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

Pancreas Endocrine –Islets of Langerhans – large spherical cellular clusters –Secrete hormones  – glucagon  – insulin  – somatostatin PP – pancreatic polypeptide Exocrine –Acini – small berry-like clusters –Secrete digestive enzymes to intestine via ducts (trypsin, chymotrypsin…)

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?

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

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

Why? –Research literature shows these cell lines being induced to express insulin by various treatments, including conophylline Natural product-derived small molecule! Encouragement to develop an assay

Assay Development 1: qPCR

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) GeneFold difference of INS-1 to AR42J Insulin7786 Gcg-- PP0.38 Pdx Glut MafA68.12 Nkx Pax Beta211.52

Testing to identify a positive control Hepatocyte Growth Factor (HGF) –Literature-based positive control Activin A –Peptide (TGF-  family) with role in endocrine function Glucagon-Like Peptide-1 (GLP-1) –Promotes  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

Treatment effects on insulin gene expression in AR42J cells

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

Untreated AR42JUntreated ARIP Assay Development 2: Insulin protein expression nuclei insulin

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

Acknowledgements Broad Chemical Biology Deepika Walpita Stefan Kubicek Tammy Gilbert Dina Fomina 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