Foxa1-Mediated Chromatin Remodeling and Enhancer Reprogramming Promotes Gemcitabine Resistance in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by a poor prognosis, and resistance to gemcitabine, a frontline chemotherapeutic agent, poses a significant challenge to effective treatment. Unraveling the molecular underpinnings of gemcitabine resistance is essential for devising novel therapeutic strategies. Here we derived gemcitabine-resistant and parental pancreatic cancer cell lines from KPC mouse models of PDAC. Employing a multi-omics approach, we integrated RNA-seq, ATAC-seq, and ChIP-seq analyses to characterize the genome-wide alterations in gene transcription, chromatin accessibility, histone modifications, and transcription factor (TF) binding in resistant cells. High-throughput chromatin conformation capture (Hi-C) was used to explore 3D chromatin remodeling. Our analyses identified differentially expressed genes (DEGs) critical for gemcitabine metabolism and resistance, notably Rrm1 and Cdadc1. We observed the upregulation of NF-kB, p53, and MAPK pathways, and the downregulation of mTORC1, MYC targets, and reactive oxygen species pathways. Differentially accessible regions (DARs) showed a significant positive correlation with the expression patterns of DEGs and histone mark H3K27 acetylation. Motif enrichment analyses revealed that TFs from the AP-1, Fox, and Hox families were significantly enriched at DARs, underscoring Foxa1 as a pivotal regulator of resistance-associated genes. We discovered that 3D chromatin reorganization, particularly the enhancement of enhancer-promoter interactions involving super-enhancer (SE) reprogramming mediated by Foxa1 and Ctcf/Smc3, drives the expression of critical resistance genes. Targeting Foxa1 or disrupting SE-associated transcription using JQ1, a bromodomain inhibitor, effectively inhibited resistant cell proliferation and enhanced gemcitabine sensitivity. In summary, this study elucidates the role of TF-mediated chromatin remodeling in gemcitabine resistance and suggests that targeting super-enhancers could be a promising therapeutic strategy to overcome resistance in PDAC, potentially revolutionizing drug therapy for this disease. Overall design: 1. Cell Culture and Resistance Induction. Maintain parental cancer cells in culture. Induce resistance by exposing cells to increasing concentrations of gemcitabine over several passages. Monitor cell viability and drug sensitivity to confirm resistance. 2. RNA-seq. Extract total RNA from both parental and resistant cells. Prepare sequencing libraries using an RNA-seq kit. Sequence libraries on a high-throughput sequencing platform to obtain transcriptome data. 3. ATAC-seq. Isolate nuclei from both cell types. Perform ATAC-seq to profile accessible chromatin regions. Prepare and sequence ATAC-seq libraries to obtain chromatin accessibility data. 4. ChIP-seq. Crosslink proteins to DNA in both cell types. Sonicate chromatin to shear DNA. Perform ChIP using antibodies against histone modifications and TFs. Prepare and sequence ChIP-seq libraries to obtain binding data. 5. Data Analysis. Process raw sequencing data to remove low-quality reads. Align RNA-seq reads to the reference genome. Align ATAC-seq and ChIP-seq reads to the reference genome. Identify differentially expressed genes, accessible chromatin regions, and differential ChIP-seq peaks between parental and resistant cells. Integrate multi-omics data to identify co-regulated genes and regulatory elements associated with gemcitabine resistance. Genome-wide landscaps of 3D chromatin architecture in parental and doxorubicin-resistant pancreatic cancer cells. In total, 6 samples were analyzed. Hi-C_Pa_1, Hi-C_Pa_2 and Hi-C_Pa_3 were three biological replicates and served as the control group. Hi-C_GR_1, Hi-C_GR_2 and Hi-C_GR_3 were three biological replicates and served as the treatment group.