Small molecule-induced pancreatic exocrine transdifferentiation: Assay development

1. Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute of MIT & Harvard Summer 2008

2. Pancreas • Endocrine • Islets of Langerhans – large spherical cellular clusters • Secrete hormones a – glucagon b – insulin d – somatostatin PP – pancreatic polypeptide • Exocrine • Acini – small berry-like clusters • Secrete digestive enzymes to intestine via ducts (trypsin, chymotrypsin…)

3. Type 1 Diabetes • Autoimmune – permanent destruction of insulin-producing β cells • No cure, lethal without insulin injections • Islet transplantation = last resort, but risk of rejection, requires lifelong immunosuppresant use • So what can we do?

4. Transdifferentiation • Process of switching between differentiated states • Goal: Increase β cell mass • Exocrine  insulin-secreting?

5. Project Cell Lines • AR42J & ARIP • Commercially available rat pancreatic exocrine • tumor cell lines • AR42J = acinar; ARIP = ductal

6. Project Cell Lines • Why? • Research literature shows these cell lines being induced to express insulin by various treatments, includingconophylline • Natural product-derived small molecule! • Encouragement to develop an assay

7. Assay Development 1: qPCR

8. Preliminary Results • Establish basal levels of expression • Quantitative comparison of untreated AR42J cells to INS-1 cells • INS-1 is a β cell-derived rat tumor cell line (insulinoma)

9. Testing to identify a positive control • Hepatocyte Growth Factor (HGF) • Literature-based positive control • Activin A • Peptide (TGF-b family) with role in endocrine function • Glucagon-Like Peptide-1 (GLP-1) • Promotes b cell function • Exendin-4 • Peptide analog of GLP-1, isolated from saliva of the Gila monster, a poisonous lizard • Trichostatin A (TSA) • Small-molecule histone deacetylase (HDAC) inhibitor

10. Treatment effects on insulin gene expression in AR42J cells

11. Assay Development 2: Insulin protein expression (1°) (2°) MIN6 cells (mouse beta) nuclei insulin

12. Assay Development 2: Insulin protein expression Untreated AR42J Untreated ARIP nuclei insulin

13. Summary and future directions • Plan: Screen for compounds to induce insulin expression in exocrine cells • First step: Assay development and identification of positive controls • Approach 1: gene expression • HGF+activin and exendin-4 increase ins mRNA in AR42J • Need to optimize TSA concentration • Need to optimize GLP-1 treatment • Approach 2: protein expression • Immunofluorescence-based approach • Ideal for high-throughput screening • But more stringent screening condition (all the way to protein) • Working on positive control conditions

14. Acknowledgements Broad Chemical Biology Deepika Walpita Stefan Kubicek Tammy Gilbert Dina Fomina Yuan Yuan Danny Chou Alex Gitlin Yiying Xu Bridget Wagner Stuart Schreiber Broad/Summer Research Program in Genomics Maura Silverstein Lucia Vielma Shawna Young Bruce Birren Eric Lander