A novel Bcl-2 Inhibitor CB6 Attenuates Intestinal Fibrosis in a Myofibroblast cell model and the Mouse S. Typhimurium Model Yongjia Feng, Laura A Johnson, Meng Zhang, Steven Collins, Shaomeng Wang, Peter D.R. Higgins MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

Is Intestinal Fibrosis Reversible? Crohn’s strictures are no more likely to occur at the site of strictureplasty than anywhere else. The biology of the myofibroblast can be changed. Can we develop a ”strictureplasty in a pill”? MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

Background • A program of fibrotic gene expression at the time of diagnosis was the strongest predictor of future stricture & surgery in Crohn’s disease (RISK study in children, Kugathasan, et al., Lancet, 2017) • Intestinal myofibroblasts are activated by tissue stiffness, are resistant to apoptosis (FasL), and generate more EC matrix (positive feedback – progression of fibrosis) • Inhibitors of Bcl-2 (a mitochondrial anti-apoptotic protein) make activated myofibroblasts more sensitive to apoptosis. • Hypothesis: A gut-selective Bcl-2 inhibitor could act as an intestinal anti-fibrotic in vitro and in rodent models of Crohn’s disease. MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

Bcl-2 Inhibitor Candidate Pathway • In development for cancer therapy • To sensitize cancer cells to death signals • Bcl inhibitors: Anti-fibrotic efficacy seen for • Apoptosis sensitization of myofibroblasts • Fibrogenic gene expression program • Systemic off-target effects (thrombocytopenia, etc.) are acceptable in cancer (can we do better?) SMAC release Mito Outer Membrane Pores Form from Oligomers Of Bax and Bak (proapoptotic) Bax polymerized into a toroid pore as seen with Atomic Force Microscopy FEBS Lett doi.org/10.1111/febs.14186 B a k B a x MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT Bim 

Previous Bcl-2 inhibitor Data in our lab • 5 different Bcl-2 inhibitors all inhibit the stiffness-induced fibrosis phenotype in intestinal myofibroblasts • These include ABT-199, ABT-263, and ABT-737 • ↓ fibrogenic gene and protein expression • These also inhibit fibrogenesis in TGF-b models in cells and in human intestinal organoids • Bcl-2 inhibitor compounds can overcome the intestinal myofibroblast resistance to cell death • Not gut-selective, anti-fibrotic efficacy is only moderate. MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

Iterative Drug Candidate Development Lead candidate molecule Identify target Synthesize structural variants Screen for efficacy in vitro Test solubility, Caco2 permeability Evaluate stability, metabolism (rapid) Toxicity testing and pK in rodents Rodent model efficacy testing Learn Structure-Activity Relationships (SAR) Best Compounds for Testing in Humans Developing BH3-mimetic compounds to be more potent and gut-selective MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

Compound # exp Co1A1 gene Fn1 gene ACTA2 gene MYLK gene Cleaved-PARP protein CB-1 2 X 10 uM X X weak CB-2 2 X X 10 µM trending CB-3 1 X X X X CB-4 2 X X X X .3,1 uM,++ CB-5 3 X X X 10 uM .1,.3,1 uM CB-6 3 X 10 µM 10 µM 1,3,10 uM Meta: trend @ 10 µM .1,.3,1 uM +++ CB-10 3 X X X 10 uM 1 uM,+ CB-11 3 X X 10 µM 10 µM CB-12 2 X X X 10 µM CB-13 1 X X X CB-14 1 +/- X X CB-15 2 X X trending 10 µM 0.1,0.3,1uM, ++ Compound screening results in vitro, testing gene expression of fibrogenic genes & cleaved PARP 

- + + + + + + + + TGFβ SMA spiro CB6 CB15 GAPDH 0.1 0.3 1 0.1 0.3 1 um CB6 (and CB15) reversed TGFβ-driven aSMA increase in vitro 0.019 0.0011 0.12 0.0013 0 1 2 3 4 ratio to control SMA Group 1 2 3 4 5 6 7 8 9 Group 1 2 3 4 5 6 7 8 9 spirono CB6 CB15 a-smooth muscle actin Western blot, Normalized to GAPDH MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

MYLK - + + + + + + + + spiro CB6 CB15 0.1 0.3 1 0.1 0.3 1 GAPDH TGFβ COL1A1 0.23 0.05 0.32 0.011 0.0 0.5 1.0 1.5 2.0 ratio to control MYLK Myosin Light-chain Kinase (MYLK) and Collagen 1 Western blot, Normalized to GAPDH CB6 > CB15 reversed TGFβ-driven MYLK increase in vitro spirono CB6 CB15 Lane 1 2 3 4 5 6 7 8 9 Group 1 2 3 4 5 6 7 8 9 

Figure 1. Metabolic stability in human liver microsomes with NADPH Figure 2. Metabolic stability in mouse liver microsomes with NADPH Microsomal Stability CB6 was selected as candidate 5 24 5 24 0 2000 4000 6000 8000 Illeum-Dis compound concentration (ng/ml) Time point (hours) PK results 5 24 5 24 0 2000 4000 6000 Cecum compound concentration (ng/ml) 5 24 5 24 0 20 40 60 80 Liver compound concentration (ng/ml) 5 24 5 24 0 5 10 15 20 25 Plasma compound concentration (ng/ml) Time point (hours) Time point (hours) Time point (hours) CB6 CB15 CB6 CB15 CB6 CB15 CB6 CB15 Terminal Ileum 

Water DMSO Water CB6 1.0µM FASL DMSO FASL CB6 0.1µM FASL CB6 0.3µM FASL CB6 1.0µM Incubated for 5 hours Serum Starve overnight CCD18co cell (1X105/ML) Harvest cells Cell lysis for western blot Chamber slides for IF Cell lysis for Co-IP FASL:100nM Normal Human colon myofibroblast Can CB6 Overcome Apoptosis Resistance? MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

FASL CB6(um) - - + + + + 0 1.0 0 0.1 0.3 1.0 BAX GAPDH 3.6e−05 0.44 8.2e−06 0.001 0.00029 0.00019 0 2000 4000 6000 BAX % to Control % to Control CB6 Not only Inhibits Bcl-2, it Decreases Bcl-2 protein expression, & Increases the expression of the pro-apoptotic protein, Bax CB6 CB6 

Cleaved Cas3 Bcl-2 Merged FASL CB6 (um) _ + + + + 0 1.0 0 0.1 0.3 1.0 _ DAPI Cleaved-PARP Caspase3-total Cleaved Caspase3 GAPDH CB6 (uM) FASL - + 0 1.0 0 0.1 0.3 1.0 CB6 increased cleaved PARP and cleaved Caspase3 expression & led to apoptosis 

Testing for Bax & Bak Oligomeric Complexes • Pro-apoptotic – oligomerize into mitochondrial pores • IP for Bax, western blot for BH3 partners • Bax oligomers were increased by both FasL and CB6 • Bak and Bak-Bax oligomers slight increase by FasL, large increase by CB6 • Bax-Bim complexes increased by FasL, not by CB6 • CB6 does increase oligomerization of Bax and Bak • Likely increases activation of CytoC and SMAC 

Water DMSO Water CB6 1.0µM TGFβ DMSO TGFβ CB6 0.1µM TGFβ CB6 0.3µM TGFβ CB6 1.0µM Incubated for 48 hours Starving overnight CCD18co cell (5X104/ML) Collection cell Cell lysis for western blot Chamber slides for IF Cell lysis for Co-IP TGFβ: 0.05nM Normal Human colon myofibroblast Is CB6 Antifibrotic in vitro? 

0.039 0.053 0.36 0.47 0.04 0.0053 0 100 200 300 MYLK TGFβ CB6(uM) - - + + + + 0 1 0 0.1 0.3 1 α-SMA MYLK GAPDH TGFβ CB6(uM) - - + + + + 0 1 0 0.1 0.3 1 % to Control 0.00029 0.0016 0.017 0.042 0.011 1.9e−05 0 100 200 300 SMA - - + + + + 0 1 0 0.1 0.3 1 α-SMA/BCL2/DAPI TGFβ(-) TGFβ(+) CB6 0uM CB6 1.0uM CB6 0uM CB6 0.1uM CB6 0.3uM CB6 1.0uM CB6 0uM CB6 1.0uM TGFβ(-) TGFβ(+) CB6 0uM CB6 0.1uM CB6 0.3uM CB6 1.0uM MYLK/DAPI TGFβ CB6 decreases aSMA expression and MYLK expression 

C-PARP C-Caspase3 SMAC Cytochrome C Caspase3 CB6 TGFß - - + + + + 0 1.0 0 0.1 0.3 1.0 CB6 also increased pro-apoptotic proteins in the TGFb cell model Simillar changes with Increased oligomers of Bak and Bax were seen in the TGFb model 

Cytochrome C SMAC Active Caspase3 Apoptosis Mitochondrion CB6 Bim Bax Bak Bax/Bak Oligomers Form MOM Pores Bcl2 CB6 Model of CB6 Activity Either CB6 or Bim could activate Bax and Bak to oligomerize 

Testing in vivo Salmonella typhimurium mouse intestinal fibrosis model MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

Mouse S. Typhimurium Model Treatment (day 4-20) S. typhimurium (day 0 to day 4) Streptomycin wipeout Infect Sac (day 21) Negative Control (5 mice) CB6 100 mg/kg/d S S S CB6 100 mg/kg/d S CB-6 Therapeutic (10 mice) Fibrotic Control (10 mice) Drug Control (5 mice) Timeline and treatment arms Levo 

0 5 10 15 20 60 80 100 days after infection Probability of Survival salmonella+vehicle salmonella+CB6 p=0.3150 H B SS_Vehicle H B SS+C B 6 salm onella+vehicle salm onella+C B 6 50 100 150 200 cecum weight (mg) 0.2948 0.0005 >0.9999 0.0173 H B SS_Vehicle H B SS+C B 6 salm onella+vehicle salm onella+C B 6 100 200 300 400 500 colon weight (mg) 0.6363 <0.0001 0.0632 0.0014 Survival Cecal Weight Colon Weight CB6 reduced cecal and colon weight 

BAX BIM-XL BIM-L BIM-S BCL-XL BCL-2 GAPDH S typhimurium CB6 - + - + - - + + A. CB6 decreases Bcl-2, and increases Bax and Bim-S in cecal tissue from S. typhimurium mice, Western blots CB6 Styph 

CB6 increases Bax-Bak Oligomers In Mouse Cecum tissue – Bax IP/ Bak Western blots Input sample Anti-Bak Western Anti-Bax IP sample Anti-Bak Western S. Typhimurium CB6 - + - + - - + + - + - + + - - + + + IP antibody BAX IgG 160KD Bax- Bak Oligomers Heavy chain 20KD Bak monomer Similar increases in Bax oligomers (Mitochondrial pores), No stable binding of Bim 

SMAC Cleaved- PARP Total – Caspase3 Cleaved – Caspase3 α-SMA 60KD 43KD GAPDH S. Typhimurium CB6 - + + - + + - - Western Blots of pro-apoptotic proteins and aSMA In Mouse Cecum tissue CB6 Styph 

CB6 S. Typhimurium _ + + _ _ _ + + Circular Muscle Layer CB6 decr aSMA & incr Apoptosis, largely in circular muscle 

S. Typhimurium CB6 + + + + - + - + Fibrosis Score (0-4) Masked Pathologist Scoring CB6 reduced cecal and colon fibrosis 

Conclusion A novel, partially gut-selective Bcl-2 /Bcl-XL inhibitor, CB6, can attenuate intestinal fibrosis in myofibroblast cell models and the mouse S. typhimurium fibrosis model through Bax/Bak oligomerization and apoptosis in the circular muscle layer. MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT 

Thank You MICHIGAN IBD ● ANTI-FIBROTIC DRUG DEVELOPMENT