MICHIGAN MEDICINE The Bcl-2 Inhibitor ABT-263 Attenuates Intestinal Fibrosis in Human Intestinal Organoids and the Mouse S. Typhimurium Model Eva S. Rodansky1, Laura A. Johnson1, Benjamin Malamet1,2, Sha Huang1, Stephen G. Collins1, Lisa Hazelwood3, Jason R. Spence1, Peter D.R. Higgins1 1University of Michigan, 2Oakland University, 3Abbvie

MICHIGAN MEDICINE Conflicts of Interest • ABT-263 is a proprietary compound owned by Abbvie • Abbvie provided funds for organoid and mouse experiments • Lisa Hazelwood is an employee of Abbvie

MICHIGAN MEDICINE The Problem with Intestinal Fibrosis • Inflammation initiates intestinal fibrosis • A gene expression signature of fibrogenesis at diagnosis predicts future surgery in children with Crohn’s • Over time, fibrosis becomes independent of inflammation Preclinical inflammatory (minimal symptoms) Symptomatic inflammatory (not yet diagnosed) IBD diagnosis made (start treatment) Bowel Damage Accumulating Inflammation Improves Fibrosis Keeps Going Kugathasan, et al., Lancet. 2017;389:1710-1718 Johnson, Higgins, et al., Inflamm Bowel Dis. 2012; 18:460-71.

MICHIGAN MEDICINE The Problem with Intestinal Fibrosis • Intestinal myofibroblasts function to • Lay down extracellular matrix (in ulcers) – patch the hole • Actively contract to close edges of wounds (ulcers) • Intestinal myofibroblasts become resistant to apoptosis in a stiff extracellular matrix – just keep generating scar • Anti-inflammatory therapy can treat inflammation – but can’t stop this. • Often biologics arrive too late to prevent surgery.

MICHIGAN MEDICINE Background and Hypotheses • Inhibitors of pro-survival Bcl2 proteins sensitize activated myofibroblasts to apoptosis and reduce pro-fibrotic gene expression • We hypothesized that ABT-263 would: • Reduce fibrogenic gene expression in intestinal myofibroblasts • Sensitize intestinal myofibroblasts to FasL-induced apoptosis • Reduce fibrogenic gene and protein expression in the human intestinal organoid (HIO) model of intestinal fibrosis. • Reduce fibrogenic gene and protein expression in the mouse Salmonella typhimurium model of intestinal fibrosis. Weder, B, Rogler, G, et al., IBD. 2018; 24: 1953-66.

MICHIGAN MEDICINE 1. Gene Expression in CCD-18co Human Intestinal Myofibroblasts Gene Percent Reduction From Peak Induction vs. Baseline Myosin Light Chain Kinase 131% Collagen 1 94% Fibronectin 36% a-Smooth Muscle Actin 84%

MICHIGAN MEDICINE 2. Apoptosis • Methods: • CCD-18Co human intestinal myofibroblasts were co-treated with 100 ng/mL Fas ligand (FASL) and a range of doses of ABT-263 for 5 hours, followed by protein isolation and western blot analysis of cleaved PARP. FASL µM ABT-263 - + + + + - 0 0 0.1 0.3 1 1 PARP GAPDH

MICHIGAN MEDICINE 2. Apoptosis • Results: • 100-300 nM ABT-263 sensitizes CCD-18Co myofibroblasts to FasL-mediated apoptosis over FasL alone, by cleaved PARP protein quantitation.

MICHIGAN MEDICINE 3. Organoid Fibrosis Model: “Stricture in a Petri Dish” • Modeling the scarring and obstruction of Crohn’s disease Some intestinal organoids have a robust mesenchymal layer Treatment with TGF-b activates an expansion of mesenchyme and the ECM fibrogenic gene program: Collagen, fibronectin, a smooth muscle actin, and myosin light chain kinase are all activated Rodansky, Higgins, et al. 2015. Exp Mol Path 98:346-51. MICHIGAN IBD ● IN VITRO MODELS OF FIBROSIS

MICHIGAN MEDICINE 3. Human Intestinal Organoid Fibrosis Model • Methods: • Human intestinal organoids (HIO) were co-treated with 2 ng/mL TGF-β and a low micromolar dose range of ABT-263 for 4 days • followed by isolation of RNA and qRT-PCR analysis • or by isolation of protein for western blot to analyze expression of fibrogenic genes and proteins.

MICHIGAN MEDICINE 3. ABT-263 Anti-fibrotic Efficacy vs. TGF-β induced Human Intestinal Organoid Gene Expression Gene expression: Data presented as random-effects meta-analysis of 4 distinct biologic replicates. Significant inhibition of expression of collagen and myosin light chain kinase is seen vs. TGFβ-mediated induction, with 10 uM ABT-263. ECM genes Fibronectin (FN1) Collagen 1(col1A1) Contractile genes Myosin light chain Kinase (MYLK) Alpha smooth muscle Actin (aSMA,acta2)

MICHIGAN MEDICINE 3. ABT-263 Anti-fibrotic Efficacy in HIO: αSMA Protein Expression Significant inhibition vs TGF-β-induction at 10 and 30 µM ABT-263.

MICHIGAN MEDICINE 3. HIO Fibrosis Model • Results: • ABT-263 at 10 µM significantly (p < 0.05) reduced the expression of COL1A1 and MYLK genes. • No significant reduction seen for FN or ACTA2. • αSMA protein expression was significantly reduced with 10 uM (p=0.046) and 30 uM ABT-263 (p=0.0099).

MICHIGAN MEDICINE 4. Mouse Salmonella typhimurium Model Day -1: Eradicate normal microbiota w/ streptomycin Day 0: Inoculate with 3 x 106 S. typhimurium Grassl, et al., Gastroenterology 2008; 134:768; Johnson, et al. J Crohns Colitis. 2017 Jun 1;11(6):724-736. • Infectious colitis & fibrosis • Control inflammation (day 8 eradication of S. typh by levofloxacin) Day 21: Harvest tissue NL S. typh MICHIGAN IBD ● RODENT FIBROSIS MODELS

MICHIGAN MEDICINE 4. Mouse S. typhimurium Fibrosis Model

MICHIGAN MEDICINE 4. Mouse S. typhimurium Fibrosis Model Colon weight 109% reduction

MICHIGAN MEDICINE 3. Mouse S. typhimurium Fibrosis Model Gene Expression 25% reduction

MICHIGAN MEDICINE 4. Mouse S. typhimurium Fibrosis Model Gene Expression 58% reduction

MICHIGAN MEDICINE 3. Mouse S. typhimurium Fibrosis Model Gene Expression 78% reduction

MICHIGAN MEDICINE 3. Mouse S. typhimurium Fibrosis Model Gene Expression 36% reduction

MICHIGAN MEDICINE 4. ABT-263 Anti-fibrotic Efficacy in mouse S. typhimurium: αSMA Protein 51% reduction

MICHIGAN MEDICINE 4. ABT-263 Anti-fibrotic Efficacy in mouse S. typhimurium: Collagen 1 Protein 41% reduction

MICHIGAN MEDICINE 4. ABT-263 Anti-fibrotic Efficacy in mouse S. typhimurium model of intestinal fibrosis • Gross Pathology • Gross colon weight decreased by ABT-263 at high dose • Gene expression • TGF-β and CTGF gene expression decreased >50% (low and high dose) by ABT-263 • Col1A1 (25%) and IGF-1 (36%) also decreased to a lesser extent • Protein expression • αSMA protein expression decreased 51% (high dose) • Collagen 1 protein expression decreased 41% (high dose)

MICHIGAN MEDICINE Conclusions • ABT-263 shows promise as an anti-fibrotic drug candidate in vitro, in 3D human organoid culture, and in a rodent model of intestinal fibrosis. • This compound appears to act in 2 ways: • Sensitizing activated myofibroblasts to apoptosis. • Reducing gene and protein expression of pro-fibrotic proteins • This compound has been shown to be safe in phase 1 trials in humans, now in phase 2 for solid tumors and ALL. • A prospective phase 2a study in stenotic CD is being planned.

MICHIGAN MEDICINE Thanks to • Laura A. Johnson • Benjamin Malamet • Stephen G. Collins • Eva S. Rodansky • Jason R. Spence • Sha Huang • Lisa Hazelwood & Abbvie

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MICHIGAN MEDICINE 3. Mouse S. typhimurium Fibrosis Model Histology

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