Expression of gemcitabine metabolizing enzymes and stromal components reveal complexities of preclinical pancreatic cancer models for therapeutic testing.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) poorly responds to antineoplastic agents. Discrepancies between preclinical success and clinical failure of compounds has been a continuous challenge and major obstacle in PDAC research. AIM: To investigate the association of the tumor microenvironment (TME) composition and gemcitabine metabolizing enzyme (GME) expression in vitro and several in vivo models. METHODS: mRNA expression and protein levels of GME (cytosolic 5'-nucleotidase 1 A; NT5C1A, cytidine deaminase; CDA, deoxycytidine kinase; DCK), gemcitabine transporters (ENT1, ENT2, RRM1, RRM2) and stromal components (hyaluroninc acid, podoplanin, masson trichrome, picrosirius) were assessed by qRT-PCR and immunohistochemistry in murine LSL-KrasG12D/+;LSL-Trp53R172 H/+; Pdx-1-Cre (KPC), orthotopically transplanted mice (OTM), human primary resected PDAC tissue (hPRT), corresponding patient-derived xenograft (PDX) mice, and KPC-SPARC-/- mice. mRNA expression of GME was analyzed in PDAC cell lines (Panc-1, MIA PaCa, BXPC3 and L3.6) upon incubation on collagen or pancreatic stellate cell (PSC) conditioned media by qRT-PCR. RESULTS: Endogenous KPC tumors exhibited significantly higher levels of GME compared to OTM. However, GME levels did not differ between hPRT and corresponding PDX mice. Using Kendalls Tau correlation coefficient we did not show a significant correlation of GME and components of the TME except for NT5C1A and hyaluronic acid in PDX mice (p=0.029). GME were not significantly altered upon SPARC depletion in vivo, and upon treatment with PSC-conditioned media or incubation on collagen plated dishes in vitro. CONCLUSIONS: Our findings suggest that the expression of GME is independent from the deposition of stromal components. KPC mice are most appropriate to study stromal composition whereas PDX mice maintain GME expression of the corresponding hPRT and could be best suited for pharmacokinetic studies.

A single-cell strategy for the identification of intronic variants related to mis-splicing in pancreatic cancer.

Most clinical diagnostic and genomic research setups focus almost exclusively on coding regions and essential splice sites, thereby overlooking other non-coding variants. As a result, intronic variants that can promote mis-splicing events across a range of diseases, including cancer, are yet to be systematically investigated. Such investigations would require both genomic and transcriptomic data, but there currently exist very few datasets that satisfy these requirements. We address this by developing a single-nucleus full-length RNA-sequencing approach that allows for the detection of potentially pathogenic intronic variants. We exemplify the potency of our approach by applying pancreatic cancer tumor and tumor-derived specimens and linking intronic variants to splicing dysregulation. We specifically find that prominent intron retention and pseudo-exon activation events are shared by the tumors and affect genes encoding key transcriptional regulators. Our work paves the way for the assessment and exploitation of intronic mutations as powerful prognostic markers and potential therapeutic targets in cancer.

An NFATc1/SMAD3/cJUN Complex Restricted to SMAD4-Deficient Pancreatic Cancer Guides Rational Therapies.

BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.

Gut microbiota predicts severity and reveals novel metabolic signatures in acute pancreatitis.

OBJECTIVE: Early disease prediction is challenging in acute pancreatitis (AP). Here, we prospectively investigate whether the microbiome predicts severity of AP (Pancreatitis-Microbiome As Predictor of Severity; P-MAPS) early at hospital admission. DESIGN: Buccal and rectal microbial swabs were collected from 424 patients with AP within 72 hours of hospital admission in 15 European centres. All samples were sequenced by full-length 16S rRNA and metagenomic sequencing using Oxford Nanopore Technologies. Primary endpoint was the association of the orointestinal microbiome with the revised Atlanta classification (RAC). Secondary endpoints were mortality, length of hospital stay and severity (organ failure >48 hours and/or occurrence of pancreatic collections requiring intervention) as post hoc analysis. Multivariate analysis was conducted from normalised microbial and corresponding clinical data to build classifiers for predicting severity. For functional profiling, gene set enrichment analysis (GSEA) was performed and normalised enrichment scores calculated. RESULTS: After data processing, 411 buccal and 391 rectal samples were analysed. The intestinal microbiome significantly differed for the RAC (Bray-Curtis, p value=0.009), mortality (Bray-Curtis, p value 0.006), length of hospital stay (Bray-Curtis, p=0.009) and severity (Bray-Curtis, p value=0.008). A classifier for severity with 16 different species and systemic inflammatory response syndrome achieved an area under the receiving operating characteristic (AUROC) of 85%, a positive predictive value of 67% and a negative predictive value of 94% outperforming established severity scores. GSEA revealed functional pathway units suggesting elevated short-chain fatty acid (SCFA) production in severe AP. CONCLUSIONS: The orointestinal microbiome predicts clinical hallmark features of AP, and SCFAs may be used for future diagnostic and therapeutic concepts. TRIAL REGISTRATION NUMBER: NCT04777812.

Time to Deliver on Promises: The Role of ERBB2 Alterations as Treatment Options for Colorectal Cancer Patients in the Era of Precision Oncology.

Receptor tyrosine kinase erythroblastic oncogene B2 (ERBB2), also known as human epidermal growth factor receptor 2 (HER2), represents an oncogenic driver and has been effectively targeted in breast and gastric cancer. Recently, next-generation sequencing (NGS) discovered ERBB2 as a promising therapeutic target in metastatic colorectal cancer (mCRC), where it is altered in 3-5% of patients, but no therapies are currently approved for this use. Herein, we present the experience of a single center in diagnosing actionable genetic ERBB2 alterations using NGS and utilizing the latest therapeutic options. Between October 2019 and December 2022, a total of 107 patients with advanced CRC underwent molecular analysis, revealing actionable ERBB2 mutations in two patients and ERBB2 amplifications in two other patients. These findings correlated with immunohistochemical (IHC) staining. Of these four patients, two were treated with trastuzumab-deruxtecan (T-DXd). We present two exemplary cases of patients with actionable ERBB2 alterations to demonstrate the effectiveness of T-DXd in heavily pretreated ERBB2-positive mCRC patients and the need for early molecular profiling. To fully exploit the potential of this promising treatment, earlier molecular profiling and the initiation of targeted therapies are essential.

Visualization of deglutition and gastroesophageal reflux using real-time MRI: a standardized approach to image acquisition and assessment.

This study aims to develop a standardized algorithm for gastroesophageal image acquisition and diagnostic assessment using real-time MRI. Patients with GERD symptoms undergoing real-time MRI of the esophagus and esophagogastric junction between 2015 and 2018 were included. A 10 ml bolus of pineapple juice served as an oral contrast agent. Patients performed Valsalva maneuver to provoke reflux and hiatal hernia. Systematic MRI assessment included visual presence of achalasia, fundoplication failure in patients with previous surgical fundoplication, gastroesophageal reflux, and hiatal hernia. A total of 184 patients (n = 92 female [50%], mean age 52.7 ± 15.8 years) completed MRI studies without adverse events at a mean examination time of 15 min. Gastroesophageal reflux was evident in n = 117 (63.6%), hiatal hernia in n = 95 (52.5%), and achalasia in 4 patients (2.2%). Hiatal hernia was observed more frequently in patients with reflux at rest (n = 67 vs. n = 6, p < 0.01) and during Valsalva maneuver (n = 87 vs. n = 8, p < 0.01). Real-time MRI visualized a morphologic correlate for recurring GERD symptoms in 20/22 patients (90%) after fundoplication procedure. In a large-scale single-center cohort of patients with GERD symptoms undergoing real-time MRI, visual correlates for clinical symptoms were evident in most cases. The proposed assessment algorithm could aid in wider-spread utilization of real-time MRI and provides a comprehensive approach to this novel imaging modality.

Endoscopic mucosal resection and endoscopic submucosal dissection with an external additional working channel (EMR+ and ESD+) are equivalent to using a double-channel endoscope: a systematic evaluation in a porcine ex vivo model.

BACKGROUND AND AIMS: With an external additional working channel (AWC) endoscopic mucosal resection (EMR) as well as endoscopic submucosal dissection (ESD) can be extended to techniques termed "EMR+" and "ESD+." These novel techniques are systematically compared to EMR and ESD under the use of a double-channel endoscope (DC). METHODS: Our trial was conducted prospectively in a pre-clinical porcine animal model (EASIE-R simulator) with standardized gastric lesions measuring 3 or 4 cm. RESULTS: EMR+ and EMR DC showed both good results for 3 cm lesions with no adverse events and an en bloc resection rate of 73.33% (EMR+) and 60.00% (EMR DC, p = 0.70). They came to their limits in 4 cm lesions with muscularis damages of 20.00% (EMR+), 13.33% (EMR DC, p ≥ 0.99) and decreasing en bloc resection rates of 60.00% (EMR+) and 46.67% (EMR DC, p = 0.72). ESD+ and ESD DC were both reliable concerning en bloc resection rates (100% in all groups) and adverse events (0.00% in 3 cm lesions, 12.50% muscularis damages in both ESD+ and ESD DC in 4 cm lesions). Resection time was slightly shorter in all groups with the AWC compared to DC although only reaching significance in 3 cm ESD lesions (p < 0.05*). CONCLUSIONS: With the AWC, a standard endoscope can easily be transformed to double-channel functionality. We could show that EMR+ and ESD+ are non-inferior to EMR and ESD under the use of a double-channel endoscope. Consequently, the AWC presents an affordable alternative to a double-channel endoscope for both EMR and ESD.

Dynamics of intestinal and intratumoral microbiome signatures in genetically engineered mice and human pancreatic ductal adenocarcinoma.

BACKGROUND: Emerging evidence has recently revealed a prominent role of the microbiome in pancreatic ductal adenocarcinoma (PDAC). However, while most observations were made in patients, mouse models still require a precise characterization of their disease-related microbiome to employ them for mechanistic and interventional preclinical studies. METHODS: To investigate the fecal and tumoral microbiome of LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) and control (CTRL) mice, Oxford Nanopore sequencing was applied. Feces were collected from 10 KPC mice and 10 CTRLs at 3 timepoints (6 weeks, 12 weeks, and when tumor-bearing (KPC) or 6 months (CTRL), respectively). Metagenomic sequencing was performed on feces DNA. KPC tumor and healthy pancreas DNA samples were subjected to 16S rRNA gene sequencing. Bacterial marker components were detected in KPC tumor tissue over time by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). RESULTS: Murine fecal samples showed a significantly different microbiome compared to age-matched healthy CTRLs regarding beta diversity (p = 0.001, R2 = 0.2-0.25 for Bray-Curtis). Adjusted human PDAC classifiers predicted disease status from feces of KPC mice achieving area under the receiver operating characteristic (AUROC) values of 80%. Furthermore, KPC tumors harbored significantly more bacterial components than healthy pancreas. Also the microbial composition differs significantly between KPC tumors and healthy pancreas tissue (p = 0.042 for Bray-Curtis). Microbiota found highly abundant in human PDAC samples were considerably more abundant in KPC tumors as compared to healthy pancreas samples (p-value <0.001). CONCLUSION: KPC fecal samples show similarities with the microbial composition of stool samples from human PDAC patients.

Optimizing the structure of interdisciplinary tumor boards for effective cancer care.

Introduction: Multi-professional interdisciplinary tumor boards (ITB) are essential institutions to discuss all newly diagnosed, relapsed or complex cancer patients in a team of specialists to find an optimal cancer care plan for each individual patient with regard to national and international clinical practice guidelines, patient´s preference and comorbidities. In a high-volume cancer center, entity-specific ITBs take place at least once a week discussing a large number of patients. To a high level of expertise and dedication, this also requires an enormous amount of time for physicians, cancer specialists and administrative support colleagues, especially for radiologists, pathologists, medical oncologists and radiation oncologists, who must attend all cancer-specific boards according to certification requirements. Methods: In this 15-month prospective German single-center analysis, we examined the established structures of 12 different cancer-specific ITBs at the certified Oncology Center and demonstrate tools helping to optimize processes before, during and after the boards for optimal, time-saving procedures. Results: By changing pathways, introducing revised registration protocols and new digital supports we could show that the workload of preparation by radiologists and pathologists could be reduced significantly by 22.9% (p=<0.0001) and 52.7% (p=<0.0001), respectively. Furthermore, two questions were added to all registration forms about the patient´s need for specialized palliative care support that should lead to more awareness and early integration of specialized help. Discussion: There are several ways to reduce the workload of all ITB team members while maintaining high quality recommendations and adherence to national and international guidelines.

Urokinase-Type Plasminogen Activator Receptor (uPAR) Cooperates with Mutated KRAS in Regulating Cellular Plasticity and Gemcitabine Response in Pancreatic Adenocarcinomas.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Given the currently limited therapeutic options, the definition of molecular subgroups with the development of tailored therapies remains the most promising strategy. Patients with high-level gene amplification of urokinase plasminogen activator receptor (uPAR/PLAUR) have an inferior prognosis. We analyzed the uPAR function in PDAC to understand this understudied PDAC subgroup's biology better. METHODS: A total of 67 PDAC samples with clinical follow-up and TCGA gene expression data from 316 patients were used for prognostic correlations. Gene silencing by CRISPR/Cas9, as well as transfection of uPAR and mutated KRAS, were used in PDAC cell lines (AsPC-1, PANC-1, BxPC3) treated with gemcitabine to study the impact of these two molecules on cellular function and chemoresponse. HNF1A and KRT81 were surrogate markers for the exocrine-like and quasi-mesenchymal subgroup of PDAC, respectively. RESULTS: High levels of uPAR were correlated with significantly shorter survival in PDAC, especially in the subgroup of HNF1A-positive exocrine-like tumors. uPAR knockout by CRISPR/Cas9 resulted in activation of FAK, CDC42, and p38, upregulation of epithelial makers, decreased cell growth and motility, and resistance against gemcitabine that could be reversed by re-expression of uPAR. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly, and transfection of mutated KRAS in BxPC-3 cells rendered the cell more mesenchymal and increased sensitivity towards gemcitabine. CONCLUSIONS: Activation of uPAR is a potent negative prognostic factor in PDAC. uPAR and KRAS cooperate in switching the tumor from a dormant epithelial to an active mesenchymal state, which likely explains the poor prognosis of PDAC with high uPAR. At the same time, the active mesenchymal state is more vulnerable to gemcitabine. Strategies targeting either KRAS or uPAR should consider this potential tumor-escape mechanism.

NFATc1 Is a Central Mediator of EGFR-Induced ARID1A Chromatin Dissociation During Acinar Cell Reprogramming.

BACKGROUND & AIMS: Loss of AT-rich interactive domain-containing protein 1A (ARID1A) fosters acinar-to-ductal metaplasia (ADM) and pancreatic carcinogenesis by down-regulating transcription programs controlling acinar cell identity. However, how ARID1A reacts to metaplasia-triggering environmental cues remains elusive. Here, we aimed to elucidate the role of ARID1A in controlling ductal pancreatic gene signatures and deciphering hierarchical signaling cues determining ARID1A-dependent chromatin regulation during acinar cell reprogramming. METHODS: Acinar cell explants with differential ARID1A status were subjected to genome-wide expression analyses. The impact of epidermal growth factor receptor (EGFR) signaling, NFATc1 activity, and ARID1A status on acinar reprogramming processes were characterized by ex vivo ADM assays and transgenic mouse models. EGFR-dependent ARID1A chromatin binding was studied by chromatin immunoprecipitation sequencing analysis and cellular fractionation. RESULTS: EGFR signaling interferes with ARID1A-dependent transcription by inducing genome-wide ARID1A displacement, thereby phenocopying ARID1A loss-of-function mutations and inducing a shift toward ADM permissive ductal transcription programs. Moreover, we show that EGFR signaling is required to push ARID1A-deficient acinar cells toward a metaplastic phenotype. Mechanistically, we identified the transcription factor nuclear factor of activated T cells 1 (NFATc1) as the central regulatory hub mediating both EGFR signaling-induced genomic ARID1A displacement and the induction of ADM-promoting gene signatures in the absence of ARID1A. Consequently, pharmacologic inhibition of NFATc1 or its depletion in transgenic mice not only preserves genome-wide ARID1A occupancy, but also attenuates acinar metaplasia led by ARID1A loss. CONCLUSIONS: Our data describe an intimate relationship between environmental signaling and chromatin remodeling in orchestrating cell fate decisions in the pancreas, and illustrate how ARID1A loss influences transcriptional regulation in acinar cell reprogramming.

[Acute necrotizing pancreatitis with hemorrhagic shock in secondary splenic rupture: a case report and literature review]. / Akute nekrotisierende Pankreatitis mit hämorrhagischem Schock bei sekundärer Milzruptur: Ein Fallbericht und Literaturübersicht.

Atraumatic splenic rupture is a rare complication of acute and chronic pancreatitis. It arises due to its anatomical proximity to the pancreas, for instance, due to erosion of large pseudocysts or walled-of-necrosis (WON).Following we describe the case of a 62-year-old woman who presented for further diagnostics and treatment of acute pancreatitis with the development of large walled-of necrosis (WON) in the pancreatic corpus and tail. During the course, the patient developed a hemorrhagic shock. An emergency computer tomography (CT) of the abdomen revealed a ruptured spleen with a large capsular hematoma with no evidence of active bleeding. In contrast to previous published case reports, our treatment was exclusively minimal-invasive: by radiological guided embolization of the splenic artery and by endosonographic guided implantation of a lumen apposing metal stent (LAMS). The splenic hematoma was spontaneously regressive without secondary drainage.

Comparison between traumatic and atraumatic over-the-scope clips in patients with duodenal ulcer bleeding: a retrospective analysis with propensity score-based matching.

BACKGROUND AND AIMS: Over-the-scope clips (OTSCs) substantially improved the endoscopic armamentarium for the treatment of severe GI bleeding and can potentially overcome limitations of standard clips. Data indicate a superiority of OTSCs in hemostasis as first- and second-line therapy. However, the impact of the OTSC designs, in particular the traumatic (-t) or atraumatic (-a) type, in duodenal ulcer bleeding has not been analyzed so far. METHODS: This was a retrospective analysis of a prospective collected database from 2009 to 2020 of 6 German endoscopic centers. All patients who underwent emergency endoscopy and were treated using an OTSC for duodenal ulcer bleeding were included. OTSC-t and OTSC-a patients were compared by the Fisher exact test, χ2 test, or Mann-Whitney U test as appropriate. A propensity score-based 1:1 matching was performed to obtain equal distribution of baseline characteristics in both groups. RESULTS: The entire cohort comprised 173 patients (93 OTSC-a, 80 OTSC-t). Age, gender, anticoagulant therapy, Rockall score, and treatment regimen had similar distributions in the 2 groups. However, the OTSC-t group showed significantly more active bleeding ulcers (Forrest Ia/b). Matching identified 132 patients (66 in both groups) with comparable baseline characteristics. Initial bleeding hemostasis (OTSC-a, 90.9%; OTSC-t, 87.9%; P = .82) and 72-hour mortality (OTSC-a, 4.5%; OTSC-t, 6.0%; P > .99) were not significantly different, but the OTSC-t group revealed a clearly higher rate of recurrent bleeding (34.9% vs 7.6%, P < .001) and necessity of red blood cell transfusions (5.1 ± 3.4 vs 2.5 ± 2.4 concentrates, P < .001). CONCLUSIONS: For OTSC use, the OTSC-a should be the preferred option for duodenal ulcer bleeding.

[Metastasis or sarcoidosis - a diagnostic pitfall in metastatic rectal carcinoma]. / Metastase oder Sarkoidose ­ eine diagnostische Herausforderung beim metastasierten Rektumkarzinom.

BACKGROUND: Granulomatous diseases as sarcoidosis can impair the staging of metastatic diseases since metastasis are hard to distinguish from granulomas in standard imaging. This case report describes the diagnostic workup and therapy in a patient with simultaneous sarcoidosis and rectal cancer with hepatic metastasis and how a curative stadium was achieved. CASE DESCRIPTION: A 71-year old male patient was diagnosed with an adenocarcinoma of the rectum after presenting with involuntary weight loss and anemia. Further diagnostics raised strong suspicion of hepatic, pulmonary and splenic metastasis. Histology after bronchoscopy surprisingly discovered non-caseating granulomas, leading to the diagnosis of sarcoidosis. Due to an obstructive tumor, a rectum resection was performed. Due to a high suspicion of splenic metastasis during surgery, the spleen was removed. Histology revealed no metastasis in the spleen but multiple granulomas due to sarcoidosis. After surgery, biopsy of a suspicious lesion in the liver revealed both metastasis and sarcoidosis in the same sample. The patient was treated with a pseudo(neo)adjuvant chemotherapy with 5-Fluorouracil, Leukovorin, Oxaliplatin (FOLFOX) and Panitumumab (Anti-EGF-antibody). After treatment, CT scan revealed two hepatic lesions decreasing in size, while all other lesions were metrically stable. A right hemihepatectomy followed and histology revealed both sarcoidosis and metastasis. The curated patient was sent to aftercare and there is no suspicion for a relapse (13 month after last surgery). DISCUSSION: The simultaneous appearance of metastatic tumors and sarcoidosis creates a diagnostic dilemma since both manifestations can barely be distinguished in regular imaging technologies. This case report demonstrates that the histological work-up of affected organs with consecutive resections can cure a patient of a metastatic tumor disease, even in the context of simultaneous sarcoidosis.

MCU controls melanoma progression through a redox-controlled phenotype switch.

Melanoma is the deadliest of skin cancers and has a high tendency to metastasize to distant organs. Calcium and metabolic signals contribute to melanoma invasiveness; however, the underlying molecular details are elusive. The MCU complex is a major route for calcium into the mitochondrial matrix but whether MCU affects melanoma pathobiology was not understood. Here, we show that MCUA expression correlates with melanoma patient survival and is decreased in BRAF kinase inhibitor-resistant melanomas. Knockdown (KD) of MCUA suppresses melanoma cell growth and stimulates migration and invasion. In melanoma xenografts, MCUA_KD reduces tumor volumes but promotes lung metastases. Proteomic analyses and protein microarrays identify pathways that link MCUA and melanoma cell phenotype and suggest a major role for redox regulation. Antioxidants enhance melanoma cell migration, while prooxidants diminish the MCUA_KD -induced invasive phenotype. Furthermore, MCUA_KD increases melanoma cell resistance to immunotherapies and ferroptosis. Collectively, we demonstrate that MCUA controls melanoma aggressive behavior and therapeutic sensitivity. Manipulations of mitochondrial calcium and redox homeostasis, in combination with current therapies, should be considered in treating advanced melanoma.

Axon guidance receptor ROBO3 modulates subtype identity and prognosis via AXL-associated inflammatory network in pancreatic cancer.

Metastatic pancreatic cancer (PDAC) has a poor clinical outcome with a 5-year survival rate below 3%. Recent transcriptome profiling of PDAC biopsies has identified 2 clinically distinct subtypes - the "basal-like" (BL) subtype with poor prognosis and therapy resistance compared with the less aggressive and drug-susceptible "classical" (CLA) subtype. However, the mechanistic events and environmental factors that promote the BL subtype identity are not very clear. Using preclinical models, patient-derived xenografts, and FACS-sorted PDAC patient biopsies, we report here that the axon guidance receptor, roundabout guidance receptor 3 (ROBO3), promotes the BL metastatic program via a potentially unique AXL/IL-6/phosphorylated STAT3 (p-STAT3) regulatory axis. RNA-Seq identified a ROBO3-mediated BL-specific gene program, while tyrosine kinase profiling revealed AXL as the key mediator of the p-STAT3 activation. CRISPR/dCas9-based ROBO3 silencing disrupted the AXL/p-STAT3 signaling axis, thereby halting metastasis and enhancing therapy sensitivity. Transcriptome analysis of resected patient tumors revealed that AXLhi neoplastic cells associated with the inflammatory stromal program. Combining AXL inhibitor and chemotherapy substantially restored a CLA phenotypic state and reduced disease aggressiveness. Thus, we conclude that a ROBO3-driven hierarchical network determines the inflammatory and prometastatic programs in a specific PDAC subtype.

TP53-Status-Dependent Oncogenic EZH2 Activity in Pancreatic Cancer.

Pancreatic Ductal Adenocarcinoma (PDAC) represents a lethal malignancy with a consistently poor outcome. Besides mutations in PDAC driver genes, the aggressive tumor biology of the disease and its remarkable therapy resistance are predominantly installed by potentially reversible epigenetic dysregulation. However, epigenetic regulators act in a context-dependent manner with opposing implication on tumor progression, thus critically determining the therapeutic efficacy of epigenetic targeting. Herein, we aimed at exploring the molecular prerequisites and underlying mechanisms of oncogenic Enhancer of Zeste Homolog 2 (EZH2) activity in PDAC progression. Preclinical studies in EZH2 proficient and deficient transgenic and orthotopic in vivo PDAC models and transcriptome analysis identified the TP53 status as a pivotal context-defining molecular cue determining oncogenic EZH2 activity in PDAC. Importantly, the induction of pro-apoptotic gene signatures and processes as well as a favorable PDAC prognosis upon EZH2 depletion were restricted to p53 wildtype (wt) PDAC subtypes. Mechanistically, we illustrate that EZH2 blockade de-represses CDKN2A transcription for the subsequent posttranslational stabilization of p53wt expression and function. Together, our findings suggest an intact CDKN2A-p53wt axis as a prerequisite for the anti-tumorigenic consequences of EZH2 depletion and emphasize the significance of molecular stratification for the successful implementation of epigenetic targeting in PDAC.