Figures and data
Expression of Wnt Signaling Pathway Components in Alcohol-Associated Cirrhotic and Control Livers
(A) QPCR expression analysis of 11 WNT, (B) 4 RSPO and (C) 10 FZD family members, (D) QPCR expression analysis of RNF43, ZNRF3 (H) ASGR1 and ASGR2 in control and alcohol-associated cirrhosis samples. Results are presented as (e(dCt)) to reflect the relative abundance of different family members. (E) Bulk RNAseq expression analysis of 3 LGR4, LGR5 and LGR6 in control and alcohol-associated cirrhosis samples. FDR is the false discovery rate, FDR sig = -1 means that there is a significant reduction in alcohol-associated cirrhosis samples using FDR < 0.05, FDR sig = 1 means that there is a significant increase in alcohol-associated cirrhosis samples using FDR < 0.05, Av Rs is the average number of readings (F) Schematic representation of the mechanism by which the RSPO mimetic, SZN-043, induces hepatocyte-targeted Wnt signaling (created with BioRender.com). (From left to right) In absence of RSPO, the E3 ligases, RNF43 and ZNRF3, induce the ubiquitination of FZD receptors and subsequent degradation. Upon binding of RSPO to the E3 ligases and its co-receptor LGR, RSPO prevents FZD ubiquitination and thereby induces the stabilization of FZD receptors. SZN-043 is a fusion antibody-based ligand containing a mutated RSPO which does not bind the LGR receptor, but still binds E3 ligases and an anti-ASGR1 binding domain. Upon binding to ASGR1, expressed predominantly in hepatocytes, and E3 ligases, SZN-043 induces Wnt activity, via the stabilization of its receptors. SZN-043 may be internalized along with ASGR1 into lysosomes. (G) Histoimmunochemistry of control and alcohol-associated cirrhosis samples liver sections using anti-human ASGR1 antibodies.
Selected genes comparative RNAseq analysis in two cohorts of control and alcoholic cirrhosis liver samples. FDR: false discovery rate, FDRsig = 1, significant fold increase when using FDR < 0.05, FDRsig = -1, significant fold decrease when using FDR < 0.05. Genes that were significantly elevated or reduced in both cohorts are highlighted in bold.
Expression of the Wnt target gene, CYP1A2, and (PSR) Staining in Alcohol-Associated Cirrhotic and Control Livers
(A) QPCR expression analysis of CYP1A2. (B) PSR histological staining and CYP1A2 immunostaining of control and alcohol-associated cirrhosis liver sections.
Dose Response Analysis of SZN-043 effect on the Expression of Wnt Target Genes, its Feedback Inhibitors and Proliferation Markers in Mice.
(A) Serum exposure of SZN-043 over time after a single intravenous administration of various SZN-043 doses ranging from 0.3 to 200 mg/kg. (B-G) Time course qPCR expression analysis of (B) Cyp1a2, (C) Axin2, (D) Notum, (E) Ccnd1, (F) Mki67and (G) Alpl and (H) serum ALP level.
Pharmacokinetic Parameters of SZN-043 in Mice Following a Single IV Bolus Dose. AUCinf, area under the serum concentration curve from time 0 to time infinity; C0, estimated serum concentration at time zero; CL, clearance; MRT, mean residence time; Terminal t1/2, terminal half-life; Vc, central compartment volume of distribution.
SZN-043 Induced a Transient Wave of Proliferation in Human Hepatocytes.
(A) Study design. FRG mice received a single dose, i.p., of either the negative control anti-GFP (10 mg/mL) or the SZN-043 test article at a 3 or 10 mg/kg dose. The left lateral and medial liver lobes were collected upon termination on Day 1, 2, 3, 5 or 7 for immunofluorescence analysis. (B) Left lateral liver tissue sections were doubly stained with the human hepatocyte specific, anti-human ASGR1 (red), and the proliferation marker, anti-Ki67 (green), antibodies. (C) The percentage of doubly positive cells was calculated using HALO imaging analysis software. * p < 0.05, ** p < 0.01
Effect of SZN-043 on Wnt Target Genes and Proliferation Markers in a Chronic-Binge Alcohol-Induced Injury Model in Aging Mice.
(A) Study Design. Aging C57BL/6J female mice were acclimated to the Lieber-DeCarli diet for 5 days, followed by an ethanol-supplemented liquid diet for 8 weeks. A control group was pair-fed with an equicaloric diet during this 8-week period. Starting on week 2 of the ethanol-supplemented diet, mice received 20% ethanol by oral gavage twice weekly for the remaining 7 weeks. The pair fed control group received an equivalent volume of ethanol-free equicaloric dose by oral gavage twice weekly also. Ethanol administration was then discontinued, and mice were returned to an ethanol-free liquid diet. The pair-fed control group and an ethanol-fed were euthanized upon ethanol discontinuation, on Day 0, and liver tissue was collected for analysis. Two hours later and daily thereafter until Day 6, mice received either SZN-043 (30 mg/kg), a control antibody anti-GFP (30 mg/kg) or a vehicle control. On Day3 and on Day 7, upon termination, liver tissue was collected for analysis. (B) QPCR expression analysis of Axin2, Mki67, Cyp1a2, Asgr1and Asgr2 genes. (C) Immunofluorescence staining with the proliferation marker, anti-Ki67, and the hepatocyte-specific marker, anti-HNF4A antibodies. (D) Percentage of double positive HNF4A and Ki67 cells as determined by quantitative image analysis.
Effect of Ethanol and SZN-043 on the Expression of Wnt Family Members in Mice.
(A) Study design. Aging C57BL/6J female mice were acclimated to the Lieber-DeCarli diet for 5 days, followed by an ethanol-supplemented liquid diet for 8 weeks. A control group was pair-fed with an equicaloric diet during this 8-week period. Starting on week 2 of the ethanol-supplemented diet, mice received 20% ethanol by oral gavage twice weekly for the remaining 7 weeks. The pair fed control group received an equivalent volume of ethanol-free equicaloric dose by oral gavage twice weekly also. Ethanol administration was then discontinued, and mice were returned to an ethanol-free liquid diet. The pair-fed control group and an ethanol-fed were euthanized upon ethanol discontinuation, on Day 0, and liver tissue was collected for analysis. 24 hours later, mice received either a single dose of SZN-043 at 10, 30 or 100 mg/kg, or daily dosing (30 mg/kg), or a single dose of a control antibody anti-GFP (30 mg/kg). On Day3, upon termination, liver tissue was collected for analysis. (B) QPCR expression analysis of Wnt family members in ethanol-treated or pair-fed controls. Results are presented as (e(dCt)) to reflect the relative abundance of different family members. (C) Effect of SZN-043 on Wnt2, Wnt4, Wnt5a and Wnt9b genes expression, determined by qPCR analysis.
Description of Human Healthy and Cirrhotic Liver Samples Used for Expression Analysis in Cohort #1.
