Steven D. Leach - US grants

Recent investigations have suggested that preoperative chemoradiation may offer improved local control in patients with resectable adenocarcinoma of the pancreatic head. However, current chemoradiation protocols are characterized by poor response rates and no instances of complete tumor regression. It is now apparent that the resistance of many human malignancies to the effects Of chemoradiation may be due to protective cell cycle responses following DNA damage. Recent investigations have demonstrated that taxol is capable of altering the cell cycle response to DNA damage in pancreatic adenocarcinoma cells, and also to induce apoptosis. Thus taxol-baeed chemoradiation may provide a novel mechanism for manipulating the cell cycle in pancreatic cancer cells, thereby allowing for effective tumor downsizing prior to surgery. A recent phase I study of taxol-based chemoradiation in patients with unresectable pancreatic cancer suggested a response rate approaching 30%, far greater than that observed with previous fluorouracil (5FLJ)based protocols. We therefore propose to investigate taxol-baeed chemoradiation in patients with potentially resectable adenocarcinoma of the pancreatic head under the auspices of a Phase III clinical trial. This proposed study emphasizes strict pre-treatment staging to include only those patients with localized, resectable disease, as well as careful assessment of treatment response using radiographic (i.e. CT, endoscopic ultrasound), metabolic (FDG- PET scanning), and molecular techniques (TUNEL assay for treatment-induced apoptosis).

DESCRIPTION (provided by applicant): Previous work supported by this RO1 has suggested that the initiation of pancreatic tumorigenesis may involve cells with an undifferentiated precursor phenotype. The long-term goal of this research program remains the identification and eventual therapeutic manipulation of exocrine precursor cells as the probable "cell of origin" for pancreatic ductal cancer. In developing mouse and zebrafish pancreas, exocrine precursor cells are characterized by expression of a panel of markers, including nestin, hesl1 and ptf1-p48. Additional data now suggest that these cells also express vertebrate orthologs of Drosophila musashI an RNA binding protein known to be required for maintenance of precursor phenotypes in the developing nervous system. In developing pancreas, musashi is expressed in undifferentiated pancreatic epithelium, but not in differentiated pancreatic cell types. These observations generate the hypothesis that musashi is also required to maintain an undifferentiated precursor population in developing pancreas, and that undifferentiated musashi-positive epithelial cells represent direct precursors for the exocrine lineage. To test these hypotheses, the following ISpecific Aims will be pursued: First, to characterize temporal and spatial patterns of musashi expression in developing mouse and zebrafish pancreas, and to determine gain-of-function and loss-of-function phenotypes associated with altered musashi expression; Second, to determine the differentiation potential of! musashi-expressing precursor cells in developing mouse pancreas using formal lineage tracing techniques; and Third, to identify and characterize target RNA's bound by musashi in developing mouse and zebrafish pancreas, anticipating that these targets will represent positive regulators of pancreatic epithelial differentiation. Together, these studies will provide important information regarding exocrine precursor populations in developing pancreas. The pursuit of these studies in both mouse and zebrafish model systems will allow development of insights not easily ascertained using any single approach. By delineating mechanisms by which an undifferentiated precursor pool is maintained in developing pancreas, these studies will further advance our understanding of pancreatic development, pancreatic regeneration and pancreatic cancer.

The aims of the Pancreas Morphology core are threefold: 1) To provide centralized access to fixed, embedded, and sectioned pancreatic tissue, including timed harvest form mouse embryos; 2) to perform immunohistochemical /immunofluorescent staining and in situ hybridization services in support of the different projects; and 3) to perform isolation and in vitro organ culture of embryonic pancreatic tissue. A central tenet of this research program is that genes regulating pancreatic beta cell function must be considered in the context of an integrated tissue, leading to a heavy reliance on morphologic evaluation. It has also become clear that many genes required for normal beta cell function (e.g. Pdx1, Nkx6.1, Pax4, Pax6) also play critical role sin embryonic pancreatic development (1-3), requiring ready access to both adult and embryonic pancreatic tissue. By providing shared services for evaluation of tissues generated by the different projects, this core will serve as an important resource for the entire program. The provision of embryonic pancreatic tissue will enable each new mouse line to be characterized in terms of developmental phenotype. Application of a detailed battery of islet, acinar, and ductal lineage markers will provide a comprehensive analysis of phenotypes arising in different targeted mouse strains. In addition, the ability to propagate embryonic epithelial tissue in vitro will further extend studies regarding proliferation and differentiation in the different models.

The signaling pathways regulating differentiation of the exocrine pancreas during embryonic development are poorly understood. These pathways may play a critical role in several human disease states, including chronic pancreatitis and pancreatic ductal adenocarcinoma. The long term goal of this research project is to identify molecular pathways responsible for controlling precursor cells in exocrine pancreas. Preliminary studies suggest that changes in acinar cell differentiation may be regulated by novel interactions between the EGF and Notch signaling pathways. Specifically, it has been demonstrated that TGF-alpha alters the differentiation status of acinar cells in mouse pancreas, reactivating a developmental program characterized by expansion of embryonic-like epithelium. The effects of TGF-alpha on exocrine differentiation appear to be mediated by activation of Notch signaling pathways, as evidenced by transactivation of Notch target genes and the ability of activated Notch-1 to induce similar effects. Moreover, selective inactivation of the Notch partner RBP-J-kappa in exocrine precursor cells results in pancreatic hypoplasia and a failure to generate mature acinar and ductal cell types. Based on these findings, we propose that Notch signaling is iteratively utilized to regulate normal development of the exocrine pancreas, and that abnormal Notch activation may be responsible for generating developmental plasticity and epithelial proliferation in the adult gland. To test this hypothesis, the following Specific Aims will be pursued: First, to investigate the role of Notch signaling in acinar cell differentiation during normal pancreatic development. Second, to examine the role of Notch signaling during transdifferentiation events in adult pancreas. Finally, to determine the effects of lineage-specific manipulation of Notch signaling in mouse exocrine pancreas, utilizing both transgenic and Cre/lox-based approaches. To accomplish these studies, the investigators have established a multidisciplinary team of scientists with noted expertise in the fields of pancreatic cell biology, Notch signaling, and mouse gene targeting. It is anticipated that these studies will provide important new insights regarding regulation of exocrine pancreatic precursor cells, and potentially generate new therapeutic strategies for diseases of the exocrine pancreas.

DESCRIPTION (provided by applicant): Recent advances in automated robotics, high-resolution digital imaging and image deconvolution software now allow real-time documentation of dynamic processes in living cells and tissues. To take advantage of this progress, we have assembled an accomplished group of senior, mid-career, and junior investigators who wish to apply this emerging technology to their NIH-funded research projects. Together, these six Johns Hopkins faculty members serve as principle investigators on ten different NIH grants including nine R01 grants and one T32 training grant, together accounting for 51 years of cumulative uninterrupted NIH funding. These research projects are funded by six Jdifferent NIH institutes, reflecting a a wide array of research areas, including epithelial and hematopoietic stem cell biology, development biology, developmental neurobiology, macrophage function, and tumor biology. In order to apply recent advances in real time imaging to these projects, the investigators seek to purchase a fully integrated state-of-the-art imaging system comprised of a Zeiss Axiovert-200M motorized inverted fluorescent microscope, a Zeiss AxioCam high-resolution digital camera, a temperature / humidity / CO2-controlled stage chamber for maintenance of viable cell and tissue explants, and sophisticated Zeiss Axioversion software with 3D image deconvolution capability. Together, this Cell Observer System allows dynamic cellular processes to be observed at high resolution using multiple fluorescent channels over extended periods of time, generating new perspectives regarding integrated biologic processes in living tissues. This proposal outlines the scientific rational for use of this sophisticated imaging system, and further describes available institutional support as well as administrative and laboratory resources designed to promote optimal use of this shared instrumentation.

[unreadable] DESCRIPTION (provided by applicant): The identity and location of epithelial precursor cells in exocrine pancreas remain unknown. Further characterization of this precursor population would represent a significant advance in our understanding of pancreatic development, pancreatic regeneration and pancreatic cancer. While studies in mice and other rodents have provided important initial insights, these species have significant limitations related to many aspects of genetic manipulation. The zebrafish (Danio rerio) has recently emerged as a central model organism in the study of vertebrate development. The increasingly widespread use of the zebrafish reflects significant advantages with respect to embryo access, simplified techniques for gain-of-function and loss-of-function analysis, and forward genetic capabilities. Recent studies have suggested that many aspects of pancreas development in the zebrafish are conserved with respect to other vertebrate species. Based on a recent NIDDK program announcement requesting proposals for innovative and exploratory research involving pancreatic stem cells and/or novel animal models (PA-01-129), we have embarked on initial studies of zebrafish exocrine pancreas development. The long-term goal of this research program is to identify and rigorously characterize exocrine pancreatic precursors in zebrafish pancreas, ultimately allowing use of this organism as a novel model system for study of human pancreatic disease. Our preliminary studies have involved cloning and characterization of the zebrafish orthologue for PTF1a-p48, a basic helix-loop-helix transcription factor required for normal pancreas development in the mouse. Initial gene expression and p48 morpholino knock-down studies have suggested fundamental and informative differences between zebrafish and mouse with respect to the relationship between endocrine and exocrine precursor pools. Based on these findings, we propose that identification and characterization of p48-positive precursors in zebrafish pancreas will allow investigation of conserved regulatory mechanisms not discernable in the mouse, and that novel regulators of pancreatic development will be identified among genes differentially expressed in wild-type vs. p48 morphant embryos. To test these hypotheses, the following Specific Aims will be pursued: First, to rigorously determine the identity, location, and gene expression profiles of p48-positive precursor cells in developing zebrafish pancreas. Second, to determine the contribution of p48-positive precursors to endocrine and exocrine lineages; and finally, to identify novel p48 target genes responsible for mediating the effects of p48 on early pancreas development. It is anticipated that these studies will provide important new insights regarding regulation of exocrine pancreatic precursor cells, and further establish the zebrafish as a novel model system for the study of epithelial differentiation in exocrine pancreas. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Pancreatic progenitor cells represent an important resource for cell replacement therapy in diabetes, and may also represent the cell-of-origin for pancreatic cancer. Among the many transcription factors regulating pancreatic development and differentiation, expression of the basic helix-loop-helix transcription factor, ptf1a-p48, represents the defining feature of pancreatic progenitor cells. Several laboratories, including our own, have identified distinct early and late effects of ptf1a-p48 during pancreatic development. Early in development, ptf1a-p48 is widely expressed in the emerging pancreatic epithelium, where it is required for specification of pancreatic identity, as well as for early growth and morphogenesis. Later in development, ptf1a-p48 becomes restricted to the exocrine compartment, where it induces cell cycle exit and acinar cell differentiation. However, the mechanisms by which ptf1a-p48 exerts its different developmental stage-specific effects remain unknown. We now propose to comprehensively characterize both early and late ptf1a-p48 functions, taking advantage of our unique ability to study pancreas development in both mouse and zebrafish. This work will be based on the following central hypotheses: First, that the early and late effects of ptf1a-p48 on pancreatic development are mediated through entirely different sets of ptf1a-p48 target genes; second, that different structural domains of the ptf1a-p48 molecule may be required for induction of these early and late effects; and third, that ptf1a-p48 target genes may include both coding and non-coding elements, including functionally important microRNA's. To test these hypotheses, the following Specific Aims will be pursued: 1) To identify discrete domains of the ptf1a-p48 molecule responsible for mediating its developmental stage-specific effects on pancreatic development, using newly developed in vivo assays; 2) To identify novel ptf1a-p48 target genes through genome-wide ChIP-on-chip analysis, and determine gain-of-function and loss-of-function phenotypes associated with altered expression of these genes; and 3) To identify and functionally characterize novel ptf1a-p48-regulated microRNA's in developing mouse and zebrafish pancreas. Together, these studies will determine the mechanisms by which ptf1a-p48 exerts its multiple influences in developing mouse and zebrafish pancreas. In so doing, it is likely that we will also identify important new regulators of pancreatic specification, morphogenesis and differentiation. By clarifying the role of this important transcription factor in regulating the pancreatic progenitor pool, these studies will contribute to the eventual therapeutic manipulation of these cells in the context of pancreatic cancer and diabetes. Public Health Relevance: Pancreatic progenitor cells represent an important resource for cell replacement therapy in diabetes, and may also represent the cell-of-origin for pancreatic cancer. Studies in this proposal focus on how Ptf1a-p48, a pancreatic transcription factor, regulates the initial specification, growth and differentiation of pancreatic progenitor cells. We have recently completed two genome-wide screens for genes that may act downstream of Ptf1a-p48. In so doing, we have identified a number of additional coding and non-coding genes that may play important roles in regulating the pancreatic progenitor pool. Using both mouse and zebrafish model systems, we now plan to functionally characterize these novel Ptf1a-p48 target genes. By better clarifying how Ptf1a-p48 regulates progenitor cell growth and differentiation, these studies will contribute to the eventual therapeutic manipulation of pancreatic progenitors in the context of pancreatic cancer and diabetes.

DESCRIPTION (provided by applicant): The past decade has witnessed unprecedented progress in our understanding of the genetic changes underlying human pancreatic cancer. Led by a long series of important contributions by Program investigators, the genetic and epigenetic basis for pancreatic cancer has slowly been unraveled, with over 30 genetic loci now recognized to undergo mutation in this disease, and characteristic changes in gene expression reported for an even larger number of coding genes and non-coding microRNAs. However, the functional contribution of these mutations to the pancreatic cancer phenotype remains largely uninvestigated. The central Aim of this Program Project Grant is to narrow the gap between genetic and functional data, by determining the functional significance of molecular alterations identified in human pancreatic cancer. This functional annotation will be pursued by examining the impact of candidate dominant and recessive mutations, as well as characteristic changes in microRNA expression, using novel cell-, zebrafish- and mouse-based assays developed by members of our group. Four projects will be pursued: Project 1: Functional annotation of human pancreatic cancer genes in a zebrafish system (Leach);Project 2: Discovery and evaluation of prioritized mutations in human pancreatic cancer (Kern);Project 3: The Hippo signaling pathway in pancreatic cancer (Maitra);and Project 4: Functional evaluation of microRNAs in pancreatic neoplasia (Mendell). These projects will be supported by three shared Cores: Core A: Cellular and Transgenic Phenotyping Core (Huso);Core B: Zebrafish Core (Parsons), and Core C: Administrative Core (Leach). The investigators pursuing these projects are all leaders in the field of pancreatic cancer research, and have an outstanding track record of synergistic interaction. The Program also leverages many already existing resources available at Johns Hopkins, including aspects of the NCI Gl SPORE grant, core resources provided by the Johns Hopkins Comprehensive Cancer Center, and the independently funded Pancreatic Cancer Genome Project. Together, these studies will dramatically accelerate the functional annotation of the pancreatic cancer genome, setting the stage for future therapeutic applications.

Pancreatic cancer remains one of the most deadly human malignancies. During the past decade, unprecedented progress has been made identifying the genetic basis for this disease, including the discovery of a number of common somatic mutations now confirmed to play important pathogenic roles. However, the recent application of whole genome deletion analysis and high throughput DNA sequencing has accelerated the rate of novel mutation detection well beyond any ability to functionally evaluate identified candidate genes. This mismatch between gene discovery and functional annotation will only increase with the completion of the already in-progress sequencing of the pancreatic cancer genome, an effort currently being pursued by investigators here at Johns Hopkins. In order to alleviate this bottleneck, and provide a system for higher throughput annotation of the pancreatic cancer genome, we have generated the first zebrafish model of exocrine pancreatic cancer. Based on the low costs and modest floorspace required to maintain adult zebrafish, as well as the ability to rapidly generate large numbers of transgenic lines, this organism offers many advantages in evaluating the molecular basis of human cancer. When an oncogenic version of human KRAS is expressed in developing zebrafish pancreas, pancreatic progenitor cells fail to undergo normal exocrine differentiation, leading to the subsequent formation of invasive pancreatic cancer. Zebrafish pancreatic cancers invade and metastasize, and exhibit many features in common with the human form of the disease, including abnormal activation of hedgehog signaling. In addition creating the first zebrafish model of exocrine pancreatic cancer, we have successfully generated transgenic lines in which a modified Gal4 transcriptional activator is expressed in pancreatic progenitor cells. Using transposon technology to insert UAS-regulated transgenes into the zebrafish genome, we now have the opportunity to functionally evaluate a wide variety of genetic lesions for their ability to modify pancreatic cancer initiation and/or progression, achieving a level of throughput not technically feasible in the mouse. Using these techniques, we now plan to pursue the following Specific Aims: First, to functionally annotate candidate dominant mutations identified in the pancreatic cancer genome, through their modular introduction into the zebrafish tumorigenesis model; second, to study the effects of graded changes in hMYC expression in pancreatic tumorigenesis, using an inducible Gal4/UAS system targeting progenitor cells in zebrafish exocrine pancreas; and third, to develop Cre-based models of KRAS-mediated pancreatic neoplasia in zebrafish. Together, these studies will provide important new information regarding the genetic basis for pancreatic cancer, allowing for the more rapid development of effective targeted therapies.

The Administrative Core will provide logistic, administrative and budgetary support for all projects and cores supported by this P01 grant. The core will be directed by Dr. Steven Leach, PI for the overall grant, and staffed by Ms. Jennifer Holcomb, a supervisory-level administrative assistant with over fifteen years of experience here at Johns Hopkins. The Core will be responsible for fiscal management of overall program, and generating budgetary reports for each of the other Projects and Cores, in conjunction with research administrative personnel and financial managers from the Departments of Surgery, Oncology and Pathology, and also the Institute for Genetic Medicine. In addition, the Core will provide clerical support assisting investigators in the preparation of manuscripts, preparation of non-competing and competing renewals, and coordinating the distribution of plasmids, cell lines and transgenic animals requested by outside investigators. The Core will also be responsible for insuring that all investigators maintain strict compliance with relevant regulatory bodies here at Johns Hopkins, including the Animal Care and Use Committee, the Institutional Review Board, and the Radiation Safety Committee. The Core will also be responsible for establishing and maintaining the Johns Hopkins Pancreatic Cancer Program Project Grant Website, which will serve as a focal point for sharing information both among Program Participants, and also with the wider scientific community. This will be provided as a direct link from the already established Sol Goldman Pancreatic Cancer Research Center website (http://www.path.ihu.edu/pancreas). and will include both secure and non-secure portals for data and reagent sharing. Finally, the Core will broadly support communication and interaction between Program investigators, by coordinating the monthly Steering Committee meeting, and also arranging the monthly Sol Goldman Seminar Series, including assisting with travel arrangements, lodging and reimbursement for outside speakers. The Administrative Core will act as the primary liaision between Program Participants and the Internal and External Advisory Boards, and will schedule and provide logistical support for the Annual Program Retreat and Advisory Board meeting. The Core will be house in 200 sq. ft. of dedicated administrative space immediately adjoining the offices of Dr. Leach, Dr. Mendell and Dr. Parsons.

DESCRIPTION (provided by applicant): This S10 Shared Instrumentation Grant application seeks funding in support of confocal live imaging resources for the Johns Hopkin University's Center for Functional Investigation in Zebrafish, also known as FinZ Center. FinZ Center was established in 2008 under joint funding from the School of Medicine, the Institute for Cellular Engineering (ICE), and the Johns Hopkins McKusick-Nathans Institute of Genetic Medicine (IGM). The Center is currently led by three scientific co-directors, Steven Leach, Andrew McCallion and Michael Parsons, each of whom is a noted investigator in zebrafish genetics and zebrafish developmental biology. Together with Dr. Joshua Mendell, another IGM faculty member pursuing work in zebrafish, this group will comprise the Major User Group for the proposed Zeiss LSM 710 confocal microscope. These four Major Users are all highly regarded investigators in a variety of scientific fields with direct impact on human health, including pancreatic development, pancreatic cancer, neuronal differentiation, b-cell regeneration, computational prediction of gene regulatory sequences, neural crest biology and the role of microRNAs in human malignancy. Together, they serve as PI's or co-Pis on seven NIH R01 or P01 grants, funded by four different NIH Institutes (NIGM, NICHD, NIDDK and NCI). Based on the Major User Group's common need for live imaging of zebrafish embryos and other tissues in support of their NIH-funded work, this application seeks funding for a Zeiss LSM 710 3-channel spectral confocal system, with an environmental control system to support long-term imaging of zebrafish embryos, tissues and cells. Justification for this request is based on the following documented elements: 1) the Zeiss 710 system offers an unprecedented signal-to-noise ratio associated with a unique scan head design, providing the opportunity to apply high resolution spectral imaging to deep-seated zebrafish tissues;2) the fact that Johns Hopkins Microscopy Core resources here at Johns Hopkins are already fully subscribed and therefore cannot accommodate the 10-36 hour time lapse imaging protocols required by the Major User Group;and 3) consistent with the broader goals of the American Recovery and Reinvestment Act, the ordering, shipping, installation, maintenance and daily use of this microscope will directly stimulate multiple segments of the U.S. economy, and lead to the direct hiring of new technical staff.

DESCRIPTION (provided by applicant): Endogenous pancreatic progenitor cells represent a potentially important resource for cell replacement therapy in diabetes, and may also act as cells of origin for pancreatic cancer. However, the presence, location and identity of multipotent progenitor cells in adult vertebrate pancreas remain unknown. Among the various pancreatic cell types proposed as candidate progenitors, centroacinar and terminal duct cells remain perhaps the most poorly characterized. We have developed a novel strategy to both visualize and isolate a low abundance subset of these cells from adult mouse and zebra fish pancreas, and generated exciting preliminary data suggesting that these cells carry a multipotent progenitor capacity. This strategy involves isolating cells based upon high expression of the retinoic acid-synthesizing enzyme, Aldehyde Dehydrogenase 1 (ALDH1). During development, ALDH1-expressing cells are located in the "tips" of the branching pancreatic epithelium, a location recently associated with progenitor activity. In adult pancreas, they are frequently observed in centroacinar and terminal duct locations. When isolated by FACS and analyzed by RT-PCR, ALDH1-expressing centroacinar and terminal duct cells showed no expression of differentiated lineage markers, but did show enrichment of transcripts for both c-Met and SDF1, markers previously associated with progenitor cells in pancreas and other tissues. When placed into suspension culture, ALDH1-expressing cells are uniquely able to form "pancreatospheres", and over time these spheres generate functional endocrine and exocrine cells. In addition, the spheres maintain a subpopulation of cells expressing ALDH1, Hes1 and Sox9, suggesting a capacity for self-renewal. When ALDH1-expressing cells are isolated from adult mouse pancreas and injected into micro-dissected E12.5 pancreatic dorsal buds, they are uniquely able to contribute to the emerging endocrine and exocrine lineages. Based on these exciting studies, we hypothesize that ALDH1-expressing pancreatic epithelial cells may function as multipotential progenitors in both in mouse and zebra fish pancreas, and also serve as important local signaling centers by virtue of their ability to synthesize retinoic acid. We plan to test these hypotheses through the application of rigorous in vivo lineage tracing techniques, as well as the independent ablation of both ALDH1 enzymatic activity and ALDH1-expressing cells, using both mouse and zebra fish model systems. These studies will set the stage for the eventual expansion and direct manipulation of these cells, potentially providing a novel pancreatic progenitor population that can be harnessed to generate new b-cells in the context of either endogenous regeneration and/or exogenous replacement therapy. PUBLIC HEALTH RELEVANCE: Pancreatic progenitor cells represent a potentially important resource for cell replacement therapy in diabetes. This project will characterize a novel population of progenitor cells in mouse and zebra fish pancreas, in hopes that they will provide a renewable resource for replacement b-cells.

DESCRIPTION (provided by applicant): Consistent with the multiple objectives of PA-08-208, "Pilot Studies in Pancreatic Cancer", we are proposing to develop a novel experimental model of human pancreatic cancer that will facilitate the identification of novel genetic and epigenetic aberrations underlying PanIN formation. The central hypothesis guiding this work is that by studying molecular and cellular changes at the very earliest time points following oncogenic Kras activation, even prior to the onset of morphologic PanIN formation, we will gain unique insight into the mechanisms underlying the true initiation of pancreatic cancer, in a manner that will allow effective chemoprevention and/or pharmacologic termination of PanIN progression. In pursuit of this goal, we are proposing to study the initiation and progression of pancreatic cancer with unprecedented temporal and spatial resolution. We are currently generating a new mouse model in which a lox-stop-lox-GFP::KrasG12D cassette encoding a GFP-KrasG12D fusion is knocked into the endogenous Kras locus. Unlike prior models, this approach will allow for the direct visualization and FACS-based isolation of specific cell populations in which oncogenic Kras has been activated, even prior to the onset of morphologic change. This will not only allow genetic, epigenetic and functional analyses to be performed with unprecedented temporal resolution, it will also allow for these analyses to be carried out on the level of individual cells, allowing an entirely novel view of cellular heterogeneity within both forming PanINs and invasive later lesions. To test the above hypothesis, we will pursue the following Specific Aims: First, we will visualize the earliest cellular responses to LSL-GFP::KrasG12D activation in the acinar and ductal compartments using a novel in vitro culture system, and will determine the signaling pathways mediating these responses;second, we will generate and compare high resolution temporal mapping of the pre-PanIN and PanIN transcriptomes following cell type-specific activation of LSL-GFP::KrasG12D in acinar and ductal lineages;and third, we will generate and compare high resolution temporal mapping of surface marker expression in individual cells following activation of LSL-GFP::KrasG12D in the acinar and ductal lineages. Together, these Aims will provide highly innovative and important insights into the earliest events in PanIN formation, as well as an initial glimpse at the onset of cellular heterogeneity during Kras-mediated neoplasia. PUBLIC HEALTH RELEVANCE: This application seeks funding to generate a novel murine model of human pancreatic cancer, in a manner that will allow the direct visualization, isolation and characterization of tumor cells with unprecedented spatial and temporal resolution.

DESCRIPTION (provided by applicant): The current rapid pace of scientific discovery in cancer research, particularly at the cellular, genomic, and molecular levels, offers unprecedented opportunities for rapid clinical application of basic scientific findings. This progress will be optimized by providing rigorous basic and clinical research training to a select group of surgical oncologists who are already directed towards academic leadership. The Specific Aim of this program is to provide formal training in scientific thought and technique to this select group of individuals. Two trainees per year will be competitively selected among trainees pursuing highly sought-after subspecialty fellowship training in either General or Thoracic Surgical Oncology. The selection process will identify and match the most qualified applicant, without regard to whether they are an internal or an external candidate. Trainees will elect to enter either the Laboratory Research Track or the Clinical Research Track. Each fellow will be guided to choose a senior preceptor/mentor from our list of participating faculty, all of whom have demonstrated an outstanding record of achievement in either laboratory or clinical cancer research, as well as an established record of successful trainee mentorship. The formal research training program begins after the required 18 months of post-residency surgical oncology training (our priority) or after the PG3 year of surgical residency, and would last for 2-3 consecutive years. This training period is uninterrupted by clinical duties, and includes formal training in research ethics as well as additional mandatory course work tailored to individual interests and capabilities. All trainees are assigned an Individual Fellowship Committee charged with critiquing the trainee's research, monitoring long-term progress towards an independent investigative career, and aiding in the selection of appropriate course work. The research training environment includes the graduate program in Cellular and Molecular Medicine, in which selected trainees may be awarded Ph.D. degrees, as well as the Graduate Training Program in Clinical Investigation, in which either Masters or PhD programs may be pursued. The specific recruitment of underrepresented minorities to this program is enhanced by broad representation of women and minorities among our Surgical Oncology and Thoracic Surgery faculty and proactive strategies described in the grant. In this manner, the program is designed to generate a highly selected group of General and Thoracic Surgical Oncologists who will develop original and significant research programs and provide academic leadership throughout the surgical and oncologic communities. RELEVANCE: Our proposed training program seeks to support formal research training for young surgical oncologists who will pursue careers in either laboratory or clinical cancer research, as they prepare for leadership roles in academic surgical oncology. This training grant will be a vital component in the national transition to a streamlined training program for academic surgical oncologists.

DESCRIPTION (provided by applicant): The current rapid pace of scientific discovery, particularly at the cellular, genomic, and molecular levels, offers unprecedented opportunities for direct application of basic scientific findings to the problem of clinical gastrointestinal disease. This progress will be optimized by providing rigorous training in research thought and technique to a select group of gastrointestinal surgeons who are already directed towards academic leadership. The Specific Aim of this program, currently in its fourteenth year of funding, is to provide formal training in either clinical or laboratory research to this select group of individuals as an integral component of their postgraduate clinical training. One trainee per year is competitively selected from those entering the Advanced GI Surgery training program at Johns Hopkins. During the first three years of postgraduate clinical training, this trainee is guided to choose a senior investigator/mentor from our list of participating faculty, all of whom have demonstrated an outstanding record of independent basic scientific achievement, as well as an established record of successful trainee mentorship. The formal scientific training period begins after the third clinical year, and lasts for two-three consecutive years. This training period is uninterrupted by clinical duties, and includes formal training in research ethics as well as other elective course work tailored to individual interests and capabilities. Many of our trainees complete our program with additional Masters or Doctoral degrees. All trainees are assigned an Individual Progress Committee charged with critiquing the trainee's research, monitoring long-term progress towards an independent investigative career, and aiding in the selection of appropriate course work. Our trainees are immersed in an outstanding educational environment, which includes the Johns Hopkins Bloomberg School of Public Health, the McKusick-Nathans Institute of Genetic Medicine, the NIDDK-funded Johns Hopkins Digestive Diseases Research Core Center, and the NCI- funded Sidney Kimmel Comprehensive Cancer Center. The specific recruitment of underrepresented minorities to this program is enhanced by the activities of the School of Medicine's Office for Diversity, as well networking and advertising through the Society of Black Academic Surgeons. To date, eight program trainees have completed their combined clinical and research training. All eight have achieved full-time faculty positions in prominent institutions, including Johns Hopkins, UPenn, University of Michigan, UCSD, UCSF and Thomas Jefferson. Four of the eight have already achieved extramural research funding. In this manner, the program is designed to generate a highly selected group of GI surgeon-scientists who will provide academic leadership for the 21st century.!

DESCRIPTION (provided by applicant): The recent explosion of interest in adult tissue-specific progenitor cells has reinforced the principle that progenitor cells never function autonomously; their behavior is always governed by signals emanating from their unique spatial niche. Translating this principle to the pancreas, we propose that successful therapeutic targeting of pancreatic stem/progenitor cells will require effective characterization and manipulation of their corresponding mesenchymal niche. We are therefore proposing an innovative and highly integrated research program to identify, characterize and manipulate the pancreatic progenitor mesenchymal niche in adult and embryonic pancreas. The proposal is based on our recent identification of both a low-abundance, self-renewing, multi-lineage epithelial progenitor cell population in adult mouse pancreas, and a corresponding mesenchymal niche cell capable of promoting progenitor expansion. Based on these exciting findings, we are proposing the following central hypotheses: First, that unique population of mesenchymal niche cells exist embryonic and adult mouse pancreas. Second, that niche identify is defined by the production and secretion of specific soluble morphogens. Third, that characterization of the niche cell secretome will allow the identification of specific morphogens responsible for regulating the proliferation, differentiation and self-renewal of embryonic and adult pancreatic progenitor cells. To test these hypotheses, we are proposing three Specific Aims. Specific Aims 1 and 2 share parallel approaches applied to the embryonic pancreas in Aim 1 and to normal and regenerating adult pancreas in Aim 2. For both of these Aims, we will characterize and quantify the relative abundance of different pancreatic mesenchymal cell subpopulations, test their ability to regulate the proliferative expansion, differentiation and sel-renewal of pancreatic epithelial progenitor cells, and identify relevant components of the niche secretome responsible for these effects. Having identified specific niche cell populations and associated secreted morphogens responsible for niche function, experiments in Aim 3 will seek to genetically manipulate adult pancreatic mesenchymal niche cells, using a combination of Cre/lox and rtTA/TRE technology for mesenchyme-specific, doxycycline-inducible expression of ActivinA and other secreted morphogens. Together, these Aims will provide an entirely novel and integrated view of pancreatic mesenchymal development and pancreatic niche biology, setting the stage for eventual therapeutic manipulation of the adult pancreatic progenitor mesenchymal niche.