Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and metastatic malignancies worldwide. Migrating cancer stem cells (miCSCs), marked by CD133⁺CXCR4⁺ expression is a key driver of PDAC progression, which currently lack effective therapeutic targets. Activated pancreatic stellate cells (PSCs) within the tumor microenvironment secrete CXCL12, the ligand for CXCR4, thereby promoting stemness, epithelial-to-mesenchymal transition (EMT), and chemoresistance in miCSCs. Despite advances in understanding PDAC biology, clinically effective strategies that target CXCR4⁺ CSC populations remain limited. In order to investigate the molecular mechanisms sustaining miCSCs, we performed protein–protein interaction network analysis, which identified the transcription factor BMI1 as a key downstream effector of the CXCL12/CXCR4 axis. Functional studies using shRNA-mediated knockdown of CXCR4 and BMI1 were conducted to assess their roles in miCSC migration, EMT, and self-renewal. We further evaluated the therapeutic potential of the endogenous CXCR4 antagonist EPI-X4 and its optimized derivative JM#21 in PDAC cell lines. We addressed the peptide stability by encapsulating JM#21 into mesoporous silica nanoparticles (MSNs) designed for improved half-life and sustained release under physiological conditions. BMI1 was confirmed as a critical mediator of CXCL12/CXCR4-driven stemness and EMT. Knockdown of CXCR4 or BMI1 significantly impaired miCSC maintenance and migration towards CXCL12. Both EPI-X4 and JM#21 potently inhibited CXCL12-mediated signaling, reduced EMT and stemness markers, and suppressed miCSC migratory potential. JM#21 displayed superior efficacy and re-sensitized previously resistant PDAC cell lines to gemcitabine and paclitaxel. Functional assays demonstrated that nanoparticle-loaded JM#21 more effectively suppressed EMT markers and self-renewal than the free peptide, highlighting the advantage of nanoparticle delivery in therapeutic applications. Given their biocompatibility and modularity, silica nanoparticles offer a promising platform for stabilizing peptide drugs. Our findings reveal that tumor–stroma crosstalk via the CXCL12/CXCR4/BMI1 axis plays a central role in sustaining miCSC-driven metastasis and therapy resistance in PDAC. Targeting this signaling pathway with nanoparticle-stabilized JM#21 represents a novel and clinically promising therapeutic strategy to disrupt PDAC progression and improve the efficacy of existing combination treatments.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows resistance to any therapeutic strategy used. Here we tested small-molecule inhibitors targeting chromatin regulators as possible therapeutic agents in PDAC. We show that JQ1, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, suppresses PDAC development in mice by inhibiting both MYC activity and inflammatory signals. The histone deacetylase (HDAC) inhibitor SAHA synergizes with JQ1 to augment cell death and more potently suppress advanced PDAC. Finally, using a CRISPR-Cas9-based method for gene editing directly in the mouse adult pancreas, we show that de-repression of p57 (also known as KIP2 or CDKN1C) upon combined BET and HDAC inhibition is required for the induction of combination therapy-induced cell death in PDAC. SAHA is approved for human use, and molecules similar to JQ1 are being tested in clinical trials. Thus, these studies identify a promising epigenetic-based therapeutic strategy that may be rapidly implemented in fatal human tumors.

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

Despite constitutive Ras/Raf/MAPK pathway activation in most pancreatic ductal adenocarcinomas (PDACs), treatment approaches targeting this pathway have primarily been unsuccessful. We conduct a drug library screen on an MEK inhibitor (MEKi)-resistant PDAC model and perform complementary pathway analysis to identify cellular resistance phenotypes. We use syngeneic models to investigate the molecular determinants of identified drug synergism. Our study reveals an enrichment for the hallmarks of G2/M checkpoints in MEKi-resistant phenotypes from all investigated models. We find overexpression of Polo-like kinase 1 (PLK1) and other G2/M checkpoint-related proteins in MEKi-resistant cells. We identify synergistic activity between MEK and PLK1 inhibition both in vitro and in vivo and mechanistically show that dual inhibition of the PLK1 and MEK pathways activates the JNK/c-JUN pathway. This causes the accumulation of DNA damage, ultimately leading to apoptotic cell death. Dual PLK1/MEK inhibition emerges as a promising targeted approach 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.

Verification of P63-dependence and B-STEP enhancer-gene regulations.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and metastatic malignancies worldwide. Migrating cancer stem cells (miCSCs) marked by CD133+CXCR4+ expression drives metastasis but lacks effective drug targets. Here, we show that activated pancreatic stellate cells secrete the CXCR4 ligand CXCL12 to foster stemness, epithelial-to-mesenchymal transition (EMT), and chemoresistance. Protein interaction network analyses links CXCL12/CXCR4 signaling axis and the downstream transcription factor BMI1. Knockdown experiments confirmed BMI1's role in (mi)CSCs maintenance and survival. Novel CXCR4 inhibitors, i.e., the endogenous human peptide EPI-X4 and its derivatives (e.g., JM#21) strongly inhibited the in vitro migration of miCSCs. In particular, the most potent EPI-X4 derivate JM#21 sufficiently suppressed EMT, stemness, and self-renewal of human PDAC cell lines. In addition, JM#21 sensitized cell lines towards gemcitabine and paclitaxel. Overall, our study reveals that (mi)CSCs are enhanced and maintained via a tumor-stroma crosstalk through BMI1, ultimately promoting metastases and therapeutic resistance in PDAC. Peptide targeting of the CXCL12/CXCR4/BMI1 signaling axis via JM#21 could enhance PDAC combination therapies, offering a promising strategy against this deadly cancer. ### Competing Interest Statement M.H., and J.M. are co-inventors of pending and issued patents that claim to use EPI-X4 (ALB408-423) and derivatives for the therapy of CXCR4-associated diseases.

Supplementary Table S5 contains list of genes associated with L3.6pl TP63 dependent enhancers.

The file contains additional supplementary methods

Supplementary Table S2 contains GöPdx4 super enhancers regions overlapping TP63

Supplementary Table S6 contains list of qPCR primers used in the study.

HER2 is a crucial therapeutic target in breast cancer, and the survival rate of breast cancer patients has increased because of this receptor's inhibition. However, tumors have shown resistance to this therapeutic strategy due to oncogenic mutations that decrease the binding of several HER2-targeted drugs, including lapatinib, and confer resistance to this drug. Neratinib can overcome this drug resistance and effectively inhibit HER2 signaling and tumor growth. In the present study, we examined the efficacy of lapatinib and neratinib using breast cancer cells by Raman microscopy combined with a deep wavelet scattering-based multivariate analysis framework. This approach discriminated between control cells and drug-treated cells with high accuracy, compared to classical principal component analysis. Both lapatinib and neratinib induced changes in the cellular biochemical composition. Furthermore, the Raman results were compared with the results of several in vitro assays. For instance, drug-treated cells exhibited (i) inhibition of ERK and AKT phosphorylation, (ii) inhibition of cellular proliferation, (iii) cell-cycle arrest, and (iv) apoptosis as indicated by western blotting, real-time cell analysis (RTCA), cell-cycle analysis, and apoptosis assays. Thus, the observed Raman spectral changes are attributed to cell-cycle arrest and apoptosis. The results also indicated that neratinib is more potent than lapatinib. Moreover, the uptake and distribution of lapatinib in cells were visualized through its label-free marker bands in the fingerprint region using Raman spectral imaging. These results show the prospects of Raman microscopy in drug evaluation and presumably in drug discovery. Raman micro-spectroscopy is applied as a label-free analytical tool to monitor the efficacy and spatial distribution of tyrosine kinase inhibitors in cells. This method has a prospective in drug evaluation and presumably in drug discovery.

The novel Oxathiazinane derivative GP-2250 (Misetionamide) displays antineoplastic activity in vitro and in vivo, as previously shown in pancreatic cancer cells and in patient-derived mouse xenografts (PDX). Currently, GP 2250 is under phase I clinical trial in pancreatic ductal adenocarcinoma (PDAC). GP-2250 in combination with Gemcitabine displays a high synergistic capacity in various primary and established pancreatic cancer cell lines. Additionally, in the eight PDX models tested, the drug combination was superior in reducing tumor volume with an aggregate tumor regression (ATR) of 74% compared to Gemcitabine alone (ATR: 10%). Similarly, in a PDX maintenance setting following two weeks of treatment with nab-Paclitaxel plus Gemcitabine, the combination of GP-2250 plus Gemcitabine resulted in outstanding tumor control (ATR: 79%) compared to treatment with Gemcitabine alone (ATR: 60%). Furthermore, GP-2250 reduced the ratio of tumor-initiating CD133+ markers on the surface of PDAC cells in spheroid cultures, indicating a possible mechanism for the synergistic effect of both substances. Considering the high tolerability of GP 2250, these results may open up a new approach to maintenance therapy with GP-2250/Gemcitabine combination following nab-Paclitaxel plus Gemcitabine as first-line treatment.

GP-2250, a novel anticancer agent, severely limits the energy metabolism, as demonstrated by the inhibition of hexokinase 2 and glyceraldehyde-3-phosphate dehydrogenase and a decrease of ATP. Rescue experiments with supplementary pyruvate or oxaloacetate demonstrated that a TCA cycle deficit largely contributed to cytotoxicity. Activation of the energy-deficit sensor, AMP-dependent protein kinase, was associated with increased phosphorylation of acetyl-CoA carboxylase and Raptor, pointing to a possible deficit in the synthesis of fatty acids and proteins as essential cell components. Binding of p65 to DNA was dose-dependently reduced in nuclear lysates. A transcriptional deficit of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) was substantiated by the downregulation of cyclin D1 and of the anti-apoptotic Bcl2, in line with reduction in tumour cell proliferation and induction of apoptosis, respectively. The upregulation of p53 concomitant with an excess of ROS supported apoptosis. Thus, the anticancer activity of GP-2250 is a result of disruption of energy metabolism and inhibition of tumour promotion by NF-κB.

Although approximately half of all metastatic colorectal cancers (mCRCs) harbour mutations in KRAS or NRAS, hardly any progress has been made regarding targeted treatment for this group over the last few years. Here, we investigated the efficacy of vertical inhibition of the RAS‐pathway by targeting epidermal growth factor receptor (EGFR) and mitogen‐activated protein kinase kinase (MEK) in patient‐derived xenograft (PDX) tumours with primary KRAS mutation. In total, 19 different PDX models comprising 127 tumours were tested. Responses were evaluated according to baseline tumour volume changes and graded as partial response (PR; ≤ − 30%), stable disease (SD; between −30% and +20%) or progressive disease (PD; ≥ + 20%). Vertical inhibition with trametinib and cetuximab induced SD or PR in 74% of analysed models, compared to 24% by monotherapy with trametinib. In cases of PR by vertical inhibition (47%), responses were lasting (as long as day 137), with a low incidence of secondary resistance (SR). Molecular analyses revealed that primary and SR was driven by transcriptional reprogramming activating the RAS pathway in a substantial fraction of tumours. Together, these preclinical data strongly support the translation of this combination therapy into clinical trials for CRC patients.

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.

Chimeric antigen receptors (CARs) have improved cancer immunotherapy in recent years. Immune cells, such as Natural killer cells (NK-cells) or T cells, are used as effector cells in CAR-therapy. NK92-cells, a cell line with known cytotoxic activity, are of particular interest in CAR-therapy since culturing conditions are simple and anti-tumor efficacy combined with a manageable safety profile was proven in clinical trials. The major pathways of immune effector cells, including NK92-cells, to mediate cytotoxicity, are the perforin/granzyme and the death-receptor pathway. Detailed knowledge of CAR-effector cells' cytotoxic mechanisms is essential to unravel resistance mechanisms, which potentially arise by resistance against apoptosis-inducing signaling. Since mutations in apoptosis pathways are frequent in lymphoma, the impact on CAR-mediated cytotoxicity is of clinical interest. In this study, knockout models of CD19-CAR-NK92 cells were designed, to investigate cytotoxic pathways in vitro. Knockout of perforin 1 (Prf1) and subsequent abrogation of the perforin/granzyme pathway dramatically reduced the cytotoxicity of CD19-CAR-NK92 cells. In contrast, knockout of FasL and inhibition of TRAIL (tumor necrosis factor-related apoptosis-inducing ligands) did not impair cytotoxicity in most conditions. In conclusion, these results indicate the perforin/granzyme pathway as the major pathway to mediate cytotoxicity in CD19-CAR-NK92 cells.