancreas, or in the inflamed pancreas of a CAE-treated mouse. For each condition, two fields of view (FOV) are shown, representative of three mice for each condition. Enlarged images of an mCherrypos normal duct in the healthy pancreas and an mCherrypos ADM lesion in the inflamed pancreas are included as insets. Scale bars, 50um. d, IHC for Klf5 performed in the same conditions as (c) in the healthy pancreas (n=3), or in the inflamed pancreas (n=2), scale bars, 50um.
Extended Data Fig. 2 mCherry FACS isolates normal ductal and ADM cells.
a, Flow cytometry plot showing mCherry-positive cells in an untreated mouse. mCherrypos and mCherryneg cells were collected and subjected to quantitative real-time PCR to determine the enrichment of ductal (Klf5 and Krt19) and acinar (Mist1) markers. Results representative of three independent experiments. b, ATAC-seq at the Ctrb1 (acinar) and Krt19 (ductal) loci in sorted mCherrypos and mCherryneg cells. c, Control flow cytometry plots to illustrate gating strategy for FACS experiments. d, Scheme for lineage tracing experiment for double-labeling of acinar and ductal cells prior to induction of inflammation (left panel). These mice were first treated with TAM, to label acinar and ductal cells, then treated with CAE two weeks later to induce inflammation. Pancreases were subject to flow cytometry on CAE D2 (right panel). e, As above, but omitting the Ptf1a-CreERT2 allele.
Extended Data Fig. 3 ADM cells are transient acinar progenitors.
a, IF for YFP and Amylase in a Klf5-KI; R26RLSL-YFP mouse treated with CAE and TAM (D55 post-CAE, n=1). Scale bars, 20um. b, IHC showing YFP staining in a normal pancreatic duct after a Klf5-KI; R26RLSL-YFP mouse was treated with TAM only, and YFP-positive acinar cells after dual TAM/CAE treatment (n=2, representative images shown). Scale bars, 100um. c, Examination of progenitor activity in the pancreas when TAM treatment coincides with CAE treatment, or when TAM administration is delayed until 7 days after CAE treatment. On D20 post-CAE mice were sacrificed (n=3) and pancreases were dissociated into single cells and subjected to flow cytometry. The ability to yield YFP-only cells was used as a proxy for the differentiation potential (progenitor activity) of the cells. Ratio of YFP-only to double-positive quantified at right. All error bars are defined as mean +/- SD,. d, H&E stain of a PDA tumor that emerged in TAM/CAE-treated Klf5-KI; LSL-KrasG12D; Cdkn2afl/fl mouse (n=3 mice, representative images shown). Scale bars, 100um.
Extended Data Fig. 4 PDLP-linked gene expression changes are maintained in human tumors.
a, H&E staining of samples derived from mice treated with TAM to induce KrasG12D used for RNA-seq and ATAC-seq experiments described in Figs. 2 and 3. Representative images from each of two replicate mice shown. Scale bars, 200um. b, H&E staining of Klf5-KI (KrasWT) and Klf5-KI; Pdx1-Cre; LSL-KrasG12D pancreases used for RNA-seq and ATAC-seq experiments in Figs. 2 and 3. One D0 Krasmut mouse (replicate 2) contained a small PanIN lesion, marked with an asterisk. Scale bars, 200um. c, GSEA analysis comparing enrichment of the PDA KrasG12D-dependent geneset (ref. 16) in mCherrypos cells compared to mCherryneg pancreatic cells in the inflamed pancreas of mice 2 days after CAE treatment. d, GSEA analysis comparing enrichment of the PDA KrasG12D-dependent geneset in mCherrypos cells in mice treated with combined CAE and LPS compared with mCherrypos cells in NT mice (normal ductal). e, Flow cytometry plot showing YFP expression in the mCherrypos cell population in a Klf5-KI; LSL-KrasG12D; R26RLSL-YFP mouse on D2 post-CAE treatment. f, GSEA analysis showing downregulation of cilium morphogenesis genes in PDLPs compared with normal ductal cells. g, mRNA expression levels of ductal terminal differentiation TFs throughout inflammation. h, Overlap of K-LOCKEDUP genes with genes upregulated in tumor ductal type 2 cells compared with normal ductal cells. P-value calculated by hypergeometric distribution. i, Genesets were created using genes upregulated or downregulated in malignant ductal type 2 cells compared with intratumoral normal ductal cells (ductal 1). We used GSEA to examine the enrichment of these genesets in the transcriptomes of mCherrypos cells from mice treated as indicated compared to terminally differentiated ductal cells (mCherrypos cells of NT mice). n=3 biologically independent animals for D2 WT, D7 WT and D7 KrasG12D groups, n=2 biologically independent animals for NT group. P-values obtained by permutation test, ***, FDR<.001, **, FDR <.01, *, FDR <.05, ns, FDR >=.05.
Extended Data Fig. 5 KrasG12D locks in PDLP-associated chromatin changes.
a, Additional ATAC-seq tracks of key KrasG12D-regulated genes. b, ATAC-seq signal intensity at the PDLP-derived K-LOCKEDOPEN sites in the indicated samples. P-values obtained by a Kolmogorov–Smirnov test, two sided. ****, p<1x10-5. c, ATAC-seq tracks showing PDLP-linked chromatin closing at K-LOCKEDDOWN genes. d, ATAC-seq signal intensity at the PDLP-derived K-LOCKEDCLOSED sites in the indicated samples. P-values obtained by a Kolmogorov–Smirnov test, two sided. ****, p<1x10-5, ***, p<.001, **, p<.01. e, Peak heatmap of K-LOCKEDCLOSED regions throughout the course of inflammation.
Extended Data Fig. 6 Human ADM is readily identified using scATAC-seq.
a, H&E sections of patient samples used for scATAC-seq. Scale bars, 100um. b, Aggregate scATAC-seq tracks depicting chromatin opening at the ductal marker gene KRT19 and the acinar marker PTF1A. Subpopulations correspond to those indicated in Fig. 3f, lower panel. c, Aggregate scATAC-seq tracks depicting aggregate chromatin opening at K-LOCKEDCLOSED genes in the cancer and normal ductal subpopulations. d, Tracks depicting scATAC-seq chromatin opening at the K-LOCKEDCLOSED gene FOXJ1 in the human ADM subpopulation compared with cancer and normal ductal cells.
Extended Data Fig. 7 MAPK dependence and conservation of Kras-regulated sites.
a, Growth of two separate TO-KrasG12D lines cultured in the presence or absence of doxycycline. b, H3K27ac ChIP-seq intensity after the indicated treatments at KrasG12D-dependent enhancers identified as described in Fig. 4. Treatments were performed with an AKT inhibitor (MK2206, 2.5 µM) or separate treatment with two different MEK inhibitors (Refametinib and PD0325901, each 500 nM) for 12 hours prior to ChIP-seq. P-values derived from a Kolmogorov–Smirnov test, two sided.****, p<1x10-5. c, ATAC-seq intensity in mCherrypos cells isolated from the indicated mice at sites of MAPK-dependent H3K27ac. P-values derived from a Kolmogorov–Smirnov test, two sided.****, p<1x10-5. d, Top five enriched TF motifs at open chromatin sites enriched in human ADM cells compared to normal ductal cells (hADMOPEN) identified by scATAC-seq. e-h, Top five enriched TF motifs at the following regions: (e) sites of open chromatin enriched in terminally differentiated normal ductal cells compared to PDLPs identified by ATAC-seq in mCherrypos cells in Klf5-KI mice (K-LOCKEDCLOSED sites), (f) sites of open chromatin enriched in normal ductal cells compared with PDA ductal cells in human samples identified by scATAC-seq (hPDACLOSED sites), (g) sites of increased H3K27ac upon KrasG12D depletion identified in TO-KrasG12D mPDA cells (KrasG12D-repessed sites), and (h) sites of open chromatin enriched in normal ductal cells compared with ADM cells in human samples identified by scATAC-seq (hADMCLOSED sites). i, Normalized mean ATAC-seq intensity signals in pancreatic and other lineages at TSS peaks to genes in the “cell cycle” KEGG pathway. Samples are as described in Fig. 4g.
Extended Data Fig. 8 Lineage TFs and KrasG12D-dependent TFs participate in an oncogenic transcriptional program.
a, Growth competition assay to validate essential TF screen hits. Two sgRNAs for each gene were separately transduced into TO-Cas9 PDA cells in an RFP-labeled vector at 30-40% efficiency. Cells were grown on doxycycline and the proportion of remaining RFP-positive cells was determined by flow cytometry at the indicated time points. n=3 sgRNAs for sgNC, sgJunb, sgFosl1, sgKlf5 groups, n=2 sgRNAs for sgFoxa2 group. P-values were obtained by an unpaired t-test, **, p<.01, *, p<.05. All error bars are defined as mean +/- SD. b, Immunoblot using anti-Junb and anti-Fosl1 antibodies showing overexpression of the two proteins in TO-KrasG12D in the presence of doxycycline or 24 hours after removal of doxycycline. Results representative of three independent experiments. β-Actin load control run on a separate gel due to size similarities. c, Photograph showing tumor sizes after 6 weeks of growth after TO-KrasG12D cells transduced with empty vector, or vectors overexpressing Junb and/or Fosl1, were implanted in mice on or off doxycycline. For the empty vector control, no tumors were found when mice were not fed doxycycline. Injections that failed to result in tumors are indicated by an “X”. d, Cross-tissue expression of KLF5 and FOXA2. GTEx data downloaded from the Human Protein Atlas (proteinatlas.org). e, CRISPRi-mediated depletion of the indicated TFs or Kras in PDA cells. Functional effect of Kras depletion was evaluated by phospho-Erk1/2 immunoblotting. An irrelevant lane was cropped out of the pErk1/2 immunoblot, indicated by a break. Immunoblots representative of two independent experiments. β-Actin load control run on a separate gel due to size similarities. f, mRNA expression levels of Fosl1 throughout inflammation. g, Immunohistochemistry showing Klf5 expression patterns throughout inflammation. Images representative of two independent expeirments are shown. Scale bars, 100um. h, Immunofluorescence for Foxa2 in NT mice, or throughout CAE-induced inflammation in the presence and absence of KrasG12D. Images representative of two independent expeirments are shown. Scale bars, 100um.
Extended Data Fig. 9 In vivo CRISPR-KO of Fosl1, Junb, and Klf5 block tumorigenesis driven by Krasmut and TSG KO.
a, Initial infection rates of pancreatic cells determined by flow cytometry five days after injection of the indicated sgRNA-expressing AAV-Cre-sgRNA viruses in LSL-Cas9-eGFP mice. b, H&E sections of pancreases from mice injected with AAV-Cre-sgRNA on D18, images representative of two sgRNAs are shown. Results for Klf5 and Rosa are representative of two independent experiments, while Fosl1 and Junb KO was performed once, with two independent sgRNAs. Scale bars, 100um. c, Relative efficiency of LSL cassette removal at the LSL-KrasG12D allele in eGFP-positive cells on D18 after AAV-Cre-sgRNA injection, evaluated by PCR from genomic DNA obtained from FACS-sorted cells. d, Efficiency of CRISPR/Cas9-induced deletion/insertions at the indicated sgRNA-targeted sites. To calculate this, genomic DNA was isolated from eGFP-positive cells on D18 after AAV-Cre-sgRNA injection, followed by PCR amplification of the targeted region. PCR products were subjected to Sanger sequencing, and the resulting trace files were analyzed using the Tracking of Indels by Decomposition (TIDE) algorithm (https://tide.nki.nl/). e, Normalized ATAC-seq intensity at K-LOCKEDOPEN sites after infection with the indicated AAV-Cre-sgRNA compared with sgROSA controls. P-values calculated by Kolmogorov–Smirnov test, two sided. ****, p<1x10-5, **, p<.001.
Extended Data Fig. 10 KrasG12D depletion results in genomic redistribution of lineage TFs.
a, Normalized mean in vivo ATAC-seq signal at sites of KrasG12D-repressed Klf5 and Foxa2 binding sites in PDA cells. P-values calculated by Kolmogorov–Smirnov test, two sided. ****, p<1x10-5, ***, p<1x10-4,**, p<1x10-3,*, p<1x10-2, ns, p>=.05.
Supplementary information
Supplementary Data 1
Source FACS data. a, FACS sequential gating strategies for Fig. 1c–f and Extended Data Fig. 2d,e. b, FACS sequential gating strategies for Figs. 2b–d,g and 3a–d, Extended Data Figs. 2c,d,f,h–I and 5a–e. c, FACS sequential gating strategies for Fig. 5b. d, FACS sequential gating strategies for Fig. 6b–g and Extended Data Fig. 9a–e. e, FACS sequential gating strategies for Extended Data Fig. 3c. f, FACS sequential gating strategies for Extended Data Fig. 8a
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Li, Y., He, Y., Peng, J. et al. Mutant Kras co-opts a proto-oncogenic enhancer network in inflammation-induced metaplastic progenitor cells to initiate pancreatic cancer. Nat Cancer 2, 49–65 (2021). https://doi.org/10.1038/s43018-020-00134-z
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DOI: https://doi.org/10.1038/s43018-020-00134-z
