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Tumour growth upon ATRi and Gemcitabine Treatment in Allografts model

Tumour growth upon Olaparib and Gemcitabine Treatment in Allografts model

Pancreatic ductal adenocarcinomas (PDAC) harbor recurrent functional mutations of the master DNA damage response kinase ATM, which has been shown to accelerate tumorigenesis and epithelial–mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements, and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Cancer Res; 77(20); 5576–90. ©2017 AACR.

Description of the genomic alterations in KC and AKC PDAC cell lines

GSEA Analysis

Tumour growth upon Olaparib and Gemcitabine Treatment in Allografts model

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with high potential of metastases and therapeutic resistance. Although genetic mutations drive PDAC initiation, they alone do not explain its aggressive nature. Epigenetic mechanisms, including aberrant DNA methylation and histone modifications, significantly contribute to inter- and intratumoral heterogeneity, disease progression and metastasis. Thus, increased understanding of the epigenetic landscape in PDAC could offer new potential biomarkers and tailored therapeutic approaches. In this review, we shed light on the role of epigenetic modifications in PDAC biology and on the potential clinical applications of epigenetic biomarkers in liquid biopsy. In addition, we provide an overview of clinical trials assessing epigenetically targeted treatments alone or in combination with other anticancer therapies to improve outcomes of patients with PDAC.

Background Minimally invasive surgery is a field of rapid development. Evidence from randomized controlled trials in visceral surgery however still falls short of attesting unequivocal superiority to laparoscopic procedures over conventional open approaches with regard to postoperative outcome. The aim of this study was to explore the perioperative immune status of patients undergoing hybrid minimally invasive or conventional open pancreatoduodenectomy in a prospective cohort study. Material and methods Subtyping, quantification and functional analysis of circulating immune cells and determination of cytokine-levels in blood samples from patients receiving either hybrid minimally invasive (laPD) or conventional open pancreatoduodenectomy (oPD) was performed. Samples were taken from 29 patients (laPD: n = 14, oPD: n = 15) prior, during and up to six weeks after surgery. Cells were analyzed by flow cytometry, cytokines/chemokines were measured by proximity extension and enzyme-linked immunoassays. Results Open surgery induced higher levels of circulating inflammatory CD14++CD16+ intermediate monocytes. In contrast, hybrid minimally invasive resection was accompanied by increased numbers of circulating regulatory CD4+CD25+CD127low T-cells and by a reduced response of peripheral blood CD3+CD4+ T-cell populations to superantigen stimulation. Yet, rates of postoperative morbidity and infectious complications were similar. Conclusions In summary, the results of this exploratory study may suggest a more balanced postoperative inflammatory response and a better-preserved immune regulation after hybrid minimally invasive pancreatoduodenectomy when compared to open surgery. Whether these results may translate to or be harnessed for improved patient outcome needs to be determined by future studies including larger cohorts and fully laparoscopic or robotic procedures.

Hyperactivation of the RAS-RAF-MEK-ERK cascade - a mitogen-activated protein kinase pathway – has a well-known association with oncogenesis of leading tumor entities, including non-small cell lung cancer, colorectal carcinoma, pancreatic ductal adenocarcinoma, and malignant melanoma. Increasing evidence shows that genetic alterations leading to RAS-RAF-MEK-ERK pathway hyperactivation mediate contact- and soluble-dependent crosstalk between tumor, tumor microenvironment (TME) and the immune system resulting in immune escape mechanisms and establishment of a tumor-sustaining environment. Consequently, pharmacological interruption of this pathway not only leads to tumor-cell intrinsic disruptive effects but also modification of the TME and anti-tumor immunomodulation. At the same time, the importance of ERK signaling in immune cell physiology and potentiation of anti-tumor immune responses through ERK signaling inhibition within immune cell subsets has received growing appreciation. Specifically, a strong case was made for targeted MEK inhibition due to promising associated immune cell intrinsic modulatory effects. However, the successful transition of therapeutic agents interrupting RAS-RAF-MEK-ERK hyperactivation is still being hampered by significant limitations regarding durable efficacy, therapy resistance and toxicity. We here collate and summarize the multifaceted role of RAS-RAF-MEK-ERK signaling in physiology and oncoimmunology and outline the rationale and concepts for exploitation of immunomodulatory properties of RAS-RAF-MEK-ERK inhibition while accentuating the role of MEK inhibition in combinatorial and intermittent anticancer therapy. Furthermore, we point out the extensive scientific efforts dedicated to overcoming the challenges encountered during the clinical transition of various therapeutic agents in the search for the most effective and safe patient- and tumor-tailored treatment approach.

Introduction Pancreatic trauma (PT) involving the main pancreatic duct is rare, but represents a challenging clinical problem with relevant morbidity and mortality. It is generally classified according to the American Association for the Surgery of Trauma (AAST) and often presents as concomitant injury in blunt or penetrating abdominal trauma. Diagnosis may be delayed because of a lack of clinical or radiological manifestation. Treatment options for main pancreatic duct injuries comprise highly complex surgical procedures. Patients and Methods We retrospectively analyzed clinical data from 12 patients who underwent surgery in two tertiary centers in Germany during 2003–2016 for grade III-V PT with affection of the main pancreatic duct, according to the AAST classification. Results The median age was 23 (range: 7-44) years. In nine patients blunt abdominal trauma was the reason for PT, whereas penetrating trauma only occurred in three patients. MRI outperformed classical trauma CT imaging with regard to detection of duct involvement. Complex procedures as i.e. an emergency pancreatic head resection, distal pancreatectomy or parenchyma sparing pancreatogastrostomy were performed. Compared to elective pancreatic surgery the complication rate in the emergency setting was higher. Yet, parenchyma-sparing procedures demonstrated safety. Conclusions Often extension of diagnostics including MRI and/or ERP at an early stage is necessary to guide clinical decision-making. If, due to main duct injuries, surgical therapy for PT is required, we suggest consideration of an organ preservative pancreatogastrostomy in grade III/IV trauma of the pancreatic body or tail.

Mutant KRAS is present in over 90% of pancreatic as well as 30-40% of lung and colorectal cancers and is one of the most common oncogenic drivers. Despite decades of research and the recent emergence of isoform-specific KRASG12C-inhibitors, most mutant KRAS isoforms, including the ones frequently associated with pancreatic ductal adenocarcinoma (PDAC), cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive mechanisms leading to tumor recurrence or resistance. We report here on the combined inhibition of SHP2, a non-receptor tyrosine phosphatase upstream of KRAS, and ERK, a serine/threonine kinase and a key molecule downstream of KRAS in PDAC. This combination shows synergistic anticancer activity in vitro, superior disruption of the MAPK pathway, and significantly increased apoptosis induction compared to single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination. Concurrent inhibition of SHP2 and ERK induces significant tumor regression in multiple PDAC mouse models. Finally, we show evidence that 18F-FDG PET scans can be used to detect and predict early drug responses in animal models. Based on these compelling results, we will investigate this drug combination in a clinical trial (SHERPA, SHP2 and ERK inhibition in pancreatic cancer, NCT04916236), enrolling patients with KRAS-mutant PDAC.

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity in vitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC., This work was funded by the American Association for Cancer Research, Lustgarten Foundation, and Stand Up to Cancer as a Pancreatic Cancer Collective New Therapies Challenge grant (grant no. SU2C-AACR-PCC-01-18).

Immune checkpoint molecules represent physiological brakes of the immune system that are essential for the maintenance of immune homeostasis and prevention of autoimmunity. By inhibiting these negative regulators of the immune response, immune checkpoint blockade can increase anti-tumor immunity, but has been primarily successful in solid cancer therapy and Hodgkin lymphoma so far. Allogeneic hematopoietic cell transplantation (allo-HCT) is a well-established cellular immunotherapy option with the potential to cure hematological cancers, but relapse remains a major obstacle. Relapse after allo-HCT is mainly thought to be attributable to loss of the graft-versus-leukemia (GVL) effect and hence escape of tumor cells from the allogeneic immune response. One potential mechanism of immune escape from the GVL effect is the inhibition of allogeneic T cells via engagement of inhibitory receptors on their surface including PD-1, CTLA-4, TIM3, and others. This review provides an overview of current evidence for a role of immune checkpoint molecules for relapse and its treatment after allo-HCT, as well as discussion of the immune mediated side effect graft-vs.-host disease. We discuss the expression of different immune checkpoint molecules on leukemia cells and T cells in patients undergoing allo-HCT. Furthermore, we review mechanistic insights gained from preclinical studies and summarize clinical trials assessing immune checkpoint blockade for relapse after allo-HCT.

Pancreatic ductal adenocarcinomas (PDAC) harbor recurrent functional mutations of the master DNA damage response kinase ATM, which has been shown to accelerate tumorigenesis and epithelial–mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements, and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Cancer Res; 77(20); 5576–90. ©2017 AACR.

Carcinosarcomas are rare biphasic neoplasms with distinct malignant epithelial and mesenchymal components. Most commonly, carcinosarcomas arise in the uterus as malignant mixed müllerian tumors, but also infrequently appear in other organs such as the ovaries and breast, the prostate and urinary tract, the lungs, or in the gastrointestinal system, among others. Pancreatic carcinosarcomas are exceedingly rare; only a few cases are reported in the English literature. Their pathogenesis remains to be fully clarified. We present here the case of a pancreatic carcinosarcoma with evidence for monoclonality via determination of Kras mutational status after microdissection and suggest a common origin of the 2 tumor components. Comprehensive review of the available literature allows the conclusion that most pancreatic carcinosarcomas appear to be of monoclonal origin and seem to have arisen from a carcinoma via metaplastic transformation of 1 part or subclone of the tumor, probably by epithelial-mesenchymal transition. All reported patients were treated with surgery. Adjuvant therapy, if administered, consisted predominantly of gemcitabine. Prognosis for this neoplasm occurs to be similar or even worse compared with classic pancreatic ductal adenocarcinoma. Despite the lack of evidence-based recommendations for its treatment, resection should be performed, if possible.

Regulation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)/Rel transcription factors (TFs) is extremely cell-type-specific owing to their ability to act disparately in the context of cellular homeostasis driven by cellular fate and the microenvironment. This is also valid for tumor cells in which every single component shows heterogenic effects. Whereas many studies highlighted a per se oncogenic function for NF-κB/Rel TFs across cancers, recent advances in the field revealed their additional tumor-suppressive nature. Specifically, pancreatic ductal adenocarcinoma (PDAC), as one of the deadliest malignant diseases, shows aberrant canonical-noncanonical NF-κB signaling activity. Although decades of work suggest a prominent oncogenic activity of NF-κB signaling in PDAC, emerging evidence points to the opposite including anti-tumor effects. Considering the dual nature of NF-κB signaling and how it is closely linked to many other cancer related signaling pathways, it is essential to dissect the roles of individual Rel TFs in pancreatic carcinogenesis and tumor persistency and progression. Here, we discuss recent knowledge highlighting the role of Rel TFs RelA, RelB, and c-Rel in PDAC development and maintenance. Next to providing rationales for therapeutically harnessing Rel TF function in PDAC, we compile strategies currently in (pre-)clinical evaluation.

[Background & Aims]: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. [Methods]: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. [Results]: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. [Conclusions]: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches., This work was supported by the Deutsche Forschungsgemeinschaft (grants AL 1174/4-1, AL1174/4-2, and Collaborative Research Center 1321 “Modeling and Targeting Pancreatic Cancer” to Hana Algül; SFB824 Z2 to Katja Steiger), the Deutsche Krebshilfe (grant 111646 to Hana Algül), a Ramon y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain (to Bruno Sainz Jr), a Coordinated Grant from Fundación Asociación Española Contra el Cáncer (GC16173694BARB to Bruno Sainz Jr), funding from The Fero Foundation (to Bruno Sainz Jr), and a Proyecto de Investigacion de Salud, ISCIII, Spain (no. PI18/00757 to Bruno Sainz Jr). Jiaoyu Ai is supported by the “China Scholarship Council” grant program.

Pancreatic ductal adenocarcinoma is a devastating malignancy, which is the result of late diagnosis, aggressive disease, and a lack of effective treatment options. Thus, pancreatic ductal adenocarcinoma is projected to become the second leading cause of cancer-related death by 2030. This review summarizes recent developments of oncological therapy in the palliative setting of metastatic pancreatic ductal adenocarcinoma. It further compiles novel targets and therapeutic approaches as well as promising treatment combinations, which are presently in preclinical evaluation, covering several aspects of the hallmarks of cancer. Finally, challenges to the implementation of an individualized therapy approach in the context of precision medicine are discussed.

The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors1. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signaling have established SHP2 as an essential player in oncogenic signaling pathways1-7. Recently, a novel potent allosteric SHP2 inhibitor was presented as a viable therapeutic option for receptor tyrosine kinase-driven cancers, but was shown to be ineffective in KRAS-mutant tumor cell lines in vitro8. Here, we report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors. Genetic deletion of Ptpn11 profoundly inhibited tumor development in mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumors delayed tumor progression but was not sufficient to achieve tumor regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumor growth control in murine and human patient-derived organoids and xenograft models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. Our data indicate the clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS-mutant cancers., This work was supported by grants from Deutsche Forschungsgemeinschaft (DFG AL1174/5-1 to H.A. and LE3222/1-1 to M.L.), Deutsche Krebshilfe (no. 111646 and no. 111464 to H.A.; Max Eder Program no. 111273 to M.R.), the Wilhelm Sander Stiftung (2014.052.1 to H.A.) and the Fundación Asociación Española Contra el Cáncer (to B.S.).

[Background and Aims]: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. [Methods]: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/–;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. [Results]: A5+/–;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/–;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/–;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/–;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. [Conclusions]: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells., This study was supported in part by the Mildred-Scheel-Professur der Deutschen Krebshilfe 111464, DFG AL 1174/6-1 to H.A., DFG DI 2299/1-1 to K.N.D., DFG SFB1321 (S01) to K.S. and W.W., and the German Federal Ministry of Education and Research to the German Center for Diabetes Research (DZD e.V.) to J.A.

Introduction The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signalling have established SHP2 as an essential player in oncogenic signalling pathways. Recently, novel potent allosteric SHP2-inhibitors have been presented as a viable therapeutic option for RTK-driven cancers, but were shown to be ineffective in KRAS mutant tumour cell lines in vitro. Material and methods Various mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) were employed to investigate the contribution of Ptpn11/SHP2 to carcinogenesis and tumour maintenance, and to determine its utility as a therapeutic target for treatment of established tumours in vivo. CRISPR/Cas9 mediated knockout of PTPN11 in human PDAC cell lines, drug-synergy screening and human PDAC organoid and tissue xenograft model therapeutic trials substantiated the rationale of dual SHP2/MEK inhibition in a human context. Results and discussions We report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumours. Genetic deletion of Ptpn11 profoundly inhibited tumour development in mutant KRAS-driven murine models PDAC and NSCLC. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumours delayed tumour progression but was not sufficient to achieve tumour regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumour growth control in murine and human patient-derived organoids and xenograft models of PDAC and NSCLC. Conclusion Our data suggest clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS mutant cancers.

Background Histone deacetylases (HDAC) catalyze N-terminal deacetylation of lysine-residues on histones and multiple nuclear and cytoplasmic proteins. In various animal models, such as trauma/hemorrhagic shock, ischemic stroke or myocardial infarction, HDAC inhibitor (HDACi) application is cyto- and organoprotective and promotes survival. HDACi reduce stress signaling, cell death and inflammation. Hepatic ischemia-reperfusion (I/R) injury during major liver resection or transplantation increases morbidity and mortality. Assuming protective properties, the aim of this study was to investigate the effect of the HDACi VPA and SAHA on warm hepatic I/R. Material and Methods Male Wistar-Kyoto rats (age: 6–8 weeks) were randomized to VPA, SAHA, vehicle control (pre-) treatment or sham-groups and underwent partial no-flow liver ischemia for 90 minutes with subsequent reperfusion for 6, 12, 24 and 60 hours. Injury and regeneration was quantified by serum AST and ALT levels, by macroscopic aspect and (immuno-) histology. HDACi treatment efficiency, impact on MAPK/SAPK-activation and Hippo-YAP signaling was determined by Western blot. Results Treatment with HDACi significantly enhanced hyperacetylation of Histone H3-K9 during I/R, indicative of adequate treatment efficiency. Liver injury, as measured by macroscopic aspect, serum transaminases and histology, was delayed, but not alleviated in VPA and SAHA treated animals. Importantly, tissue destruction was significantly more pronounced with VPA. SAPK-activation (p38 and JNK) was reduced by VPA and SAHA in the early (6h) reperfusion phase, but augmented later on (JNK, 24h). Regeneration appeared enhanced in SAHA and VPA treated animals and was dependent on Hippo-YAP signaling. Conclusions VPA and SAHA delay warm hepatic I/R injury at least in part through modulation of SAPK-activation. However, these HDACi fail to exert organoprotective effects, in this setting. For VPA, belated damage is even aggravated.

Background & Aims Under conditions of inflammation in the absence of micro-organisms (sterile inflammation), necrotic cells release damage-associated molecular patterns that bind to Toll-like receptors on immune cells to activate a signaling pathway that involves activation of IκB kinase and nuclear factor κB (NF-κB). Little is known about the mechanisms that control NF-κB activity during sterile inflammation. We analyzed the contribution of B-cell CLL/lymphoma 3 (BCL3), a transcription factor that associates with NF-κB, in control of sterile inflammation in the pancreas and biliary system of mice. Methods Acute pancreatitis (AP) was induced in C57BL/6 (control) and Bcl3 −/− mice by intraperitoneal injection of cerulein or pancreatic infusion of sodium taurocholate. We also studied Mdr2 −/− mice, which develop spontaneous biliary inflammation, as well as Bcl3 −/− Mdr2 −/− mice. We performed immunohistochemical analyses of inflamed and noninflamed regions of pancreatic tissue from patients with AP or primary sclerosing cholangitis (PSC), as well as from mice. Immune cells were characterized by fluorescence-activated cell sorting analysis. Control or Bcl3 −/− mice were irradiated, injected with bone marrow from Bcl3 −/− or control mice, and AP was induced. Results Pancreatic or biliary tissues from patients with AP or PSC had higher levels of BCL3 and phosphorylated RelA and IκBα in inflamed vs noninflamed regions. Levels of BCL3 were higher in pancreata from control mice given cerulein than from mice without AP, and were higher in biliary tissues from Mdr2 −/− mice than from control mice. Bcl3 −/− mice developed more severe AP after administration of cerulein or sodium taurocholate than control mice; pancreata from the Bcl3 −/− mice with AP had greater numbers of macrophages, myeloid-derived suppressor cells, dendritic cells, and granulocytes than control mice with AP. Activation of NF-κB was significantly prolonged in Bcl3 −/− mice with AP, compared with control mice with AP. Bcl3 −/− Mdr2 −/− mice developed more severe cholestasis and had increased markers of liver injury and increased proliferation of biliary epithelial cells and hepatocytes than Mdr2 −/− mice. In experiments with bone marrow chimeras, expression of BCL3 by acinar cells, but not myeloid cells, was required for reduction of inflammation during development of AP. BCL3 inhibited ubiquitination and proteasome-mediated degradation of p50 homodimers, which prolonged binding of NF-κB heterodimers to DNA. Conclusions BCL3 is up-regulated in inflamed pancreatic or biliary tissues from mice and patients with AP or cholangitis. Its production appears to reduce the inflammatory response in these tissues via blocking ubiquitination and proteasome-mediated degradation of p50 homodimers.

BACKGROUND & AIMS: One treatment strategy for pancreatic ductal adenocarcinoma is to modify, rather than deplete, the tumor stroma. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) is associated with progression of pancreatic and other solid tumors. We investigated whether loss of P53 function contributes to persistent activation of STAT3 and modification of the pancreatic tumor stroma in patients and mice. METHODS: Stat3, Il6st (encodes gp130), or Trp53 were disrupted, or a mutant form of P53 (P53R172H) or transgenic sgp130 were expressed, in mice that developed pancreatic tumors resulting from expression of activated KRAS (KrasG12D, KC mice). Pancreata were collected and analyzed by immunohistochemistry, in situ hybridization, quantitative reverse-transcription polymerase chain reaction (qPCR), or immunoblot assays; fluorescence-activated cell sorting was performed to identify immune cells. We obtained frozen pancreatic tumor specimens from patients and measured levels of phosphorylated STAT3 and P53 by immunohistochemistry; protein levels were associated with survival using Kaplan-Meier analyses. We measured levels of STAT3, P53, ligands for gp130, interleukin 6, cytokines, sonic hedgehog signaling, STAT3 phosphorylation (activation), and accumulation of reactive oxygen species in primary pancreatic cells from mice. Mice with pancreatic tumors were given gemcitabine and a Janus kinase 2 (JAK2) inhibitor; tumor growth was monitored by 3-dimensional ultrasound. RESULTS: STAT3 was phosphorylated constitutively in pancreatic tumor cells from KC mice with loss or mutation of P53. Tumor cells of these mice accumulated reactive oxygen species and had lower activity of the phosphatase SHP2 and prolonged phosphorylation of JAK2 compared with tumors from KC mice with functional P53. These processes did not require the gp130 receptor. Genetic disruption of Stat3 in mice, or pharmacologic inhibitors of JAK2 or STAT3 activation, reduced fibrosis and the numbers of pancreatic stellate cells in the tumor stroma and altered the types of immune cells that infiltrated tumors. Mice given a combination of gemcitabine and a JAK2 inhibitor formed smaller tumors and survived longer than mice given control agents; the tumor stroma had fewer activated pancreatic stellate cells, lower levels of periostin, and alterations in collagen production and organization. Phosphorylation of STAT3 correlated with P53 mutation and features of infiltrating immune cells in human pancreatic tumors. Patients whose tumors had lower levels of phosphorylated STAT3 and functional P53 had significantly longer survival times than patients with high levels of phosphorylated STAT3 and P53 mutation. CONCLUSIONS: In pancreatic tumors of mice, loss of P53 function activates JAK2-STAT3 signaling, which promotes modification of the tumor stroma and tumor growth and resistance to gemcitabine. In human pancreatic tumors, STAT3 phosphorylation correlated with P53 mutation and patient survival time. Inhibitors of this pathway slow tumor growth and stroma formation, alter immune cell infiltration, and prolong survival of mice. Transcript profiling: ArrayExpress accession number: E-MTAB-3278. ZB 0 Z8 0 ZR 0 ZS 0

Pancreatic cancer has a very dismal prognosis, because of the late detection of the entity and the scarcity of therapeutic options. While chemotherapeutics are used for standard patient care, targeted therapeutic approaches so far did not add a considerable survival benefit. Mild- to absent response to standard or targeted therapies can be attributed to the genetic-epigenetic heterogeneity of the tumor in general. On the other hand, immunotherapeutic approaches hold promise due to their potential to boost the anti-tumor immune response. Especially the adaptive immune system with its large antigen recognition repertoire can bypass an acquired genetic-epigenetic heterogeneity of tumors. However, although immunotherapy has demonstrated powerful impact on many tumor entities, its effect on pancreatic cancer is not yet convincing. The biggest obstacle for successful immunotherapy in pancreatic cancer appears to be its unique tumor microenvironment. Therefore, other than dissecting cancer cell-specific resistances, an understanding of the microenvironmental tumor immune network specifically in pancreatic cancer is required. Here in this chapter, upon an introduction to major immune cell types in the tumor microenvironment, we aim to reveal how pancreatic cancer circumvents the benefits of currently employed immunotherapeutic approaches and consider options for future improvements.