Development Of Exocrine Pancreas Research Articles

Tmed10 deficiency results in impaired exocrine pancreatic differentiation in zebrafish larvae

Transmembrane p24 trafficking protein 10 (TMED10) is a conserved vesicle trafficking protein. It is dysregulated in Alzheimer disease and plays a pivotal role in the pathogenesis of Alzheimer disease. In addition to the brain, TMED10 is highly expressed in the exocrine pancreas; however, its biological functions and underlying mechanisms remain largely unknown. We studied reduced Tmed10 in zebrafish embryos by morpholino oligonucleotide knockdown and CRISPR-Cas9 mutagenesis. Tmed10-deficient embryos showed extensive loss of acinar mass and impaired acinar differentiation. TMED10 has been reported to have an inhibitory effect on γ-secretase. As one of the substrates of γ-secretase, membrane-bound β-catenin was significantly reduced in Tmed10-deficient embryos. Increased γ-secretase activity in wild-type embryos resulted in a phenotype similar to that of tmed10 mutants. And the mutant phenotype could be rescued by treatment with the γ-secretase inhibitor, N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester (DAPT). In addition, the reduced membrane-bound β-catenin was accompanied with up-regulated β-catenin target genes in Tmed10-deficient embryos. Overexpression of β-catenin signaling inhibitor Dickkopf-1 (DKK-1) could rescue the exocrine pancreas defects. Taken together, our study reveals that Tmed10 regulates exocrine pancreatic differentiation through γ-secretase. Reduced membrane-bound β-catenin, accompanied with hyperactivation of β-catenin signaling, is an important cause of exocrine pancreas defects in Tmed10-deficient embryos. Our study reaffirms the importance of appropriate β-catenin signaling in exocrine pancreas development. These findings may provide a theoretical basis for the development of treatment strategies for TMED10-related diseases.

Peroxisomes during postnatal development of mouse endocrine and exocrine pancreas display cell-type- and stage-specific protein composition

Peroxisomal dysfunction unhinges cellular metabolism by causing the accumulation of toxic metabolic intermediates (e.g. reactive oxygen species, very -chain fatty acids, phytanic acid or eicosanoids) and the depletion of important lipid products (e.g. plasmalogens, polyunsaturated fatty acids), leading to various proinflammatory and devastating pathophysiological conditions like metabolic syndrome and age-related diseases including diabetes. Because the peroxisomal antioxidative marker enzyme catalase is low abundant in Langerhans islet cells, peroxisomes were considered scarcely present in the endocrine pancreas. Recently, studies demonstrated that the peroxisomal metabolism is relevant for pancreatic cell functionality. During the postnatal period, significant changes occur in the cell structure and the metabolism to trigger the final maturation of the pancreas, including cell proliferation, regulation of energy metabolism, and activation of signalling pathways. Our aim in this study was to (i) morphometrically analyse the density of peroxisomes in mouse endocrine versus exocrine pancreas and (ii) investigate how the distribution and the abundance of peroxisomal proteins involved in biogenesis, antioxidative defence and fatty acid metabolism change during pancreatic maturation in the postnatal period. Our results prove that endocrine and exocrine pancreatic cells contain high amounts of peroxisomes with heterogeneous protein content indicating that distinct endocrine and exocrine cell types require a specific set of peroxisomal proteins depending on their individual physiological functions. We further show that significant postnatal changes occur in the peroxisomal compartment of different pancreatic cells that are most probably relevant for the metabolic maturation and differentiation of the pancreas during the development from birth to adulthood.

IPSC-Derived Pancreatic Progenitors Lacking FOXA2 Reveal Alterations in miRNA Expression Targeting Key Pancreatic Genes

Recently, we reported that forkhead box A2 (FOXA2) is required for the development of human pancreatic α- and β-cells. However, whether miRNAs play a role in regulating pancreatic genes during pancreatic development in the absence of FOXA2 expression is largely unknown. Here, we aimed to capture the dysregulated miRNAs and to identify their pancreatic-specific gene targets in pancreatic progenitors (PPs) derived from wild-type induced pluripotent stem cells (WT-iPSCs) and from iPSCs lacking FOXA2 (FOXA2–/–iPSCs). To identify differentially expressed miRNAs (DEmiRs), and genes (DEGs), two different FOXA2–/–iPSC lines were differentiated into PPs. FOXA2–/– PPs showed a significant reduction in the expression of the main PP transcription factors (TFs) in comparison to WT-PPs. RNA sequencing analysis demonstrated significant reduction in the mRNA expression of genes involved in the development and function of exocrine and endocrine pancreas. Furthermore, miRNA profiling identified 107 downregulated and 111 upregulated DEmiRs in FOXA2–/– PPs compared to WT-PPs. Target prediction analysis between DEmiRs and DEGs identified 92 upregulated miRNAs, predicted to target 1498 downregulated genes in FOXA2–/– PPs. Several important pancreatic TFs essential for pancreatic development were targeted by multiple DEmiRs. Selected DEmiRs and DEGs were further validated using RT-qPCR. Our findings revealed that FOXA2 expression is crucial for pancreatic development through regulating the expression of pancreatic endocrine and exocrine genes targeted by a set of miRNAs at the pancreatic progenitor stage. These data provide novel insights of the effect of FOXA2 deficiency on miRNA-mRNA regulatory networks controlling pancreatic development and differentiation.Graphical

Deoxyhypusine synthase, an essential enzyme for hypusine biosynthesis, is required for proper exocrine pancreas development.

Pancreatic diseases including diabetes and exocrine insufficiency would benefit from therapies that reverse cellular loss and/or restore cellular mass. The identification of molecular pathways that influence cellular growth is therefore critical for future therapeutic generation. Deoxyhypusine synthase (DHPS) is an enzyme that post‐translationally modifies and activates the mRNA translation factor eukaryotic initiation factor 5A (eIF5A). Previous work demonstrated that the inhibition of DHPS impairs zebrafish exocrine pancreas development; however, the link between DHPS, eIF5A, and regulation of pancreatic organogenesis remains unknown. Herein we identified that the conditional deletion of either Dhps or Eif5a in the murine pancreas results in the absence of acinar cells. Because DHPS catalyzes the activation of eIF5A, we evaluated and uncovered a defect in mRNA translation concomitant with defective production of proteins that influence cellular development. Our studies reveal a heretofore unappreciated role for DHPS and eIF5A in the synthesis of proteins required for cellular development and function.

Zebrafish miR-462-731 is required for digestive organ development

MicroRNAs (miRNAs), as important regulators of post-transcriptional gene expression, play important roles in the occurrence and function of organs. In this study, morpholino (MO) knockdown of miR-462/miR-731 was used to investigate the potential mechanisms of the miR-462-731 cluster during zebrafish liver development. The results showed significant reduction of digestive organs, especially liver and exocrine pancreas after the miR-462/miR-731 knockdown, and those phenotypes could be partially rescued by corresponding miRNA duplex. Acinar cells of the exocrine pancreas were also severely affected with pancreatic insufficiency. In particular, knockdown of miR-462 caused pancreas morphogenesis abnormity with specific bilateral exocrine pancreas. Additionally, it was found that miR-731 played a role in liver and exocrine pancreas development by directly targeting dkk3b, instead of the down-regulation of Wnt/β-catenin signaling. These findings contribute significantly to our understanding of molecular mechanisms of miR-462-731 cluster in development of digestive organs.

Mypt1 regulates Bmp signaling to promote embryonic exocrine pancreas growth in zebrafish.

Myosin phosphatase targeting subunit 1 (Mypt1) is the regulatory subunit of myosin phosphatase which dephosphorylates the light chain of myosin II to inhibit its contraction. Although biochemical properties of Mypt1 have been characterized in detail, its biological functions in organisms are not well understood. The zebrafish mypt1 sq181 allele was found defective in the ventral pancreatic bud and extrapancreatic duct development, resulting in dysplasia of exocrine pancreas. In mypt1 sq181 mutant, the early growth of the ventral pancreatic bud was initiated but failed to expand due to impaired cell proliferation and increased cell apoptosis. As Mypt1 is essential for cell migration, the loss-of-function of Mypt1 in the mutant disrupted the lateral plate mesoderm migration during gut looping, therefore, altering the Bmp2a expression pattern within it, and eventually leading to impaired Bmp signaling in the adjacent exocrine pancreas. Overexpression of bmp2a could rescue the development of exocrine pancreas, suggesting that the impaired Bmp2a signaling is responsible for the pancreatic development defects. Bmp2a has been reported to promote the early specification of the ventral pancreatic bud, and our study reveals that it continues to serve as a cell proliferation/survival signal to ensure pancreatic bud growth properly in zebrafish.

Abstract 3679: A novel mechanism for pancreatic cancer stem cell maintenance: PAF1C-independent role of PAF1

Abstract Background: Cancer stem cells (CSCs), a minor subset of cancer cells, mediate aggressiveness, metastasis, and drug resistance of many cancers including the deadly pancreatic cancer (PC). Molecular governance of pancreatic CSCs remains elusive, mandating the need for their better molecular characterization to improve management of PC. PD2 (Pancreatic Differentiation 2) or RNA Polymerase II-Associated Factor 1 (Paf1) is a core component of human PAF1 complex (PAF1C), which regulates transcription elongation and mRNA processing. Human PAF1C consists of 5 members: PAF1, LEO1, CDC73, CTR9 and SKI8. Recently, PAF1 has emerged as a novel pancreatic CSC marker that enhances tumorigenic and metastatic potential of PC. Paf1 also maintains the self-renewal of mouse embryonic stem cells and ovarian CSCs via its interaction with OCT3/4, a major regulator of pluripotency. However, the mechanistic role of PAF1 in CSC maintenance and CSC-mediated PC pathogenesis is poorly understood. Hypothesis: We hypothesize that “PAF1 forms a sub-complex exclusive of PAF1C, wherein PAF1 functions as the master-regulator for maintaining pancreatic CSCs by regulating stem cell gene signature”. Experimental Design: PAF1 knockdown (KD) was used to identify its targets and role in tumorigenesis. PAF1 sub-complex in CSCs was identified using immunoprecipitation (IP) and mass spectrometry (MS). ChIP-Seq was performed to confirm binding of PAF1 on its target genes. Results: Inducible KD of PAF1 led to a significant reduction in tumor burden from orthotopically implanted human PC cells in athymic nude mice, indicating its role in pancreatic tumorigenesis. CSCs from different PC cell lines demonstrated higher expression of PAF1 along with CSC markers and PAF1 KD caused a significant decrease in CSC and self-renewal markers analyzed through Western blotting and immunofluorescence. Reciprocal co-IP and MS revealed that PAF1 interacted with Phf5a, DDX3, and hnRNP-K in CSCs. PAF1 KD in CSCs showed significant down-regulation of tumorigenic and stemness maintenance genes by RNA-Seq and PCR array analysis. PAF1 along with its binding partners occupied Nanog promoter in pancreatic CSCs. Further, KD of PAF1 in CSCs did not affect the expression of other PAF1C components, whereas individual KD of single PAF1C components decreased other remaining PAF1C members including PAF1. Moreover, other PAF1C components did not interact with Phf5a, a PHD-fold harboring nuclear protein in CSCs, suggesting that PAF1 forms a sub-complex with Phf5a, independent of PAF1C that functions in CSC maintenance. Depletion of Paf1 from mouse pancreas since birth using a CRISPR/Cas9-based conditional Paf1 knockout mouse model severely affected exocrine pancreas development with peri-ductal sclerosis and inflammation. Conclusion: Altogether, PAF1 functions as the master-regulator for CSC maintenance by forming a sub-complex, which regulates CSC-network genes in PC. Citation Format: Saswati Karmakar, Sanchita Rauth, Rama Krishna Nimmakayala, Srikanth Barkeer, Mohd W. Nasser, Satyanarayana Rachagani, Dario Ghersi, Moorthy P. Ponnusamy, Surinder K. Batra. A novel mechanism for pancreatic cancer stem cell maintenance: PAF1C-independent role of PAF1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3679.

Expression and activity of key lipases during the larval development of walking catfish (Clarias magur).

The study was conducted to investigate the expression and activity of key lipolytic enzymes during the ontogenetic development of Clarias magur. After partial characterization, the messenger RNA (mRNA) expression analysis of lipoprotein lipase (LPL), pancreatic triacylglycerol lipase (PL), and bile salt-activated lipase (BAL) genes along with the specific lipase activity were performed in larvae from Day 1 after hatching till 34-day posthatch (dph). Heterogeneous patterns of mRNA expression were shown by the important lipolytic enzymes and were detected before first exogenous feeding during the yolk-sac stage. LPL started increasing from 13 dph and peaked at 16 dph followed by a declining trend till 34 dph. However, the PL observed to be peaking at 9, 22, and 30 dph. Similarly, BAL showed an increasing trend from 11 to 22 dph with a significantly high level of mRNA expression at 16 dph. Later, the specific lipase activity was evaluated which appears at Day 1 after hatching with a progressive increase from 7 to 16 dph and a further declining trend afterwards with a peak at 22 dph. The results indicated the development of exocrine pancreas at 16 dph. Furthermore, the transcript levels and the activity of lipases were regulated with the age. Hence, the present study can be helpful in devising different strategies containing optimum lipid levels at a suitable stage of development for improving the survival during larval rearing. Furthermore, the study could be a baseline for elucidating the optimized dietary lipid levels of this catfish during its larval rearing.

Regulation of the Pancreatic Exocrine Differentiation Program and Morphogenesis by Onecut 1/Hnf6.

Background & AimsThe Onecut 1 transcription factor (Oc1, a.k.a. HNF6) promotes differentiation of endocrine and duct cells of the pancreas; however, it has no known role in acinar cell differentiation. We sought to better understand the role of Oc1 in exocrine pancreas development and to identify its direct transcriptional targets.MethodsPancreata from Oc1Δpanc (Oc1fl/fl;Pdx1-Cre) mouse embryos and neonates were analyzed morphologically. High-throughput RNA-sequencing was performed on control and Oc1-deficient pancreas; chromatin immunoprecipitation sequencing was performed on wild-type embryonic mouse pancreata to identify direct Oc1 transcriptional targets. Immunofluorescence labeling was used to confirm the RNA-sequencing /chromatin immunoprecipitation sequencing results and to further investigate the effects of Oc1 loss on acinar cells.ResultsLoss of Oc1 from the developing pancreatic epithelium resulted in disrupted duct and acinar cell development. RNA-sequencing revealed decreased expression of acinar cell regulatory factors (Nr5a2, Ptf1a, Gata4, Mist1) and functional genes (Amylase, Cpa1, Prss1, Spink1) at embryonic day (e) 18.5 in Oc1Δpanc samples. Approximately 1000 of the altered genes were also identified as direct Oc1 targets by chromatin immunoprecipitation sequencing, including most of the previously noted genes. By immunolabeling, we confirmed that Amylase, Mist1, and GATA4 protein levels are significantly decreased by P2, and Spink1 protein levels were significantly reduced and mislocalized. The pancreatic duct regulatory factors Hnf1β and FoxA2 were also identified as direct Oc1 targets.ConclusionsThese findings confirm that Oc1 is an important regulator of both duct and acinar cell development in the embryonic pancreas. Novel transcriptional targets of Oc1 have now been identified and provide clarity into the mechanisms of Oc1 transcriptional regulation in the developing exocrine pancreas. Oc1 can now be included in the gene-regulatory network of acinar cell regulatory genes. Oc1 regulates other acinar cell regulatory factors and acinar cell functional genes directly, and it can also regulate some acinar cell regulatory factors (eg, Mist1) indirectly. Oc1 therefore plays an important role in acinar cell development.

Cover 1 - Oc1 Regulation of Exocrine Pancreas Development

Cover 1 - Oc1 Regulation of Exocrine Pancreas Development

Mutation-Specific Dose Reduction in Functional SRP54 Protein Causes Isolated or Syndromic Neutropenia with Shwachman-Diamond-like Features

Sequencing analyses are increasingly performed on patients presenting with suspected congenital disease but lacking classical mutations linked to the presented phenotype. However, in case a novel mutation is found, its causal contribution to the patients' clinical symptoms is yet unclear and requires further exploration in functional studies. Here we use the zebrafish model to analyze the functional relevance of heterozygous SRP54 gene mutations identified in patients with unexplained neutropenia (T117del) or Shwachman-Diamond like disease involving severe neutropenia and exocrine pancreas insufficiency (T115A, G226E) (Carapito et al, 2017). The function of wildtype SRP54 protein was explored in hematopoiesis and pancreas development using two different antisense morpholino oligonucleotides (MO) and a zebrafish srp54 mutant. Reduced neutrophil numbers were observed in MO versus control injected fish when analyzed by WISH for mpx or using Tg(lyz:DsRed) and Tg(mpx:eGFP) lines. Morphants displayed not only quantitatively but also qualitatively impaired neutrophils, which migrated less to injury sites in tail fin injury assays. Furthermore, exocrine but not endocrine pancreas impairment was observed in MO versus control injected fish. Both neutrophiles and exocrine pancreas development could be rescued by co-injection with wildtype SRP54 mRNA.Interestingly, in the corresponding Sanger mutant, homozygous srp54-/- fish display not only most severe neutropenia but also gross developmental defects which cause early lethality at 2 days post-fertilization and thus impede assessment of pancreas development. Heterozygous srp54+/- fish are viable, show less severe neutropenia than homozygous fish and, interstingly, have no exocrine pancreas defect. The delineated experiments explore thus the effects of different dosages of residual functional SRP54 protein (wildtype > srp54+/- > morpholino treated > srp54-/-) on phenotypic manifestation. The obtained results indicate that the dose of residual SRP54 protein that can functionally act within the SRP may dictate disease phenotype in patients with SRP54 mutations (neutropenia only > more severe neutropenia and exocrine pancreas defects > most severe neutropenia, early lethality).We thus hypothesize that the different mutations observed in patients differentially affect the functionality of the residual wildtype SRP54 protein and thus associate with isolated neutropenia (T117del) or more severe disease with Shwachman-Diamond like features (T115A, G226E). To mimic the heterozygote mutation status observed in patients, we injected the different mutated mRNAs in srp54+/- zebrafish. None of the injected mRNAs was able to rescue the neutropenia phenotype, indicating that all mutated proteins are functionally impaired. Confirming our hypothesis, injection of T115A and G226E in fact further aggravated neutropenia and additionally induced exocrine pancreas defects, while T117del did not.Taken together, these analyses support the notion that full deletion of SRP54 is not compatible with life and therefore homozygous SRP54 are not observed in patients. Instead, heterozygous SRP54 mutations may cause only neutropenia or alternatively the more severe Shwachman-Diamond like syndrom, depending on the nature of the underlying mutation (either functional null or dominant negative). DisclosuresNo relevant conflicts of interest to declare.

Exocrine Pancreatic Function as a Novel Biomarker in Pre-T1D

Recent studies demonstrated reduced serum trypsinogen levels as well as pancreatic weight/volume in patients with type 1 diabetes (T1D) and nondiabetic/autoantibody (AAb)-positive subjects. Histological defects including increased CD8+ T cell infiltration and elevated C4d deposition have been observed in the exocrine T1D pancreas. Like trypsinogen, amylase and lipase are produced by the exocrine pancreas and serve as more common markers of exocrine function. We hypothesized that amylase and lipase levels would be lower in patients with T1D as well as in subjects with multiple (i.e., ≥2) T1D-related AAb compared with controls. To test this, serum amylase and lipase levels were determined in 70 patients with recent-onset T1D (duration <3 mo), 57 patients with established T1D (duration >3 mo), 56 single AAb positive (1AAb+) subjects, 42 multiple AAb positive (≥2AAb+) subjects, 96 AAb negative (AAb-) relatives of T1D patients, and 110 AAb- control subjects. Amylase levels, which are independent of age (r = -0.02, P = 0.7), were significantly lower in patients with T1D (mean ± SD 39.98 ± 17.8 ng/mL; P < 0.005) vs. AAb- subjects (47.40 ± 20.17 ng/mL). Lipase levels, which are positively correlated with age (r = 0.30, P < 0.0001), were significantly lower in T1D patients (25.95 ± 21.ng/mL; P < 0.001) as well as ≥2AAb+ subjects (22.59 ± 11.12 ng/mL; P < 0.001) compared with 1AAb+ individuals (37.60 ± 16.79 ng/mL). These results provide further evidence that exocrine pancreas abnormalities exist in patients with T1D and ≥2AAb+ subjects considered to have early-stage T1D with high risk of progressing to clinical diagnosis. These data also support the possibility that an inherent or acquired defect in exocrine pancreas development and/or function may contribute to disease pathogenesis, but cause and effect cannot yet be ascertained. Importantly, these exocrine pancreas enzymes may serve as biomarkers of T1D to better predict disease progression and/or stratify patients according to etiology. Disclosure J.J. Ross: None. C. Wasserfall: None. D.J. Perry: None. K.M. McGrail: None. A.L. Posgai: None. T.M. Brusko: Stock/Shareholder; Self; OneVax, LLC. Advisory Panel; Self; Caladrius Biosciences, Inc.. Consultant; Self; Merck & Co., Inc., Sanofi-Aventis. D. Schatz: None. M.J. Haller: None. M.A. Atkinson: Other Relationship; Self; Patent Issued.

Mig6 Is Required for Pancreas Development in Zebrafish

Strategies to promote β cell neogenesis could be used to restore functional β cell mass in both type 1 and type 2 diabetes. Here we examine the role of mitogen-inducible gene 6 (mig6, a.k.a. erffi1) in the regulation of functional β cell mass. mig6 is evolutionarily conserved and encodes a protein that inhibits the epidermal growth factor receptor (EGFR) in a classic feedback mechanism, suggesting a potential role in cellular growth and development. Based on functional studies in mouse and human, we hypothesized that mig6 is required in pancreatic ducts for pancreatogenesis and β cell function. We used morpholino knockdown in zebrafish to define the role of mig6 in both exocrine pancreas and islet development. We established that mig6 knockdown (mig6MO) embryos have a disrupted pancreas morphogenesis, particularly, a truncated exocrine pancreas and fewer intra-pancreatic duct cells. Additionally, mig6MO-injected animals had fewer β cells than controls. Together, our data lead us to hypothesize that mig6 may facilitate the expansion of the pancreas progenitor pool by blocking EGFR activation. We further investigated the role of mig6 in islet regeneration. Neogenic β cells may arise from two sources in the developing zebrafish, which can be distinguished by the retention of a fluorescent mark made at the one-cell stage. β cells derived via transdifferentiation from other postmitotic endocrine cells remain labeled whereas those derived from duct-associated progenitors lose their label via proliferation. We found that although the number of regenerated β cells was reduced overall in mig6MO-injected embryos, most new β cells were not labeled, suggesting that a significant progenitor pool remains in the knockdown embryos. Together, our data demonstrate that mig6 is essential for pancreas development and that it might be exploited as a switch between progenitor differentiation and endocrine transdifferentiation to increase β cell mass in diabetic patients. Disclosure K. El: None. P.T. Fueger: None. R.M. Anderson: None.

Functional and association analysis of an Amerindian-derived population-specific p.(Thr280Met) variant in RBPJL, a component of the PTF1 complex

PTF1 complex is critical for pancreatic development and maintenance of adult exocrine pancreas. As a part of our ongoing studies to identify genetic variation that contributes to type 2 diabetes (T2D) in American Indians, we analyzed variation in genes that form this complex, namely PTF1A, RBPJ, and its paralogue RBPJL. A c.839C>T (p.(Thr280Met)) variant (rs200998587:C>T, risk allele frequency = 0.03) in RBPJL, identified only in Amerindian-derived populations, associated with T2D (OR = 1.60[1.21–2.13] per Met allele, P = 0.001) and age of diabetes onset (HR = 1.40[1.14–1.72], P = 0.001). Knockdown of Rbpjl in mouse pancreatic acinar cells resulted in a significant decrease in the mRNA expression of genes encoding exocrine enzymes including Ctrb. CTRB1/2 is an established T2D locus where the protective allele associates with increased GLP-1-stimulated insulin secretion and higher expression of CTRB1/2. In vitro studies show that cells expressing the Met280 allele had lower RBPJL protein levels than cells expressing the Thr280 allele, despite having comparable levels of RNA, suggesting that the Met280 RBPJL is less stable. Additionally, luciferase assays in HEK293 cells which examined two different RBPJL responsive promoters, including the promoter for CTRB1, also identified reduced transactivation by the Met280 RBPJL. Similarly, overexpression of both Met280 and Thr280 RBPJL in mouse pancreatic acinar cells identified a significant impairment in the expression of Cel when transactivated by the Met280 RBPJL. In summary, we identified a functional, Amerindian-derived population-specific c.839C>T (p.(Thr280Met)) variant in the pancreas specific RBPJL that may modify T2D risk by regulating exocrine enzyme expression.

Endoderm Jagged induces liver and pancreas duct lineage in zebrafish

Liver duct paucity is characteristic of children born with Alagille Syndrome (ALGS), a disease associated with JAGGED1 mutations. Here, we report that zebrafish embryos with compound homozygous mutations in two Notch ligand genes, jagged1b (jag1b) and jagged2b (jag2b) exhibit a complete loss of canonical Notch activity and duct cells within the liver and exocrine pancreas, whereas hepatocyte and acinar pancreas development is not affected. Further, animal chimera studies demonstrate that wild-type endoderm cells within the liver and pancreas can rescue Notch activity and duct lineage specification in adjacent cells lacking jag1b and jag2b expression. We conclude that these two Notch ligands are directly and solely responsible for all duct lineage specification in these organs in zebrafish. Our study uncovers genes required for lineage specification of the intrahepatopancreatic duct cells, challenges the role of duct cells as progenitors, and suggests a genetic mechanism for ALGS ductal paucity.

Abstract B45: Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell and patient-derived tumor organoids

Abstract There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells (PSCs) into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53R175H induced cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. Culture conditions are also defined for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture, phenotypic heterogeneity of the primary tumor, and retain patient-specific physiologic changes including hypoxia, oxygen consumption, epigenetic marks, and differential sensitivity to EZH2 inhibition. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies. Citation Format: Ling Huang, Audrey Holtzinger, Ishaan Jagan, Cristina Nostro, Rennian Wang, Lakshmi Muthuswamy, Cheryl Arrowsmith, Sean Cleary, David Schaeffer, Michael Roehrl, Tsao Ming-Sound, Steven Gallinger, Gordon Keller, Senthil Muthuswamy.{Authors}. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell and patient-derived tumor organoids. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B45.

Abstract PR01: Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell and patient-derived tumor organoids

Abstract There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells (PSCs) into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53R175H induced cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. Culture conditions are also defined for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture, phenotypic heterogeneity of the primary tumor, and retain patient-specific physiologic changes including hypoxia, oxygen consumption, epigenetic marks, and differential sensitivity to EZH2 inhibition. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies. This abstract is also being presented as Poster B10. Citation Format: Senthil K. Muthuswamy. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell and patient-derived tumor organoids. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr PR01.

Prox1-Heterozygosis Sensitizes the Pancreas to Oncogenic Kras-Induced Neoplastic Transformation

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.

Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.

There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53(R175H) induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.

Mnk1 is a novel acinar cell-specific kinase required for exocrine pancreatic secretion and response to pancreatitis in mice

ObjectivePancreatic acinar cell maturation is dependent on the activity of the pancreas transcription factor 1 (PTF1) complex. Induction of pancreatitis leads to MAP kinase activation and transient suppression of the...