Coexisting mechanisms of luminogenesis in pancreatic cancer-derived organoids.

Lumens are crucial features of the tissue architecture in both the healthy exocrine pancreas, where ducts shuttle enzymes from the acini to the intestine, and in the precancerous lesions of the highly lethal pancreatic ductal adenocarcinoma (PDAC), similarly displaying lumens that can further develop into cyst-like structures. Branched pancreatic-cancer derived organoids capture key architectural features of both the healthy and diseased pancreas, including lumens. However, their transition from a solid mass of cells to a hollow tissue remains insufficiently explored. Here, we show that organoids display two orthogonal but complementary lumen formation mechanisms: one relying on fluid intake for multiple microlumen nucleation, swelling and fusion, and the other involving the death of a central cell population, thereby hollowing out cavities. These results shed further light on the processes of luminogenesis, deepening our understanding of the early formation of PDAC precancerous lesions, including cystic neoplasia.

Mitochondrial perturbation in the intestine causes microbiota-dependent injury and gene signatures discriminative of inflammatory disease.

Mitochondrial dysfunction is associated with inflammatory bowel diseases (IBDs). To understand how microbial-metabolic circuits contribute to intestinal injury, we disrupt mitochondrial function in the epithelium by deleting the mitochondrial chaperone, heat shock protein 60 (Hsp60Δ/ΔIEC). This metabolic perturbation causes self-resolving tissue injury. Regeneration is disrupted in the absence of the aryl hydrocarbon receptor (Hsp60Δ/ΔIEC;AhR-/-) involved in intestinal homeostasis or inflammatory regulator interleukin (IL)-10 (Hsp60Δ/ΔIEC;Il10-/-), causing IBD-like pathology. Injury is absent in the distal colon of germ-free (GF) Hsp60Δ/ΔIEC mice, highlighting bacterial control of metabolic injury. Colonizing GF Hsp60Δ/ΔIEC mice with the synthetic community OMM12 reveals expansion of metabolically flexible Bacteroides, and B. caecimuris mono-colonization recapitulates the injury. Transcriptional profiling of the metabolically impaired epithelium reveals gene signatures involved in oxidative stress (Ido1, Nos2, Duox2). These signatures are observed in samples from Crohn's disease patients, distinguishing active from inactive inflammation. Thus, mitochondrial perturbation of the epithelium causes microbiota-dependent injury with discriminative inflammatory gene profiles relevant for IBD.

A Novel AMPK Inhibitor Sensitizes Pancreatic Cancer Cells to Ferroptosis Induction.

Cancer cells must develop strategies to adapt to the dynamically changing stresses caused by intrinsic or extrinsic processes, or therapeutic agents. Metabolic adaptability is crucial to mitigate such challenges. Considering metabolism as a central node of adaptability, it is focused on an energy sensor, the AMP-activated protein kinase (AMPK). In a subtype of pancreatic ductal adenocarcinoma (PDAC) elevated AMPK expression and phosphorylation is identified. Using drug repurposing that combined screening experiments and chemoproteomic affinity profiling, it is identified and characterized PF-3758309, initially developed as an inhibitor of PAK4, as an AMPK inhibitor. PF-3758309 shows activity in pre-clinical PDAC models, including primary patient-derived organoids. Genetic loss-of-function experiments showed that AMPK limits the induction of ferroptosis, and consequently, PF-3758309 treatment restores the sensitivity toward ferroptosis inducers. The work established a chemical scaffold for the development of specific AMPK-targeting compounds and deciphered the framework for the development of AMPK inhibitor-based combination therapies tailored for PDAC.

ZEB1-mediated fibroblast polarization controls inflammation and sensitivity to immunotherapy in colorectal cancer.

The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers are reduced. Single-cell transcriptomics, histological characterization, and in vitro modeling reveal a crucial role of ZEB1 in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1-deficient CAF repertoire sensitizes to immune checkpoint inhibition, offering a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.

The Janus kinase 1 is critical for pancreatic cancer initiation and progression.

Interleukin-6 (IL-6)-class inflammatory cytokines signal through the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and promote the development of pancreatic ductal adenocarcinoma (PDAC); however, the functions of specific intracellular signaling mediators in this process are less well defined. Using a ligand-controlled and pancreas-specific knockout in adult mice, we demonstrate in this study that JAK1 deficiency prevents the formation of KRASG12D-induced pancreatic tumors, and we establish that JAK1 is essential for the constitutive activation of STAT3, whose activation is a prominent characteristic of PDAC. We identify CCAAT/enhancer binding protein δ (C/EBPδ) as a biologically relevant downstream target of JAK1 signaling, which is upregulated in human PDAC. Reinstating the expression of C/EBPδ was sufficient to restore the growth of JAK1-deficient cancer cells as tumorspheres and in xenografted mice. Collectively, the findings of this study suggest that JAK1 executes important functions of inflammatory cytokines through C/EBPδ and may serve as a molecular target for PDAC prevention and treatment.

Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2.

OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.

Exosomes define a local and systemic communication network in healthy pancreas and pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), a lethal disease, requires a grasp of its biology for effective therapies. Exosomes, implicated in cancer, are poorly understood in living systems. Here we use the genetically engineered mouse model (ExoBow) to map the spatiotemporal distribution of exosomes from healthy and PDAC pancreas in vivo to determine their biological significance. We show that, within the PDAC microenvironment, cancer cells establish preferential communication routes through exosomes with cancer associated fibroblasts and endothelial cells. The latter being a conserved event in the healthy pancreas. Inhibiting exosomes secretion in both scenarios enhances angiogenesis, underscoring their contribution to vascularization and to cancer. Inter-organ communication is significantly increased in PDAC with specific organs as most frequent targets of exosomes communication occurring in health with the thymus, bone-marrow, brain, and intestines, and in PDAC with the kidneys, lungs and thymus. In sum, we find that exosomes mediate an organized intra- and inter- pancreas communication network with modulatory effects in vivo.

Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx.

Although long noncoding RNAs (lncRNAs) dominate the transcriptome, their functions are largely unexplored. The extensive overlap of lncRNAs with coding and regulatory sequences restricts their systematic interrogation by DNA-directed perturbation. Here we developed genome-scale lncRNA transcriptome screening using Cas13d/CasRx. We show that RNA targeting overcomes limitations inherent to other screening methods, thereby considerably expanding the explorable space of the lncRNAome. By evolving the screening system toward pan-cancer applicability, it supports molecular and phenotypic data integration to contextualize screening hits or infer lncRNA function. We thereby addressed challenges posed by the enormous transcriptome size and tissue specificity through a size-reduced multiplexed gRNA library termed Albarossa, targeting 24,171 lncRNA genes. Its rational design incorporates target prioritization based on expression, evolutionary conservation and tissue specificity, thereby reconciling high discovery power and pan-cancer representation with scalable experimental throughput. Applied across entities, the screening platform identified numerous context-specific and common essential lncRNAs. Our work sets the stage for systematic exploration of lncRNA biology in health and disease.

Preclinical efficacy of carfilzomib in BRAF-mutant colorectal cancer models.

Serine/threonine-protein kinase B-raf (BRAF) mutations are found in 8-15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF-mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF-mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune-mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic-damage-associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF-mutant murine tumors and mobilized the danger-signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug-treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3)+ T-cell surface glycoprotein CD4 (CD4)+ T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF-mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF-mutant colorectal cancer patients.

Novel Tumor Organoid-Based Mouse Model to Study Image Guided Radiation Therapy of Rectal Cancer After Noninvasive and Precise Endoscopic Implantation.

PURPOSE: Preoperative (neoadjuvant) radiation therapy (RT) is an essential part of multimodal rectal cancer therapy. Recently, total neoadjuvant therapy (TNT), which combines simultaneous radiochemotherapy with additional courses of chemotherapy, has emerged as an effective approach. TNT achieves a pathologic complete remission in approximately 30% of resected patients, opening avenues for treatment strategies that avoid radical organ resection. Furthermore, recent studies have demonstrated that anti-programmed cell death protein 1 immunotherapy can induce clinical complete responses in patients with specific genetic alterations. There is significant potential to enhance outcomes through intensifying, personalizing, and de-escalating treatment approaches. However, the heterogeneous response rates to RT or TNT and strategies to sensitize patients without specific genetic changes to immunotherapy remain poorly understood. METHODS AND MATERIALS: We developed a novel orthotopic mouse model of rectal cancer based on precisely defined endoscopic injections of tumor organoids that reflect tumor heterogeneity. Subsequently, we employed endoscopic- and computed tomography-guided RT and validated rectal tumor growth and response rates to therapy using small-animal magnetic resonance imaging and endoscopic follow-up. RESULTS: Rectal tumor formation was successfully induced in all mice after 2 organoid injections. Clinically relevant RT regimens with 5 × 5 Gy significantly delayed clinical signs of tumor progression and significantly improved survival. Consistent with human disease, rectal tumor progression correlated with the development of liver and lung metastases. Notably, long-term survivors after RT showed no evidence of tumor recurrence, as demonstrated by in vivo radiologic tumor staging and histopathologic examination. CONCLUSIONS: Our novel mouse model combines orthotopic tumor growth via noninvasive and precise rectal organoid injection and small-animal RT. This model holds significant promise for investigating the effect of tumor cell-intrinsic aspects, genetic alterations of the host, and exogenous factors (eg, nutrition or microbiota) on RT outcomes. Furthermore, it allows for the exploration of combination therapies involving chemotherapy, immunotherapy, or novel targeted therapies.

Epigenetic control of pancreatic cancer metastasis.

Surgical resection, when combined with chemotherapy, has been shown to significantly improve the survival rate of patients with pancreatic ductal adenocarcinoma (PDAC). However, this treatment option is only feasible for a fraction of patients, as more than 50% of cases are diagnosed with metastasis. The multifaceted process of metastasis is still not fully understood, but recent data suggest that transcriptional and epigenetic plasticity play significant roles. Interfering with epigenetic reprogramming can potentially control the adaptive processes responsible for metastatic progression and therapy resistance, thereby enhancing treatment responses and preventing recurrence. This review will focus on the relevance of histone-modifying enzymes in pancreatic cancer, specifically on their impact on the metastatic cascade. Additionally, it will also provide a brief update on the current clinical developments in epigenetic therapies.

Phenotype screens of murine pancreatic cancer identify a Tgf-α-Ccl2-paxillin axis driving human-like neural invasion.

Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modeled in genetically engineered mouse models (GEMM) of PDAC. Here, we systematically screened for human-like NI in Europe's largest repository of GEMM of PDAC, comprising 295 different genotypes. This phenotype screen uncovered 2 GEMMs of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming growth factor α (TGF-α) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGF-α upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGF-α-expression constituted strong prognostic factors. Therefore, we believe that the TGF-α-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.

The pharmacoepigenomic landscape of cancer cell lines reveals the epigenetic component of drug sensitivity.

Aberrant DNA methylation accompanies genetic alterations during oncogenesis and tumour homeostasis and contributes to the transcriptional deregulation of key signalling pathways in cancer. Despite increasing efforts in DNA methylation profiling of cancer patients, there is still a lack of epigenetic biomarkers to predict treatment efficacy. To address this, we analyse 721 cancer cell lines across 22 cancer types treated with 453 anti-cancer compounds. We systematically detect the predictive component of DNA methylation in the context of transcriptional and mutational patterns, i.e., in total 19 DNA methylation biomarkers across 17 drugs and five cancer types. DNA methylation constitutes drug sensitivity biomarkers by mediating the expression of proximal genes, thereby enhancing biological signals across multi-omics data modalities. Our method reproduces anticipated associations, and in addition, we find that the NEK9 promoter hypermethylation may confer sensitivity to the NEDD8-activating enzyme (NAE) inhibitor pevonedistat in melanoma through downregulation of NEK9. In summary, we envision that epigenomics will refine existing patient stratification, thus empowering the next generation of precision oncology.

Learning-prolonged maintenance of stimulus information in CA1 and subiculum during trace fear conditioning.

Temporal associative learning binds discontiguous conditional stimuli (CSs) and unconditional stimuli (USs), possibly by maintaining CS information in the hippocampus after its offset. Yet, how learning regulates such maintenance of CS information in hippocampal circuits remains largely unclear. Using the auditory trace fear conditioning (TFC) paradigm, we identify a projection from the CA1 to the subiculum critical for TFC. Deep-brain calcium imaging shows that the peak of trace activity in the CA1 and subiculum is extended toward the US and that the CS representation during the trace period is enhanced during learning. Interestingly, such plasticity is consolidated only in the CA1, not the subiculum, after training. Moreover, CA1 neurons, but not subiculum neurons, increasingly become active during CS-and-trace and shock periods, respectively, and correlate with CS-evoked fear retrieval afterward. These results indicate that learning dynamically enhances stimulus information maintenance in the CA1-subiculum circuit during learning while storing CS and US memories primarily in the CA1 area.

Dual Recombinase-Based Mouse Models Help Decipher Cancer Biology and Targets for Therapy.

The advent of next-generation sequencing (NGS) and single-cell profiling technologies has revealed the complex and heterogenous ecosystem of human tumors under steady-state and therapeutic perturbation. Breakthroughs in the development of genetically engineered mouse models (GEMM) of human cancers that are based on the combination of two site-specific recombinase systems [dual-recombinase system (DRS)] offer fundamental new possibilities to elucidate and understand critical drivers of the diverse tumor phenotypes and validate potential targets for therapy. Here, we discuss opportunities DRS-based cancer GEMMs offer to model, trace, manipulate, and functionally investigate established cancers, their interactions with the host, and their response to therapy.

An integrated cellular and molecular model of gastric neuroendocrine cancer evolution highlights therapeutic targets.

Gastric neuroendocrine carcinomas (G-NEC) are aggressive malignancies with poorly understood biology and a lack of disease models. Here, we use genome sequencing to characterize the genomic landscapes of human G-NEC and its histologic variants. We identify global and subtype-specific alterations and expose hitherto unappreciated gains of MYC family members in a large part of cases. Genetic engineering and lineage tracing in mice delineate a model of G-NEC evolution, which defines MYC as a critical driver and positions the cancer cell of origin to the neuroendocrine compartment. MYC-driven tumors have pronounced metastatic competence and display defined signaling addictions, as revealed by large-scale genetic and pharmacologic screening of cell lines and organoid resources. We create global maps of G-NEC dependencies, highlight critical vulnerabilities, and validate therapeutic targets, including candidates for clinical drug repurposing. Our study gives comprehensive insights into G-NEC biology.

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation.

Cell-selective proteomics is a powerful emerging concept to study heterocellular processes in tissues. However, its high potential to identify non-cell-autonomous disease mechanisms and biomarkers has been hindered by low proteome coverage. Here, we address this limitation and devise a comprehensive azidonorleucine labeling, click chemistry enrichment, and mass spectrometry-based proteomics and secretomics strategy to dissect aberrant signals in pancreatic ductal adenocarcinoma (PDAC). Our in-depth co-culture and in vivo analyses cover more than 10,000 cancer cell-derived proteins and reveal systematic differences between molecular PDAC subtypes. Secreted proteins, such as chemokines and EMT-promoting matrisome proteins, associated with distinct macrophage polarization and tumor stromal composition, differentiate classical and mesenchymal PDAC. Intriguingly, more than 1,600 cancer cell-derived proteins including cytokines and pre-metastatic niche formation-associated factors in mouse serum reflect tumor activity in circulation. Our findings highlight how cell-selective proteomics can accelerate the discovery of diagnostic markers and therapeutic targets in cancer.

Brg1 controls stemness and metastasis of pancreatic cancer through regulating hypoxia pathway.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. We previously reported that chromatin remodeler Brg1 is essential for acinar cell-derived PDAC formation in mice. However, the functional role of Brg1 in established PDAC and its metastasis remains unknown. Here, we investigated the importance of Brg1 for established PDAC by using a mouse model with a dual recombinase system. We discovered that Brg1 was a critical player for the cell survival and growth of spontaneously developed PDAC in mice. In addition, Brg1 was essential for metastasis of PDAC cells by inhibiting apoptosis in splenic injection and peritoneal dissemination models. Moreover, cancer stem-like property was compromised in PDAC cells by Brg1 ablation. Mechanistically, the hypoxia pathway was downregulated in Brg1-deleted mouse PDAC and BRG1-low human PDAC. Brg1 was essential for HIF-1α to bind to its target genes to augment the hypoxia pathway, which was important for PDAC cells to maintain their stem-like properties and to metastasize to the liver. Human PDAC cells with high BRG1 expression were more susceptible to BRG1 suppression. In conclusion, Brg1 plays a critical role for cell survival, stem-like property and metastasis of PDAC through the regulation of hypoxia pathway, and thus could be a novel therapeutic target for PDAC.

spongEffects: ceRNA modules offer patient-specific insights into the miRNA regulatory landscape.

MOTIVATION: Cancer is one of the leading causes of death worldwide. Despite significant improvements in prevention and treatment, mortality remains high for many cancer types. Hence, innovative methods that use molecular data to stratify patients and identify biomarkers are needed. Promising biomarkers can also be inferred from competing endogenous RNA (ceRNA) networks that capture the gene-miRNA gene regulatory landscape. Thus far, the role of these biomarkers could only be studied globally but not in a sample-specific manner. To mitigate this, we introduce spongEffects, a novel method that infers subnetworks (or modules) from ceRNA networks and calculates patient- or sample-specific scores related to their regulatory activity. RESULTS: We show how spongEffects can be used for downstream interpretation and machine learning tasks such as tumor classification and for identifying subtype-specific regulatory interactions. In a concrete example of breast cancer subtype classification, we prioritize modules impacting the biology of the different subtypes. In summary, spongEffects prioritizes ceRNA modules as biomarkers and offers insights into the miRNA regulatory landscape. Notably, these module scores can be inferred from gene expression data alone and can thus be applied to cohorts where miRNA expression information is lacking. AVAILABILITY AND IMPLEMENTATION: https://bioconductor.org/packages/devel/bioc/html/SPONGE.html.