Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a dismal 5-year survival rate at 12%. The fibrotic PDAC desmoplasia is a major contributor to chemoresistance and metastasis that drive this poor prognosis. Cancer-associated fibroblasts (CAFs) generate PDAC tumour fibrosis and have been identified as therapeutic targets to remodel the stroma to a more drug-permissive microenvironment. We assessed the therapeutic potential of inhibiting the collagen chaperone, heat shock protein 47 (HSP47) in PDAC cells and CAFs. Collagen is a key component of PDAC fibrosis and requires the activity of HSP47 to ensure correct maturation and secretion. Herein, we show that HSP47 knockdown inhibits both PDAC cells and CAF proliferation in vitro. In vivo, therapeutic HSP47 knockdown in orthotopic PDAC tumours significantly reduced intratumoural fibrosis and opened intratumoural blood vessels, while stable HSP47 knockdown specifically in CAFs additionally reduced PDAC tumour growth. We observed that HSP47 is highly expressed in the stroma of >80% of patients in a PDAC cohort (Australian Pancreatic Cancer Genome Initiative), but that it was only prognostic of poorer overall survival in the tumour compartment. Functional relevance in the tumour compartment was further validated in 3D human PDAC explants. Our work demonstrates that HSP47 is a potential therapeutic target in both PDAC cells and CAFs and represents a robust target to interfere with tumour collagen deposition.

MEK1/2 inhibition in KPC KrasG12D/fl mice with established tumours reverses enrichment of immune response related gene programmes.

Release from AZD6244 treatment results in rapid acinar to ductal metaplasia in KC KrasG12D/fl.

Background and Aims The tumor microenvironment drives many malignant features of pancreatic ductal adenocarcinoma (PDAC). The fibroblasts within pancreatic tumors promote tissue remodeling, immune suppression, and resistance to therapy. However, the interactions between stromal populations and pancreatic cancer cells are less understood in the liver, the most frequent site of PDAC metastasis. Methods To address this, we employ single cell transcriptomics to compare primary pancreatic vs. liver PDAC lesions. Using a ligand-receptor interaction tool, we assess upregulated pathways in liver PDAC. Results Here, we identify the expression of hepatocyte growth factor (HGF) in fibroblasts and its receptor MET in cancer cells are both markedly increased in the PDAC liver niche. Using functional assays, we validate that mitogenic MET signaling is activated in PDAC cells by liver-derived fibroblasts. Importantly, the inhibition of MET signaling leads to reduced tumor growth in immune competent mouse models. Conclusions Collectively, our data demonstrates that liver stromal-epithelial crosstalk networks engage in signaling pathways distinct from primary pancreatic tumors, highlighting opportunities to develop new treatments for metastatic disease.

Acceleration of pancreatic tumour initiation after loss of wild-type Kras in KPC KrasG12D/fl mice.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic adenocarcinoma (PDAC) and key for early diagnosis and therapy. During PanIN, epithelial-to-mesenchymal transition (EMT) promotes invasion and early cell dissemination. Tissue mechanics change during progression, with elasticity previously modelled as a diagnostic tool. However, viscoelasticity—combining elastic and viscous properties—is increasingly recognized as critical in cancer and mechanosensitive cell behaviour. Using PDAC mouse models and 2D polyacrylamide hydrogels, this study shows that viscoelasticity shifts dynamically from healthy to PanIN tissue. By separating elasticity and viscosity, it identifies viscosity as a key driver of EMT-related phenotypic changes, suggesting an important role for facilitating invasive potential which could be a novel target for diagnostics and therapy.

Increased fibrosis in wild-type Kras deficient PanINs.

ABSTRACT Background Pancreatic ductal adenocarcinoma (PDAC) is a particularly lethal malignancy with few treatment options available. Extensive remodelling of extracellular matrix (ECM) generates a highly fibrotic tumour landscape, which impairs therapeutic response. Objective We investigated whether stromal priming via the highly specific Focal Adhesion Kinase (FAK) inhibitor narmafotinib (AMP945) in combination with the two major standard-of-care chemotherapies in PDAC, gemcitabine/Abraxane and FOLFIRINOX, reduces fibrosis and enhances treatment efficacy. Design 3D organotypic matrices, intravital imaging, and in vivo subcutaneous and orthotopic PDAC models were used to provide a rationale for a first-line priming regimen of narmafotinib prior to chemotherapy. Results Neoadjuvant chemotherapy induces fibrosis in PDAC indicating a need for upfront first-line priming of the ECM to normalise the stroma for optimal treatment response. Narmafotinib is a new potent small molecule FAK inhibitor. Phase I safety data shows excellent safety, tolerability, and pharmacokinetics following oral administration in humans. We reveal that narmafotinib treatment during early ECM remodelling (‘priming’) reduces fibrosis, while limiting subsequent PDAC invasion. Moreover, intravital imaging demonstrates real-time FAK inactivation and cell cycle stalling, leading to improved chemotherapeutic efficacy upon narmafotinib priming in vivo . Long-term assessment in patient-derived models shows that narmafotinib priming prior to gemcitabine/Abraxane or FOLFIRINOX reduces PDAC progression and extends survival in both chemotherapy settings. Conclusions Our results using these Phase II-ready drug combinations strongly support the clinical assessment of narmafotinib in PDAC. Narmafotinib is currently in Phase Ib/IIa trials, assessing a pulsed dosing regimen prior to gemcitabine/Abraxane, and warrants further clinical assessment in combination with FOLFIRINOX. SIGNIFICANCE OF THIS STUDY What is already known on this topic Pancreatic cancer (PC) is one of the most lethal malignancies and is characterised by a dense, fibrotic stroma, which impairs chemotherapy efficacy. The non-receptor tyrosine kinase FAK is known to promote cancer fibrosis and therefore represents a therapeutic target to normalise the PC stroma and to improve chemotherapy performance. What this study adds Neoadjuvant chemotherapy induces early fibrosis indicating a need for upfront first-line priming of the ECM to blunt or normalise stromal fibrosis for optimal response to therapy. The small molecule inhibitor narmafotinib (which is currently under Phase Ib/IIa clinical trial assessment) shows high specificity towards FAK as well as desirable pharmacokinetics and pharmacodynamics in healthy human volunteers. Early short-term narmafotinib priming reduces fibrosis and improves the efficacy of subsequent standard-of-care gemcitabine/Abraxane chemotherapy. FOLFIRINOX (oxaliplatin, irinotecan, leucovorin and 5-fluorouracil) is a multi-agent chemotherapy preferentially used in PDAC patients with good performance status. Our results demonstrate that narmafotinib priming also improves FOLFIRINOX efficacy, leading to extended survival in patient-derived PDAC models. How this study might affect research, practice, or policy This study supports the clinical development of narmafotinib in combination with both gemcitabine/Abraxane (ACCENT trial) and further FOLFIRINOX standard-of-care chemotherapies for PDAC patient treatment. The first-line priming strategy and early ECM normalisation used in this study may also be applicable to other combination therapy settings and warrants further investigation in ongoing clinical studies.

The microtubule protein βIII-tubulin is a prognostic, pro-survival, and chemoresistance factor in multiple malignancies, including pancreatic ductal adenocarcinoma (PDAC). However, the precise survival mechanisms controlled by βIII-tubulin in cancer remain unknown. Here, we discovered a link between βIII-tubulin and the activation of caspase 8-mediated extrinsic apoptosis. Silencing βIII-tubulin in PDAC cells activated caspase 8, leading to decreased cell viability and growth both in vitro and in vivo. βIII-tubulin knockdown also increased the sensitivity of PDAC cells to extrinsic cell death signals, including TNF-related apoptosis-inducing ligand (TRAIL), TNFα, and FasL. Furthermore, we demonstrated that βIII-tubulin knockdown in PDAC cells, in the absence or presence of TRAIL, increased diffusion and clustering of the TRAIL death receptor DR5 at the cell membrane, inducing extrinsic apoptosis. Nanoparticle delivery of βIII-tubulin siRNA to mouse PDAC tumours reduced tumour growth and increased responsiveness to TRAIL therapy. In patient-derived human PDAC explants, βIII-tubulin silencing reduced tumour cell frequency and improved sensitivity to TRAIL. Finally, we showed that high βIII-tubulin expression in the human PDAC stroma was independently prognostic for poor overall survival. Taken together, silencing βIII-tubulin represents an innovative strategy to activate a suicide signal in PDAC cells and render them more sensitive to microenvironment- and chemotherapy-derived death signals.

RAS family proteins, including HRAS, NRAS, and KRAS, are frequently mutated in cancer. Although there has been recent success in designing inhibitors that target oncogenic RAS, they elicit resistance and treating RAS-driven cancer remains difficult. Here, employing a proteomic analysis, we find that multiple spliceosome components are upregulated in the nuclei of cells undergoing RAS-induced senescence. This upregulation depends on RAS signalling and occurs in both senescent preneoplastic and fully transformed cancer cells. Spliceosome components are also highly expressed in preneoplastic and cancerous lesions in human and murine lung, liver, colorectal, and pancreatic cancers. Using siRNA screens, we identify six spliceosome components, including SF3B1 and RBM39, that are essential in cells expressing oncogenic RAS. We find that SF3B1 is required in these cells for maintaining splicing fidelity. By combining transcriptome and splicing analyses with functional screens, we identify the RNA Pol II-associated factor SPT5 as a key mediator of the SF3B1 effects. Importantly, using mouse models of liver cancer, we show that RBM39 and SF3B1 inhibitors are effective in targeting both preneoplastic lesions and aggressive tumours expressing oncogenic RAS. In summary, our study highlights the spliceosome as a promising target for RAS-driven cancers capable of inhibiting both cancer initiation and progression.

Loss of wild-type KRAS does not alter PI3K-AKT signalling.

Loss of wild-type KRAS sensitizes KPC KrasG12D/fl tumours to MEK1/2 inhibition.

Pancreatic ductal adenocarcinoma (PDAC) stands to become the second most deadly cancer by 2030. The small GTPase, KRAS, is mutated in over 90% of PDAC patients and considered the primary driver mutation. Despite being an almost ubiquitous event, KRAS mutations have been difficult to target therapeutically, particularly KRAS G12D , the most common mutation in PDAC. In addition to these pharmacological challenges, KRAS mutations have been shown to drive signaling plasticity and therapeutic resistance through phosphorylation cascades in most cancers. Protein phosphatases are master regulators of kinase signaling, however the contribution of phosphatase deregulation to mutant KRAS cancer phenotypes is poorly understood. Protein phosphatase 2A (PP2A) inhibits effectors downstream of KRAS, placing this family of enzymes as key regulators of PDAC oncogenic signaling. However, our previous studies utilizing small molecule activating compounds of PP2A show a heterogeneous response in PDAC, with some cell lines displaying increased oncogenic signaling despite induction of phosphatase activity. Similarly, specific PP2A subunits exhibit both tumor suppressive and oncogenic functions depending on the cellular context. Therefore, understanding the role of PP2A in regulating cancer phenotypes is critical for the future development of therapeutic strategies that leverage this phosphatase. Here, we determined the impact of the specific PP2A subunit, B56α, on PDAC phenotypes using both genetic and pharmacological activation strategies in human PDAC cell lines and genetic mouse models. We demonstrate that while PP2A-B56α suppresses specific oncogenic pathways, B56α activation exacerbates PDAC proliferative phenotypes and decreases overall survival in vivo , potentially through increased epidermal growth factor receptor (EGFR) signaling. EGFR is a critical signaling node in PDAC as inhibition or loss of EGFR prevents KRAS-driven tumorigenesis and increased EGFR activity is associated with poor patient outcome. The activation of EGFR by PP2A-B56α is in part mediated through increased expression and processing of EGFR ligands, specifically amphiregulin, heparin-binding EGF-like growth factor (HB-EGF), and epiregulin. Furthermore, pharmacological PP2A activation in combination with EGFR inhibitors mitigates this signaling and increases cell death. Together, these studies implicate a previously undescribed non-canonical role for PP2A-B56α in EGFR signaling that contributes to PDAC progression.

Pre-malignant transformation of pancreatic acinar cells by oncogenic Kras is dependent upon stochastic emergence of metaplastic cell states, through unknown mechanisms. We reveal that an early, transcriptionally-mediated effect of Kras is sporadic failure of proteostatic ER-phagy. Genetically-altered mice deficient in ER-phagy demonstrate that this event co-operates with Kras to drive acinar-ductal metaplasia (ADM) and subsequent cancer. Mechanistically, proteomics and high-resolution imaging uncover pathologic aggregation of a subset of ER proteins, including the injury marker REG3B, resulting from failure to physically interact with the ER-phagy receptor CCPG1. Spatial transcriptomics demonstrate that the appearance of sporadic intracellular aggregates upon Kras activation marks rare acinar cells existing in an injured, ADM-primed state. Importantly, engineered mutants of REG3B establish that aggregate formation is sufficient to directly engender this epithelial cell state. Pancreatic cancer can thus arise from stochastic pathologic protein aggregates that are influenced by and co-operate with an oncogene. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND & AIMS:The adult pancreas protects against cancer by actively expelling genetically mutated cells. Pancreatic cancer starts with cells carrying KRAS mutations; however, it is not clear how some KRAS mutant cells override cell elimination mechanisms to survive in tissues. METHODS:An in vivo mouse model of sporadic tumorigenesis was used to induce Kras and/or Tp53 mutations in low numbers of cells in the adult pancreas. The mutant cell fate was monitored over time using quantitative fluorescence imaging. Gene signatures of noneliminated mutant cell populations were identified using bulk RNA sequencing. Differential gene expression was overlapped with publicly available datasets. Key molecular pathways were validated in murine pancreas using immunofluorescence and functionally tested using inhibitor studies in vivo and epithelial coculture systems in vitro. RESULTS:Although most genetically mutant cells are eliminated from the adult pancreas, a population of KRASG12D- or p53R172H-expressing cells are stably retained. Wnt5a signaling, cell dormancy, and stemness were identified as key features of surviving KrasG12D cells in vivo. Wnt5a specifically inhibits apical extrusion of RasV12 cells by promoting stable E-cadherin-based cell-cell adhesions at RasV12: normal cell-cell boundaries in vitro. In the pancreas, Wnt signaling, E-cadherin, and β-catenin are increased at cell-cell contacts between noneliminated KrasG12D cells and normal neighbors. Active Wnt signaling is a general mechanism required to promote KrasG12D and p53R172H cell retention and cell survival in vivo. CONCLUSIONS:RAS mutant cells activate Wnt5a and cell dormancy to avoid cell expulsion and to survive in the adult pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer mortality, with minimal improvements in 5-year survival over the past decade. While PDAC originates from premalignant lesions, only a minority of these lesions advance further into malignancy. Distinguishing which lesions are likely to progress would transform enabling timely preventative surgical intervention, but remains a key challenge. In addition to the more well-characterized genetic mutations associated with PDAC onset, recent studies suggest that inflammation in the pancreas microenvironment promotes epithelial chromatin accessibility remodeling to increase their plasticity. This plasticity is thought to enhance cellular sensitivity in response to local signaling cues, which varies drastically across PDAC tumors. We propose that the cell lineage fate of the premalignant epithelial cells is heavily influenced by their surrounding microenvironment. To investigate this, we are using the CosMx spatial transcriptomics platform to profile cellular composition heterogeneity across pancreatic tissue cores from KrasLSL-G12D; Trp53LSL-R172H; Pdx1-Cre (KPC) mouse model of PDAC. By analyzing spatial heterogeneity within the tumor microenvironment, we aim to delineate how local microenvironmental differences might influence epithelial lineage fate decisions into PDAC. Our findings will help understand the factors driving progression from early lesions to invasive cancer, supporting efforts in early PDAC detection and informing clinical strategies for surgical resection. Jennifer Claire Muscat, Eva Freckmann, Andrew S. Papanastasiou, Jennifer P. Morton, Crispin J. Miller. Investigating the tumor microenvironment’s role in lineage fate determination in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B118.

Pancreatic cancer (PC) is a highly metastatic malignancy. More than 80% of patients with PC present with advanced-stage disease, preventing potentially curative surgery. The neuropeptide Y (NPY) system, best known for its role in controlling energy homeostasis, has also been shown to promote tumorigenesis in a range of cancer types, but its role in PC has yet to be explored. We show that expression of NPY and NPY1R are up-regulated in mouse PC models and human patients with PC. Moreover, using the genetically engineered, autochthonous KPR172HC mouse model of PC, we demonstrate that pancreas-specific and whole-body knockout of Npy1r significantly decreases metastasis to the liver. We identify that treatment with the NPY1R antagonist BIBO3304 significantly reduces KPR172HC migratory capacity on cell-derived matrices. Pharmacological NPY1R inhibition in an intrasplenic model of PC metastasis recapitulated the results of our genetic studies, with BIBO3304 significantly decreasing liver metastasis. Together, our results reveal that NPY/NPY1R signaling is a previously unidentified antimetastatic target in PC.

Late-stage intervention with MEK1/2 inhibition improves survival of KPC KrasG12D/fl mice. A, Experimental schematic. KPC KrasG12D/+ and KPC KrasG12D/fl mice were palpated for tumor burden, with palpable tumor burden confirmed by ultrasound imaging, with mice treated continuously from the following day with either vehicle or AZD6244. Tumor growth was monitored by ultrasound imaging once weekly to clinical endpoint. B, Relative volume of pancreatic tumors arising in KPC KrasG12D/+ and KPC KrasG12D/fl mice treated with either vehicle control or AZD6244 from palpable tumor and aged to clinical endpoint. Tumor volume was measured once weekly by high-resolution ultrasound imaging. Each line represents an individual mouse of the indicated genotype and treatment. KPC KrasG12D/fl vehicle, n = 6; KPC KrasG12D/fl AZD6244, n = 7; KPC KrasG12D/+ vehicle, n = 6; KPC KrasG12D/+ AZD6244, n = 4. FC, fold change. C, Change of tumor volume (mm3) of KPC KrasG12D/+ and KPC KrasG12D/fl mice treated with either vehicle control or AZD6244 from palpable tumor between initial ultrasound measurement and a secondary ultrasound measurement (between 6 and 13 days later). Plot represents Log2(FC) of tumor volume between first and second measurements for the full treatment cohort when more than one imaging session was possible. KPC KrasG12D/fl vehicle, n = 5; KPC KrasG12D/fl AZD6244, n = 7; KPC KrasG12D/+ vehicle, n = 4; KPC KrasG12D/+ AZD6244, n = 4. D, Kaplan–Meier survival curves for KPC KrasG12D/+ and KPC KrasG12D/fl mice treated with vehicle or AZD6244, as indicated, from palpable tumor burden and aged to clinical endpoint. KPC KrasG12D/fl vehicle, n = 5; KPC KrasG12D/fl AZD6244, n = 8; KPC KrasG12D/+ vehicle, n = 6; KPC KrasG12D/+ AZD6244, n = 5. MS, median survival. E, Representative hematoxylin and eosin images of tumors from KPC KrasG12D/+ and KPC KrasG12D/fl mice treated with vehicle or AZD6244, as indicated, from palpable tumor burden and aged until clinical endpoint. Representative of five mice per group. Scale bar, 500 μm. F, Left, volcano plot for differentially expressed genes of KPC KrasG12D/+ tumors treated with vehicle or AZD6244 from palpable tumor burden to clinical endpoint. Right, volcano plot for differentially expressed genes of KPC KrasG12D/fl tumors treated with vehicle or AZD6244 from palpable tumor burden to clinical endpoint. Red, significantly altered genes. G, Schematic representing relative impact of WT Kras deletion upon tumur outgrowth and therapeutic responses from KPC KrasG12D/+ and KrasG12D/fl comparison.