Why Animal Models of Human Disease? Gregory J. Gores, M.D.

Pros Cons Recapitulate disease process Allow genetic manipulation Allow pharmacologic studies Necessary for FDA approval processes Cons Disease processes in animals and humans can be strikingly different Few large animal models due to expense Drug metabolism is different Models are time limited (days or months) vs. humans (years)

Ideal Animal Model of PSC Would Display Inflammatory bowel disease Large bile duct strictures Progression over time Loss of bile ducts Cirrhosis Develop bile duct malignancy Unfortunately, the ideal animal model of PSC does not exist!

Animal Models of Inflammatory Bowel Disease Toxic injury of the intestinal lining Genetic models – IL10 genetic deficiency Immune cell manipulation Genetic Adaptive transfer None of these models develop PSC!

What are we doing at Mayo Clinic? Acute model of PSC in mice Mdr2-/- chronic models of PSC in mice Genetic models of bile duct cancer in mice

SMAC MIMETIC ANIMAL MODEL BV6 (0.1 mg/100 L) Saline (100 L) CBD clamp CBD clamp, Gallbladder Injection CBD clamp release sacrifice 45 min Day 5

SM Intra-biliary Injection Results in a PSC-like Phenotype Sirius red αSMA Mac2 Saline M5 BV6 100 µm ALT Alk. Phos Bile Acids Total Bilirubin 1400 * 700 14 * Saline * 250 * 1200 600 12 BV6 200 1000 500 10 units/L 800 units/L 400 umol/L 150 8 mg/dL 600 300 6 100 400 200 4 200 50 100 2 saline BV6 saline BV6 saline BV6 saline BV6 (N=8) (N=8) * p<0.05

Cholangiographic Evidence of Intrahepatic Bile Duct Alterations in Mice Injected with the SM saline BV6

CONCLUSIONS & WORKING MODEL Cholangiocyte apoptosis macrophage cIAPs stellate cell Fibro-inflammatory cholangiopathy cholangiocyte Pro-inflammatory Cytokines & Chemokines

Mdr2 Knock Out Mouse as a Model of PSC Defect in cannalicular phospholipid transport PSC-like cholangiopathy in the mouse Obliterative biliary fibrosis Primary dysfunction is in hepatocytes, abnormal bile composition  cholestasis and chronic biliary inflammation and fibrosis Lazaridis & LaRusso, NEJM 2018 Lazaridis & LaRusso, Mayo Clin Proc 2016 Mariotti et al, Biochim Biophys Acta Mol Basis Dis 2018

Reichert MC and Lammert F. Seminars in Liver Disease 2018

Cenicroviroc – CCR2/CCR5 Inhibitor Oral drug which blocks monocyte/macrophage recruitment to the liver In clinical trials for PSC NCT02653625 Completed enrolling (25 patients) 24 week study

BV6 BV6 + cenicriviroc (CVC) CD68 C57BL/6 mice 5 days after intrabiliary BV6 injection, ± CVC (15 mg/kg, s.c., for 5 days) CVC: CCR2/CCR5 antagonist

CCR2 inhibition “Cenicriviroc” Cholangiocyte insult CCL2 Monocyte-derived macrophages Circulating monocytes CCR2 inhibition “Cenicriviroc” Bile duct Fibro-inflammatory cholangiopathy

Ductular Reaction Aberrant reparative response to liver injury1 Comprise of reactive or activated cholangiocyte-like cells2 Proliferative Pro-inflammatory Pro-fibrotic Form ductules and extend into liver parenchyma Progenitor characteristics Cholangiocyte or hepatocyte derived based on injury model EpCAM, SOX9, CK7 and CK19, (also markers of cholangiocytes) are used to identify ductular reactive cells in cholestasis Systemic injection of the cytokine TWEAK induces ductular reaction in normal mice3 Sato et al, Hepatology 2018 Banales et al, Nat Rev Gastroenterol Hepatol 2019 Bird et al, PNAS 2013 PanCK SOX9 The left most panel is the schematic from the first slide showing a normal bile duct and these ductules with irregular or obliterated lumens and dyspmorphic cholangiocytes. This is reflected in the immunohistochemistry images taken from one of my study mice. And the point here is that you have ductules and then you have these cells tracking into the parenchyma.

Question: Does MCL-1 inhibition reduce ductular reaction and fibrosis in vivo? Serum analysis Immunohistochemistry SOX9, PanCK, αSMA Sirius red, Masson-Trichrome staining S63845 40 mg/kg, IV, 5 days Mdr2-/-

S63845 reduces fibrosis: Mdr2-/- mice Vehicle S63845 Trichrome α-SMA Reduction in fibrosis correlates with attenuated activation of fibroblasts

Animal Models of Cholangiocarcinoma

Summary Animal models of PSC are important to understand mechanisms of disease and test preclinical therapies PSC Partners Seeking a Cure has helped support critical research THANKS FOR EVERYTHING YOU DO TO RAISE AWARENESS OF PSC AND SUPPORT RESEARCH RELEVANT TO THIS DISEASE

Acknowledgements Gores lab members Greg Gores Steve Bronk Eugenia Guicciardi Petra Hirsova Tomohiro Katsumi Anuradha Krishnan Ayano Niibe Genome Analysis Core Biomedical Statistics and Informatics Ying Li Optical Microscopy Core