Small molecule-induced pancreatic exocrine transdifferentiation:

Assay development

Kristin RoseBridget Wagner, Ph.D.

Broad Chemical Biology, Broad Institute of MIT & HarvardSummer 2008

Pancreas

• Endocrine

– Islets of Langerhans – large spherical cellular clusters

– Secrete hormones – glucagon – insulin– somatostatinPP – 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

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)

Gene Fold difference of INS-1 to AR42J

Insulin 7786

Gcg --

PP 0.38

Pdx1 2771.91

Glut2 250.73

MafA 68.12

Nkx2.2 47.95

Pax4 15.69

Beta2 11.52

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

0

1

2

3

4

5

NT HGF+Act GLP-1 exen-4

Treatment

Fo

ld C

han

ge

Assay Development 2: Insulin protein expression

MIN6 cells (mouse beta)

(1°)

(2°)

nuclei

insulin

Untreated AR42J Untreated 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 BiologyDeepika Walpita

Stefan Kubicek

Tammy Gilbert

Dina Fomina

Yuan Yuan

Danny Chou

Alex Gitlin

Yiying Xu

Bridget Wagner

Stuart Schreiber

Broad/Summer Research Program in GenomicsMaura Silverstein

Lucia Vielma

Shawna Young

Bruce Birren

Eric Lander