Pancreas Progenitors Research Articles

Abstract B032: Sox4-dependent acinar cell plasticity in pancreatic regeneration and cancer initiation

Abstract Gene alterations play a prominent role in driving cancer initiation and progression. Yet, mutations on oncogenes (those genes that promote tumorigenesis) only transform cells under certain cellular contexts. The mechanisms controlling neoplastic transformation (oncogenic competence) are poorly understood in pancreatic ductal adenocarcinoma (PDAC). Our laboratory investigates the interplay of mutations on the Kirsten Rat Sarcoma oncogene (Kras), developmental transcriptional programs, and the tissue microenvironment PDAC initiation. Our data demonstrate that Sox4 is necessary for the specification of cellular states in preinvasive cancer lesions and regulates the characteristics and cellular compositions of the tumor microenvironment in an autochthonous genetic model of PDAC. The pancreas has a remarkable ability to regenerate and recover from acute pancreatitis. In this process, acinar cells repress the expression of genes associated with acinar function and transiently activate a gene program that resembles pancreas progenitors of development. This mechanism, defined as cellular plasticity, alleviates tissue damage, stimulates acinar proliferation, and is necessary for pancreas regeneration. However, it makes the acinar cells competent to malignant transformation mediated by Kras. To investigate SOX4 function in pancreas regeneration and cancer, we used the KCacinar mouse model (Ptf1a-CreER; Kras G12DLSL). After injury, we observed a transient four-fold increase in the expression of SOX4, which correlates with the morphological and molecular evidence of cellular plasticity. KCacinar mice rapidly develop mucinous pancreatic intraepithelial neoplasias (PanINs) after caerulein-induced pancreatitis. Histological analysis KCAcinar Sox4-depleted pancreas (KCSAcinar) reveals a distinct cystic lesion lined by cuboidal or flattened epithelium with large irregular and hyperchromatic nuclei and absence of mucin-producing cells. Mice lacking Sox4 showed a significant reduction in the number of tuft cells. Furthermore, we observed an extensive reduction of the tumor stroma surrounding epithelial acinar-derived lesions in KCSAcinar compared to KC littermates. Next, we perform gene expression analysis of lesions 21 days after the induction of pancreatitis. Principal component analysis clusters the samples according to the genotype. Differential gene expression validated our histopathological analysis and showed a significant reduction in the expression of mucins and tuft cell markers. Cumulative, our data suggest that Sox4 is necessary for the specification of cellular states in the precursor lesions of PDAC, and that the cellular state of the tumor cell of origin determines the characteristics and cellular composition of the tumor microenvironment. Our work will continue to unravel the function of Sox4 in cancer initiation and the interaction of signaling pathways activated in pancreas regeneration with Kras mutations. Citation Format: Jonathan Baldan, Juan Camacho Roda, Charlotte Vestrup Rift, Jane Preuss Hasselby, Patrick Jacquemin, Ilse Rooman, Véronique Lefebvre, Luis Arnes. Sox4-dependent acinar cell plasticity in pancreatic regeneration and cancer initiation [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B032.

Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.

The Ribosome Biogenesis Factor Ltv1 Is Essential for Digestive Organ Development and Definitive Hematopoiesis in Zebrafish.

Ribosome biogenesis is a fundamental activity in cells. Ribosomal dysfunction underlies a category of diseases called ribosomopathies in humans. The symptomatic characteristics of ribosomopathies often include abnormalities in craniofacial skeletons, digestive organs, and hematopoiesis. Consistently, disruptions of ribosome biogenesis in animals are deleterious to embryonic development with hypoplasia of digestive organs and/or impaired hematopoiesis. In this study, ltv1, a gene involved in the small ribosomal subunit assembly, was knocked out in zebrafish by clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR associated protein 9 (Cas9) technology. The recessive lethal mutation resulted in disrupted ribosome biogenesis, and ltv1Δ14/Δ14 embryos displayed hypoplastic craniofacial cartilage, digestive organs, and hematopoiesis. In addition, we showed that the impaired cell proliferation, instead of apoptosis, led to the defects in exocrine pancreas and hematopoietic stem and progenitor cells (HSPCs) in ltv1Δ14/Δ14 embryos. It was reported that loss of function of genes associated with ribosome biogenesis often caused phenotypes in a P53-dependent manner. In ltv1Δ14/Δ14 embryos, both P53 protein level and the expression of p53 target genes, Δ113p53 and p21, were upregulated. However, knockdown of p53 failed to rescue the phenotypes in ltv1Δ14/Δ14 larvae. Taken together, our data demonstrate that LTV1 ribosome biogenesis factor (Ltv1) plays an essential role in digestive organs and hematopoiesis development in zebrafish in a P53-independent manner.

Stem/progenitor cells in normal physiology and disease of the pancreas

Though embryonic pancreas progenitors are well characterised, the existence of stem/progenitor cells in the postnatal mammalian pancreas has been long debated, mainly due to contradicting results on regeneration after injury or disease in mice. Despite these controversies, sequencing advancements combined with lineage tracing and organoid technologies indicate that homeostatic and trigger-induced regenerative responses in mice could occur. The presence of putative progenitor cells in the adult pancreas has been proposed during homeostasis and upon different stress challenges such as inflammation, tissue damage and oncogenic stress. More recently, single cell transcriptomics has revealed a remarkable heterogeneity in all pancreas cell types, with some cells showing the signature of potential progenitors. In this review we provide an overview on embryonic and putative adult pancreas progenitors in homeostasis and disease, with special emphasis on in vitro culture systems and scRNA-seq technology as tools to address the progenitor nature of different pancreatic cells.

A 3D system to model human pancreas development and its reference single-cell transcriptome atlas identify signaling pathways required for progenitor expansion

Human organogenesis remains relatively unexplored for ethical and practical reasons. Here, we report the establishment of a single-cell transcriptome atlas of the human fetal pancreas between 7 and 10 post-conceptional weeks of development. To interrogate cell–cell interactions, we describe InterCom, an R-Package we developed for identifying receptor–ligand pairs and their downstream effects. We further report the establishment of a human pancreas culture system starting from fetal tissue or human pluripotent stem cells, enabling the long-term maintenance of pancreas progenitors in a minimal, defined medium in three-dimensions. Benchmarking the cells produced in 2-dimensions and those expanded in 3-dimensions to fetal tissue identifies that progenitors expanded in 3-dimensions are transcriptionally closer to the fetal pancreas. We further demonstrate the potential of this system as a screening platform and identify the importance of the EGF and FGF pathways controlling human pancreas progenitor expansion.

Chronically Elevated Exogenous Glucose Elicits Antipodal Effects on the Proteome Signature of Differentiating Human iPSC-Derived Pancreatic Progenitors.

The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing β-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is, thus, extremely valuable for managing the disease progression. Extracellular glucose is known to influence cell identity by impacting the redox balance. Here, we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex effect of exogenous glucose on differentiating pancreas progenitors characterized by a distinct proteome signature.

A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

SummaryThe notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.

2050-P: A Multiomics Investigation of a Patient-Derived HNF-1A MODY IPSC Disease Model Reveals Insights into Its Role in Pancreatic Islet Development and Function

Heterozygous loss of function mutations in HNF1A, encoding the transcription factor hepatocyte nuclear factor 1 alpha (HNF-1A) are the most common cause of monogenic diabetes. HNF-1A is involved in both beta cell development and mature cell function. However, model organisms have often failed to faithfully recapitulate the human phenotypes. As such, we used a patient-derived HNF1A Pro291fsinsC iPSC model and CRISPR-Cas9 genome editing to correct the mutation and generate an isogenic control. iPSC clones (n=6) were differentiated towards the endocrine lineage in triplicate, with RNA-seq (bulk and single-cell) and ATAC-seq performed at definitive endoderm (DE), pancreatic endoderm (PE) and beta-like cells (BLC) stages. RNA-seq data confirmed HNF1A haploinsuffiency in the patient cell lines, with corrected cell lines demonstrating restored HNF1A expression at both transcript and protein level. At the BLC stage, there was a significant overlap in differentially expressed genes and HNF1A targets identified in an independent HNF1A knock-down experiment in the EndoC-βh1 cell line (p=4.4e-132). Corrected clones demonstrated a higher expression of INS (p=7.2e-03), insulin secretion genes and effects on pancreas progenitor cell genes indicating that HNF1A may mediate the cellular composition of the pancreatic islet. Bulk RNA-seq data were also supported in the preliminary analysis of the single-cell RNA-seq data. Open chromatin, assessed by ATAC-seq was significantly different between patient and corrected lines, with >90% of the sites being more accessible at both PE and BLC stages in the corrected clones. These sites were enriched for HNF1A binding motifs, implicating HNF1A in the establishment of open chromatin. In conclusion, our patient-derived HNF1A-MODY iPSC model recapitulates a number of features of HNF1A-MODY, as well as providing insights into the cellular and molecular phenotypes caused by HNF-1A deficiency. Disclosure C. Duff: Research Support; Self; Novo Nordisk A/S. R. Jensen: None. M. Perez-Alcantara: None. M. Rasmussen: None. A. Grotz: None. M. Jansen: None. B. Holst: None. C. Clausen: None. N.A.J. Krentz: None. M. Hansson: Employee; Self; Novo Nordisk A/S. M.I. McCarthy: Advisory Panel; Self; Illumina. Consultant; Self; Eli Lilly and Company, Novo Nordisk Inc., Zoe Global Ltd. Employee; Self; Genentech, Inc. Research Support; Self; AbbVie Inc., Merck & Co., Inc., Sanofi-Aventis, Servier, Takeda UK. A.L. Gloyn: Consultant; Self; Merck Sharp & Dohme Corp. Employee; Spouse/Partner; Genentech, Inc. Research Support; Self; Novo Nordisk A/S. A. Wesolowska-Andersen: None. C. Honore: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. Funding Innovative Medicines Initiative (115439)

Loss of RE-1 silencing transcription factor accelerates exocrine damage from pancreatic injury

Regulation of pancreas plasticity is critical for preventing injury and promoting regeneration upon tissue damage. The intricate process of pancreatic differentiation is governed by an orchestrated network of positive and negative transcription factors for appropriate gene expression. While the transcriptional repressor REST is well characterized as a silencer of neuronal genes in non-neuronal cells, the role of REST in regulating exocrine pancreas cell identity remains largely unexplored. Rest expression is increased upon injury in the mouse pancreas, such as induced acute and chronic pancreatitis and ductal adenocarcinoma. At the cellular level, Rest expression is lower in mature acinar cells compared with pancreas progenitor and ductal cells. To investigate the role of REST activity in pancreatic transdifferentiation and homeostasis, we developed a novel mouse model (Cre/RESTfl/fl) with conditional knockout (KO) of Rest expression within pancreas cells. The high Cre-mediated excision efficiency of Rest exon two KO caused decreased Rest expression and activity within the pancreas. Short-term organoid cultures of pancreatic acini to undergo acinar-to-ductal metaplasia (ADM) showed that loss of REST impedes induced ADM, while overexpression of REST increases ADM. Interestingly, REST ablation accelerated acute pancreatitis in mice treated with the cholecystokinin analog caerulein, as indicated by cellular morphology, elevated serum amylase levels and pancreatic edema. Furthermore, Cre/RESTfl/fl mice were more sensitive to acute pancreatitis injury and displayed augmented tissue damage and cellular lesions. These results suggest REST has a novel protective role against pancreatic tissue damage by acting as a regulator of exocrine cell identity.

TrxG Complex Catalytic and Non-catalytic Activity Play Distinct Roles in Pancreas Progenitor Specification and Differentiation

Appropriate regulation of genes that coordinate pancreas progenitor proliferation and differentiation is required for pancreas development. Here, we explore the role of H3K4 methylation and the Trithorax group (TrxG) complexes in mediating gene expression during pancreas development. Disruption of TrxG complex assembly, but not catalytic activity, prevented endocrine cell differentiation in pancreas progenitor spheroids. Invivo loss of TrxG catalytic activity in PDX1+ cells increased apoptosis and the fraction of progenitors in the G1 phase of the cell cycle. Pancreas progenitors were reallocated to the acinar lineage, primarily at the expense of NEUROG3+ endocrine progenitors. Later in development, acinar and endocrine cell numbers were decreased, and increased gene expression variance and reduced terminal marker activation in acinar cells led to their incomplete differentiation. These findings demonstrate that TrxG co-activator activity is required for gene induction, whereas TrxG catalytic activity and H3K4 methylation help maintain transcriptional stability.

Pancreatic duct-like cell line derived from pig embryonic stem cells: expression of uroplakin genes in pig pancreatic tissue.

The isolation of a cell line, PICM-31D, with phenotypic characteristics like pancreatic duct cells is described. The PICM-31D cell line was derived from the previously described pig embryonic stem cell-derived exocrine pancreatic cell line, PICM-31. The PICM-31D cell line was morphologically distinct from the parental cells in growing as a monolayer rather than self-assembling into multicellular acinar-like structures. The PICM-31D cells were propagated for over a year at split ratios of 1:3 to 1:10 at each passage without change in phenotype or growth rate. Electron microscopy showed the cells to be a polarized epithelium of cuboidal cells joined by tight junction-like adhesions at their apical/lateral aspect. The cells contained numerous mucus-like secretory vesicles under their apical cell membrane. Proteomic analysis of the PICM-31D's cellular proteins detected MUC1 and MUC4, consistent with mucus vesicle morphology. Gene expression analysis showed the cells expressed pancreatic ductal cell-related transcription factors such as GATA4, GATA6, HES1, HNF1A, HNF1B, ONECUT1 (HNF6), PDX1, and SOX9, but little or no pancreas progenitor cell markers such as PTF1A, NKX6-1, SOX2, or NGN3. Pancreas ductal cell-associated genes including CA2, CFTR, MUC1, MUC5B, MUC13, SHH, TFF1, KRT8, and KRT19 were expressed by the PICM-31D cells, but the exocrine pancreas marker genes, CPA1 and PLA2G1B, were not expressed by the cells. However, the exocrine marker, AMY2A, was still expressed by the cells. Surprisingly, uroplakin proteins were prominent in the PICM-31D cell proteome, particularly UPK1A. Annexin A1 and A2 proteins were also relatively abundant in the cells. The expression of the uroplakin and annexin genes was detected in the cells, although only UPK1B, UPK3B, ANXA2, and ANXA4 were detected in fetal pig pancreatic duct tissue. In conclusion, the PICM-31D cell line models the mucus-secreting ductal cells of the fetal pig pancreas.

Protein Methyltransferase Inhibition Decreases Endocrine Specification Through the Upregulation of Aldh1b1 Expression.

Understanding the mechanisms that promote the specification of pancreas progenitors and regulate their self‐renewal and differentiation will help to maintain and expand pancreas progenitor cells derived from human pluripotent stem (hPS) cells. This will improve the efficiency of current differentiation protocols of hPS cells into β‐cells and bring such cells closer to clinical applications for the therapy of diabetes. Aldehyde dehydrogenase 1b1 (Aldh1b1) is a mitochondrial enzyme expressed specifically in progenitor cells during mouse pancreas development, and we have shown that its functional inactivation leads to accelerated differentiation and deficient β‐cells. In this report, we aimed to identify small molecule inducers of Aldh1b1 expression taking advantage of a mouse embryonic stem (mES) cell Aldh1b1 lacZ reporter line and a pancreas differentiation protocol directing mES cells into pancreatic progenitors. We identified AMI‐5, a protein methyltransferase inhibitor, as an Aldh1b1 inducer and showed that it can maintain Aldh1b1 expression in embryonic pancreas explants. This led to a selective reduction in endocrine specification. This effect was due to a downregulation of Ngn3, and it was mediated through Aldh1b1 since the effect was abolished in Aldh1b1 null pancreata. The findings implicated methyltransferase activity in the regulation of endocrine differentiation and showed that methyltransferases can act through specific regulators during pancreas differentiation. Stem Cells 2019;37:640–651

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.

Stepwise reprogramming of liver cells to a pancreas progenitor state by the transcriptional regulator Tgif2

The development of a successful lineage reprogramming strategy of liver to pancreas holds promises for the treatment and potential cure of diabetes. The liver is an ideal tissue source for generating pancreatic cells, because of its close developmental origin with the pancreas and its regenerative ability. Yet, the molecular bases of hepatic and pancreatic cellular plasticity are still poorly understood. Here, we report that the TALE homeoprotein TGIF2 acts as a developmental regulator of the pancreas versus liver fate decision and is sufficient to elicit liver-to-pancreas fate conversion both ex vivo and in vivo. Hepatocytes expressing Tgif2 undergo extensive transcriptional remodelling, which represses the original hepatic identity and, over time, induces a pancreatic progenitor-like phenotype. Consistently, in vivo forced expression of Tgif2 activates pancreatic progenitor genes in adult mouse hepatocytes. This study uncovers the reprogramming activity of TGIF2 and suggests a stepwise reprogramming paradigm, whereby a ‘lineage-restricted’ dedifferentiation step precedes the identity switch.

Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development

The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease.

Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters

ABSTRACTRegeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP+/+ mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP+ cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP+Ck19+ cells had derived from insulin-positive, glucose-transporter-2-low (Ins+Glut2LO) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin+Glut2LO cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin+Glut2+ cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins+Glut2LO cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future.

An insulin signaling feedback loop regulates pancreas progenitor cell differentiation during islet development and regeneration

As one of the key nutrient sensors, insulin signaling plays an important role in integrating environmental energy cues with organism growth. In adult organisms, relative insufficiency of insulin signaling induces compensatory expansion of insulin-secreting pancreatic beta (β) cells. However, little is known about how insulin signaling feedback might influence neogenesis of β cells during embryonic development. Using genetic approaches and a unique cell transplantation system in developing zebrafish, we have uncovered a novel role for insulin signaling in the negative regulation of pancreatic progenitor cell differentiation. Blocking insulin signaling in the pancreatic progenitors hastened the expression of the essential β cell genes insulin and pdx1, and promoted β cell fate at the expense of alpha cell fate. In addition, loss of insulin signaling promoted β cell regeneration and destabilization of alpha cell character. These data indicate that insulin signaling constitutes a tunable mechanism for β cell compensatory plasticity during early development. Moreover, using a novel blastomere-to-larva transplantation strategy, we found that loss of insulin signaling in endoderm-committed blastomeres drove their differentiation into β cells. Furthermore, the extent of this differentiation was dependent on the function of the β cell mass in the host. Altogether, our results indicate that modulation of insulin signaling will be crucial for the development of β cell restoration therapies for diabetics; further clarification of the mechanisms of insulin signaling in β cell progenitors will reveal therapeutic targets for both in vivo and in vitro β cell generation.

Aldehyde dehydrogenase activity is necessary for beta cell development and functionality in mice

Aims/hypothesisPancreatic beta cells maintain glucose homeostasis and beta cell dysfunction is a major risk factor in developing diabetes. Therefore, understanding the developmental regulatory networks that define a fully functional beta cell is important for elucidating the genetic origins of the disease. Aldehyde dehydrogenase activity has been associated with stem/progenitor cells and we have previously shown that Aldh1b1 is specifically expressed in pancreas progenitor pools. Here we address the hypothesis that Aldh1b1 may regulate the timing of the appearance and eventual functionality of beta cells.MethodsWe generated an Aldh1b1-knockout mouse line (Aldh1b1tm1lacZ) and used this to study pancreatic development, beta cell functionality and glucose homeostasis in the absence of Aldh1b1 function.ResultsDifferentiation in the developing pancreas of Aldh1b1tm1lacZ null mice was accelerated. Transcriptome analyses of newborn and adult islets showed misregulation of key beta cell transcription factors and genes crucial for beta cell function. Functional analyses showed that glucose-stimulated insulin secretion was severely compromised in islets isolated from null mice. Several key features of beta cell functionality were affected, including control of oxidative stress, glucose sensing, stimulus-coupling secretion and secretory granule biogenesis. As a result of beta cell dysfunction, homozygous mice developed glucose intolerance and age-dependent hyperglycaemia.Conclusions/interpretationThese findings show that Aldh1b1 influences the timing of the transition from the pancreas endocrine progenitor to the committed beta cell and demonstrate that changes in the timing of this transition lead to beta cell dysfunction and thus constitute a diabetes risk factor later in life.Gene Expression Omnibus (GEO) accession: GSE58025Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-015-3784-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Abstract 5148: NR5A2 is essential for pancreas development and affects pancreas carcinogenesis

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers today. A genome-wide association study (GWAS) has implicated the nuclear receptor NR5A2 in modulating risk for pancreatic cancer. While a role for this gene in pancreatic cancer has not been previously recognized, it plays an important role in stem cell pluripotency and metabolism. Using the zebrafish model, we demonstrate that genetic or pharmacologic inhibition of nr5a2 activity results in absence of exocrine pancreas, while leaving endocrine pancreas unaffected, indicating an essential role in pancreas organ formation. Further, we localize nr5a2 expression to the endodermal bud that gives rise to the exocrine pancreas, consistent with a role for nr5a2 in regulating differentiation of pancreas progenitors. In resected human PDAC specimens and in vitro cell lines, we find that NR5A2 expression is altered compared to normal ductal epithelium. Moreover, shRNA knockdown of NR5A2 dramatically alters proliferation of PDAC cell lines, supporting the hypothesis that NR5A2 is involved in pancreatic carcinogenesis. These data support a model that NR5A2 regulates the proliferation and differentiation of exocrine pancreas progenitor cells during development. We postulate that loss-of-function of NR5A2 results in dysregulation of these processes in the adult pancreas by driving acinar cells to acquire properties of progenitor cells, and therefore is an important step in pancreas oncogenesis. Note: This abstract was not presented at the meeting. Citation Format: Sahar Nissim, Olivia Weeks, John Hedgepeth, Julia Wucherpfennig, Xiao-Xu Wang, Alec Kimmelman, Wolfram Goessling. NR5A2 is essential for pancreas development and affects pancreas carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5148. doi:10.1158/1538-7445.AM2015-5148

Abstract LB-130: Reporter genes for a rapid in vivo screen of PDA therapeutics are required for energy homeostasis in pancreatic cancer-associated malnutrition

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths. Limited progress in developing effective therapy for PDA is partially due to the lack of a robust in vivo screen for effective drug combinations. Kras mutations (e.g. KrasG12D) are found in over 90% of human PDA and occur early in tumor progression. G Protein Coupled Receptor (GPCR) and protein kinase signaling can initiate Ras activation. Regulators of G-protein Signaling (RGS) proteins are coincidence detectors of Ras activation that feedback regulate, by virtue of their GTPase Activating Protein (GAP) activity, the intensity and duration of Gi- and Gq-coupled GPCR signaling. RGS-resistant mutations in Gq have been associated with PDA. We show a Rgs16::GFP transgene is a KrasG12D-dependent marker of all stages of neoplasia in the LSL-KrasG12D; Cdkn2af/f; p48Cre (KIC) mice. GFP is proportional to and coincident with tumor burden. Although KrasG12D is expressed in embryonic pancreas progenitor cells and in all mature acinar cells, Rgs16::GFP expression in tumors first emerges in ductal PanINs as early as 12 days post birth. The receptor tyrosine kinase Axl is highly expressed in PDA progenitor cells. The Gas6 ligand evokes Axl signaling in epithelial progenitor cells and contributes to activation of KrasG12D, PDA initiation and progression. In a proof-of-principle for drug screens, we determined that warfarin, which blocks maturation of Gas6, an Axl agonist, combined with the standard of care Gemcitabine and Abraxane (GA), significantly reduced PDA progression. In humans, partial pancreatic deficiency often precedes pancreatic cancer. Pancreatic insufficiency develops by 5 weeks in KC (LSL-KrasG12D;p48Cre) mice that express KrasG12D in all pancreas cells. KrasG12D, in the context of wild type Cdkn2a, causes dedifferentiation of acinar cells and a drastic reduction in digestive enzymes secreted by the pancreas. KC mice become malnourished but can survive over one year before succumbing to PDA. We find Intraductal Papillary Mucinous Neoplasm (IPMN) in KC mice express Rgs16::GFP by 2 weeks of age. We crossed the Rgs8-16 double knockout into KC (KC-R) mice to test if Rgs8-16 are tumor suppressor genes. Most KC-R mice die before 4 months of age because they can not maintain energy homeostasis - Rgs8-16 are required in liver to conserve energy utilization in malnourished mice. The effects of Rgs8-16 deficiency on exocrine pancreas function, acinar-to-ductal metaplasia (ADM), apoptosis and tumor progression in KC-R mice are under investigation. As a reporter gene, Rgs16::GFP faithfully tracks PDA progression and sensitivity to new drug regimens that inhibit KrasG12D mediated oncogenesis. Supported by NCI CA161624. Citation Format: Ozhan Ocal, Yalda Zolghadri, Galvin H. Swift, Rolf A. Brekken, Thomas M. Wilkie. Reporter genes for a rapid in vivo screen of PDA therapeutics are required for energy homeostasis in pancreatic cancer-associated malnutrition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-130. doi:10.1158/1538-7445.AM2015-LB-130