Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal cancer with a 5-year survival rate of 13%. Despite recent molecular stratification of tumors into distinct classical and basal-like cell states, most tumors are heterogeneous and contain both subtypes. Therefore, therapeutic approaches targeting only one subtype are unlikely to be effective as standalone PDAC treatments. Here, we integrated chromatin accessibility [assay for transposase-accessible chromatin with sequencing (ATAC-seq)], genome-wide occupancy [chromatin immunoprecipitation sequencing (ChIP-seq)] for epigenetic status (H3K27ac), and H3K4me3-anchored chromatin topology (HiChIP) to uncover subtype-independent highly interactive enhancers that interact with essential genes in PDAC. Motif analysis revealed that these common enhancers were bound by KLF5 with subsequent depletion leading to decreased cell viability via induction of apoptosis. To elucidate the transcriptional and epigenetic mechanisms by which KLF5 functions in PDAC, we used rapid depletion of KLF5 with dTAG technology and profiled the effects on the open and active chromatin landscape and transcription with nascent RNA and messenger RNA sequencing over time. Enhancer inactivation via KRAB domain Zim3-dCas9 fusion protein confirmed KLF5-bound enhancers regulate target genes, including the anti-apoptotic gene BCL2L1. Multiplex immunofluorescence confirmed costaining of KLF5 and Bcl-xL in patient samples and overexpression of Bcl-xL rescued the induction of apoptosis after KLF5 depletion. Together, this study provides insights into common mechanisms to target highly heterogeneous PDAC tumors.

A chromosome 4p score predicts poor survival.

BACKGROUND:The efficacy of pharmacological glycogen synthase kinase-3β (GSK3β) inhibition is currently being investigated in unselected cohorts of metastatic pancreatic ductal adenocarcinoma (PDAC). Here, we sought to determine the clinical significance of nuclear GSK3β accumulation in patients with resectable PDAC. OBJECTIVE:This study aimed to explore the therapeutic potential and underlying mechanisms of GSK3β pathway disruption in PDAC with enriched nuclear GSK3β levels. DESIGN:We investigated the activation and function of GSK3β and its downstream transcription factor NFATc1 in tumour recurrence, growth and resistance using human PDAC tissues, patient-derived organoids and tumour cells, PDAC explants, cell lines and murine models. GSK3β signalling was disrupted using genetic and pharmacological approaches. Live-cell imaging, proliferation, homologous recombination (HR) repair and comet assays, messenger RNA sequencing and chromatin immunoprecipitation were used to explore GSK3β-NFATc1 signalling-mediated target gene regulation in DNA repair, growth and resistance. RESULTS:Nuclear GSK3β accumulates in a subset of resected PDAC and promotes proliferation and DNA repair through NFATc1. The GSK3βhigh/NFATc1high subtype accounts for 14% of resected PDAC and is associated with rapid tumour recurrence and poor survival. The GSK3β-NFATc1 signalling pathway contributes to cisplatin resistance by inducing BRCA genes transcription, which facilitates HR-mediated DNA double-strand breaks (DSBs) repair. Disruption of the GSK3β-NFATc1 axis impairs HR-driven DSB repair, increasing cisplatin sensitivity in vitro and in preclinical PDAC models. CONCLUSION:We have identified a highly aggressive GSK3βhigh/NFATc1high subtype that predicts early recurrence, poor survival and cisplatin resistance in PDAC. This subtype reveals new treatment vulnerabilities suggesting that patients with PDAC may benefit from stratification-based tailored treatment strategies.

NF-κB signaling can be subdivided into canonical and noncanonical pathways, culminating in the transcriptional activity of RELA and RELB, respectively. However, the upstream signals that activate these transcription factors and their specific regulatory roles in pancreatic ductal adenocarcinoma (PDAC) remain incompletely understood. We investigated the differential activation and function of RELA and RELB in PDAC using transcriptome-wide gene expression profiling, genome-wide occupancy mapping, and epigenomic analysis. Temporal activation patterns were assessed following TNFα or TWEAK stimulation. Single-cell RNA sequencing and multiplex immunofluorescence staining were used to characterize activity in primary PDAC tissues. Motif enrichment and chromatin accessibility were evaluated to determine transcription factor binding dynamics and co-regulatory associations. We demonstrate that TNFα is the primary activator of canonical NF-κB signaling via RELA, while TWEAK selectively engages noncanonical signaling through RELB in PDAC. RELA and RELB display distinct temporal dynamics and regulatory activity. RELA binds to both open and closed chromatin and drives a broad transcriptional program, while RELB exclusively occupies pre-accessible chromatin regions co-enriched for AP1 motifs. Motif analysis reveals a particularly strong association of RELB with AP1 elements, suggesting selective co-regulation. Single-cell transcriptomic analysis and multiplex staining in primary tumors reveal distinct spatial and cellular distribution patterns, with RELA and RELB active in separate tumor and microenvironmental compartments. These findings underscore the distinct and complementary roles of TNFα and TWEAK in regulating NF-κB signaling in PDAC. TNFα engages a broader transcriptional program via RELA, whereas TWEAK targets a more selective set of genes marked by chromatin accessibility and AP1 co-binding through RELB. This study provides critical insight into the regulatory dynamics of NF-κB signaling in pancreatic cancer and highlights the specialized functions of RELA and RELB in modulating gene expression and tumor-microenvironment interactions.

USP44 alters the epigenome and regulates gene expression.

USP44 is expressed early in neural crest development.

USP44 loss alters H3K27ac at distal regions.

USP44 impairs retinoic acid-induced neuritic development in neuroblastoma cell lines.

USP44 expression correlates with CIN in neuroblastoma.

Adrenergic or Mesenchymal identities do not reliably correlate with USP44 status.

Despite recent medical advances, colorectal cancer (CRC) remains the second-leading cause of cancer-related death worldwide. For patients with KRAS wild-type metastatic CRC, the monoclonal antibody cetuximab, which targets the epidermal growth factor receptor (EGFR), is an approved treatment option. However, therapeutic success is often limited by the emergence of drug-resistant cancer cell populations within a few months. Therefore, alternative strategies to effectively target cetuximab-refractory CRC are urgently needed. Here, we sought to identify second-line therapeutic strategies using a CRC cell line with acquired cetuximab resistance as a model. Transcriptomic profiling of the resistant cells identified the apoptosis pathway as a potential therapeutic target, which was supported by their increased susceptibility to BH3-mimetics targeting the anti-apoptotic proteins MCL-1 and BCL-xL under both 2D and 3D culture conditions. These findings were validated in organotypic CRC slice cultures generated from cetuximab-resistant patient-derived xenografts (PDXs). Multiplex immunofluorescence staining demonstrated that BCL-xL inhibition effectively triggered apoptosis in heterogeneous PDX tumor slice models, including models harboring oncogenic BRAF mutations. Our findings suggest that cetuximab-resistant CRC retains apoptotic competence, and that BCL-xL inhibition serves as a robust alternative therapeutic strategy that is largely independent of the tumor mutational profile.

USP44 expression declines with development.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with a 5-year survival of only ∼13%. Despite incremental advances through combination chemotherapy, most patients relapse rapidly due to profound molecular heterogeneity and intrinsic resistance. Recent genomic and transcriptomic studies have defined distinct PDAC molecular subtypes, classical and basal-like, which differ in differentiation state, prognosis, and therapeutic vulnerability. Classical tumors, marked by GATA6 and hepatocyte nuclear factors, exhibit epithelial identity and relative chemosensitivity, whereas basal-like tumors driven by ΔNp63 and MYC display mesenchymal and inflammatory programs associated with resistance and poor outcome. Importantly, these subtypes are dynamic, with single-cell and spatial analyses revealing frequent coexistence and therapy-induced transitions, highlighting cellular plasticity as a major determinant of treatment response. Subtype identity is governed by lineage-defining transcription factors, chromatin regulators, and stromal cues that integrate to form reversible epigenetic states. Targeting these mechanisms with inhibitors of EZH2, BET proteins, or CDK9 can restore differentiation programs and resensitize tumors to chemotherapy. Integrating molecular subtyping with epigenetic modulation thus offers a rational path toward biomarker-guided therapy. Continued efforts combining spatially resolved profiling, organoid modeling, and liquid-biopsy monitoring will be essential to capture tumor evolution in real time. Understanding and therapeutically exploiting the transcriptional and epigenetic plasticity in PDAC may ultimately enable reprogramming of resistant states and improve clinical outcomes in this intractable disease.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal cancer with a 5-year survival rate of 13%. Despite recent molecular stratification of tumors into distinct classical and basal-like cell states, most tumors are heterogeneous contain of both subtypes. Therefore, therapeutic approaches targeting one subtype may not be suitable for PDAC therapy. Here, we integrated chromatin accessibility (ATAC-seq), genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac) and H3K4me3-anchored chromatin topology (HiChIP) to uncover subtype-independent highly interactive enhancers that interact with essential genes in PDAC. Motif analysis revealed these common enhancers were bound by KLF5 with subsequent depletion leading to decreased cell viability via induction of apoptosis. To elucidate the transcriptional and epigenetic mechanisms by which KLF5 functions in PDAC, we employed rapid depletion of KLF5 with dTAG technology and profiled the effects on the open and active chromatin landscape and transcription with nascent RNA and mRNA-seq over time. Enhancer inactivation via KRAB domain Zim3-dCas9 fusion protein confirmed KLF5-bound enhancers regulate target genes, including the anti-apoptotic gene BCL2L1. Multiplex immunofluorescence confirmed co-staining of KLF5 and Bcl-xL in patient samples and overexpression of Bcl-xL rescued the induction of apoptosis after KLF5 depletion. Taken together, this study provides new insights into common mechanisms to target highly heterogeneous PDAC tumors. Teaser KLF5 controls subtype-independent highly interactive enhancers to regulate cell viability in pancreatic cancer. ### Competing Interest Statement The authors have declared no competing interest. Robert Bosch Stiftung

Defining the basal-like A-specific enhancer landscape in PDAC

Defining the specificity and P63-dependence of B-STEP

Supplementary Table S3 contains list of genes associated with Gö4Pdx4 super enhancers overlapping TP63.

Supplementary Table S4 contains L3.6pl TP63-dependent enhancer regions.