FGF401 and vinorelbine synergistically mediate antitumor activity and vascular normalization in FGF19-dependent hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a lethal cancer with limited therapeutic options, and standard therapy with sorafenib provides only modest survival benefits. Fibroblast growth factor 19 (FGF19) has been proposed as a driver oncogene, and targeting its receptor, FGFR-4, may provide a better alternative to standard therapy for patients with FGF19-driven tumors. Sixty-three HCC patient-derived xenograft (PDX) models were screened for FGF19 expression. Mice bearing high and low FGF19-expressing tumors were treated with FGF401 and/or vinorelbine, and the antitumor activity of both agents was assessed individually and in combination. Tumor vasculature and intratumoral hypoxia were also examined. High FGF19 expression was detected in 14.3% (9 of 63) of the HCC models tested and may represent a good target for HCC treatment. FGF401 potently inhibited the growth of high FGF19-expressing HCC models regardless of FGF19 gene amplification. Furthermore, FGF401 inhibited the FGF19/FGFR-4 signaling pathway, cell proliferation, and hypoxia, induced apoptosis and blood vessel normalization and prolonged the overall survival (OS) of mice bearing high FGF19 tumors. FGF401 synergistically acted with the microtubule-depolymerizing drug vinorelbine to further suppress tumor growth, promote apoptosis, and prolong the OS of mice bearing high FGF19 tumors, with no evidence of increased toxicity. Our study suggests that a subset of patients with high FGF19-expressing HCC tumors could benefit from FGF401 or FGF401/vinorelbine treatment. A high level of FGF19 in a tumor may serve as a potential biomarker for patient selection.

H3B-6527 clinical biomarker assay development and characterization of HCC patient samples.

4121 Background: FGFR4/FGF19 signaling axis is a novel therapeutic target in HCC. Multiple covalent FGFR4 inhibitors, including H3B-6527, are under clinical development. Preclinical efficacy studies in mice (including PDX) have shown that FGF19 expression (FGF19+) is a predictive biomarker for FGFR4 inhibitor response. The mechanisms driving FGF19 expression in HCC is largely unknown however, in some cases, focal amplification of ch11q13.3 containing FGF19 gene is thought to drive the FGF19 expression. Consistent with the preclinical observations, clinical studies have also shown that FGF19+is a predictive biomarker for FGFR4 inhibitor response. However, these trials have also reported a large number of FGF19+patients failing to respond to FGFR4 inhibitors necessitating refinement of patient selection strategies. In an attempt to obtain deeper insights into the role of FGF19+as a predictive biomarker and potentially uncover additional biomarkers that will enable improvement in patient selection strategies, we have characterized a set of 258 HCC patient samples Methods: Samples were acquired from biobanks and utilized to qualify clinical assays including FGF19 copy number (FISH), mRNA expression (qRT-PCR), FGF19 protein (IHC), and a focused NGS panel for assessing both mutations and copy number. A multiplexed protein and mRNA platform enabled assessment of p-ERK and Ki67 (protein) and Cyp7A1 (mRNA) amongst other exploratory PD biomarkers from two FFPE slides. Results: FGF19 positivity rates for IHC and qRT-PCR were 18% (41/225) and 42% (87/209), respectively. The overall correlation was 60%, with 63% (22/235) IHC positive cases also being positive by qRT-PCR. For IHC+/qRT-PCR (-) cases, RNA quality may have impacted assay sensitivity. 4% (9/244) of samples were positive for FGF19 copy number. Among samples with FGF19 copy number gains, 22% (2/9) did not show positive FGF19 expression. Conclusions: Based on our data, FGF19 mRNA is a more inclusive selection strategy and offers an approach to further refine thresholds for efficacy as determined in the clinic. Multiplexed protein-mRNA assays were also validated and implemented to enable a more comprehensive clinical biomarker program.

A Clostridia-rich microbiota contributes to increased excretion of bile acids in diarrhea-predominant irritable bowel syndrome

ABSTRACTObjectiveAn excess of fecal bile acids (BAs) is thought to be one of the mechanisms for diarrhea-predominant irritable bowel syndrome (IBS-D). However, the factors causing excessive BA excretion remains unclear. Given the importance of gut microbiota in BA metabolism, we hypothesized that gut dysbiosis might contribute to excessive BA excretion in IBS-D.DesignMetabolomic and metagenomic analyses were performed of specimens from 290 IBS-D patients and 89 healthy volunteers. By transplanting human microbiota and manipulating specific microbiome species in mice, the effects of microbiota on host BA metabolism were assessed at metabolic, genetic and protein levels. Effects of individual and mixed BAs on enterohepatic feedback pathways were also tested in vitro and in vivo.ResultsTotal fecal BAs were excessively excreted in 24.5% of IBS-D patients. Their fecal metagenomes showed increased abundances of Clostridia and BA-transforming genes (hdhA and bais). The increases of Clostridia bacteria (e.g. C. scindens) were positively associated with the levels of fecal BAs and serum 7α-hydroxy-4-cholesten-3-one (C4), while being negatively correlated with serum fibroblast growth factor 19 (FGF19). Both Clostridia-rich human microbiota and C. scindens enhanced levels of serum C4 and hepatic conjugated BAs in mice recipients and reduced ileal FGF19 expression. Inhibition of Clostridium species by vancomycin yielded opposite findings. Clostridia-derived BAs (e.g. conjugated and free ursodeoxycholic acid) significantly suppressed intestinal FGF19 expression.ConclusionThe Clostridia-rich microbiota contributes to excessive BA excretion in IBS-D patients. This study provided the basis for more precise clinical diagnosis and management for IBS-D.

Upregulation of Fibroblast Growth Factor 19 Is Associated with the Initiation of Colorectal Adenoma

Fibroblast growth factor 19 (FGF19) promotes tumor growth in various types of cancer, but its function has not been investigated in the context of colorectal adenoma. Here, we report that FGF19 expression was greater in colorectal adenoma than in normal tissues, as measured by an enzyme-linked immunosorbent assay, quantitative reverse-transcription PCR and immunohistochemistry. FGF19 expression was also elevated in a subset of human colon cancer cell lines. Moreover, FGF receptor 4 (FGFR4), the cognate receptor for FGF19, was upregulated in colorectal adenoma tissues. Lipid levels and body mass index values strongly correlated with FGF19 and FGFR4 levels in patients with colon adenomas. These observations indicate that the FGF19/FGFR4 pathway may be involved in the development of neoplasia, and that FGF19 may be a valuable diagnostic marker for the identification of patients with colorectal adenomas.

Bile acid flux through portal but not peripheral veins inhibits CYP7A1 expression without involvement of ileal FGF19 in rabbits

It was proposed that CYP7A1 expression is suppressed through the gut-hepatic signaling pathway fibroblast growth factor (FGF) 15/19-fibroblast growth factor receptor 4, which is initiated by activation of farnesoid X receptor in the intestine rather than in the liver. The present study tested whether portal bile acid flux alone without ileal FGF19 could downregulate CYP7A1 expression in rabbits. A rabbit model was developed by infusing glycodeoxycholic acid (GDCA) through the splenic vein to bypass ileal FGF19. Study was conducted in four groups of rabbits: control; bile fistula + bovine serum albumin solution perfusion (BF); BF + GDCA (by portal perfusion); and BF + GDCA-f (by femoral perfusion). Compared with only BF, BF + GDCA (6 h portal perfusion) suppressed CYP7A1 mRNA, whereas BF + GDCA-f (via femoral vein) with the same perfusion rate of GDCA did not show inhibitory effects. Meanwhile, there was a decrease in ileal FGF19 expression and portal FGF19 protein levels, but an equivalent increase in biliary bile acid outputs in both GDCA perfusion groups. This study demonstrated that portal bile acid flux alone downregulated CYP7A1 expression with diminished FGF19 expression and protein levels, whereas the same bile acid flux reaching the liver through the hepatic artery via femoral vein had no inhibitory effect on CYP7A1. We propose that bile acid flux through the portal venous system may be a kind of “intestinal factor” that suppresses CYP7A1 expression.

Potent stimulation of fibroblast growth factor 19 expression in the human ileum by bile acids

Fibroblast growth factor 19 (FGF19) is proposed to be a negative feedback regulator of hepatic bile acid (BA) synthesis. We aimed to clarify the distribution of FGF19 expression in human intestine and to investigate induction in a novel explant system. Ileal and colonic mucosal biopsies were obtained at endoscopy and analyzed for FGF19 transcript expression. Primary explants were incubated with physiological concentrations of various BA for up to 6 h, and expression of FGF19 and other genes was determined. FGF19 transcripts were detected in ileum but were unquantifiable in colon. No loss of FGF19 mRNA occurred as a consequence of the explant system. Ileal FGF19 transcript expression was induced 350-fold by 50 μM chenodeoxycholate (CDCA, n = 24, P < 0.0001) and 161-fold by 50 μM glycochenodeoxycholate (GCDCA, n = 12, P = 0.0005). The responses of other genes to CDCA or GCDCA (50 μM) were smaller: median increases of ileal bile acid binding protein, organic solute transporter-α and -β, and short heterodimer partner were 2.4- to 4.0-fold; apical membrane sodium bile acid transporter and farnesoid X receptor (FXR) showed little change. The EC50 for FGF19 transcript induction by CDCA was 20 μM. FGF19 protein concentrations were significantly higher in the culture fluid from BA-stimulated explants. FGF19 induction with cholate was 81% of that found with CDCA, but deoxycholate (40%) and lithocholate (4%) were significantly less potent. The synthetic FXR agonist obeticholic acid was much more potent than CDCA with a 70-fold FGF19 stimulation at 1 μM. We concluded that FGF19 expression in human ileum is very highly responsive to BA. Changes in FGF19 induction are a potential mechanism involved in disorders of BA homeostasis.

FGF19 (fibroblast growth factor 19) as a novel target gene for activating transcription factor 4 in response to endoplasmic reticulum stress

FGF19 (fibroblast growth factor 19), expressed in the small intestine, acts as an enterohepatic hormone by mediating inhibitory effects on the bile acid synthetic pathway and regulating carbohydrate and lipid metabolism. In an attempt to identify novel agents other than bile acids that induce increased FGF19 expression, we found that some ER (endoplasmic reticulum) stress inducers were effective. When intestinal epithelial Caco-2 cells were incubated with thapsigargin, marked increases were observed in the mRNA and secreted protein levels of FGF19. This was not associated with the farnesoid X receptor. Reporter gene analyses using the 5′-promoter region of FGF19 revealed that a functional AARE (amino-acid-response element) was localized in this region, and this site was responsible for inducing its transcription through ATF4 (activating transcription factor 4), which is activated in response to ER stress. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays showed that ATF4 bound to this site and enhanced FGF19 expression. Overexpression of ATF4 in Caco-2 cells induced increased FGF19 mRNA expression, whereas shRNA (short hairpin RNA)-mediated depletion of ATF4 significantly attenuated a thapsigargin-induced increase in FGF19 mRNA.