Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4–FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4–mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease

Antibody response to the surface envelope of caprine arthritis-encephalitis lentivirus: disease status is predicted by SU antibody isotype

This study evaluated the hypothesis that the disease status of Saanen goats infected with caprine arthritis-encephalitis lentivirus (CAEV) is associated with the focus of immune responses to viral antigens, particularly the surface envelope glycoprotein (SU). Specifically, we have proposed that Th2 responses promote progressive immune-mediated arthritis, whereas Th1 responses restrict virus replication and development of clinical disease. The present study determined the isotype of SU antibodies associated with progressor and long-term nonprogressor (LTNP) status. We show that chronically infected goats that develop clinical arthritis have predominantly IgG1 antibodies to SU during both preclinical and clinical stages of disease, whereas SU antibodies of LTNP goats are relatively biased toward IgG2. Additional studies determined the isotype of SU antibodies induced initially by CAEV infection. These experiments show that initial IgG1-dominated responses to SU are associated with subsequent development of preclinical inflammatory joint lesions, whereas lack of joint pathology is associated with an IgG2 bias of initial responses to SU. Our results using the CAEV model suggest that isotype bias of SU antibodies is a reliable indicator of clinical disease caused by lentiviruses. Isotype analysis may be a useful method to screen candidate lentiviral vaccines intended to prevent disease progression

Differential effects of targeting Notch receptors in a mouse model of liver cancer

UnlabelledPrimary liver cancer encompasses both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). The Notch signaling pathway, known to be important for the proper development of liver architecture, is also a potential driver of primary liver cancer. However, with four known Notch receptors and several Notch ligands, it is not clear which Notch pathway members play the predominant role in liver cancer. To address this question, we utilized antibodies to specifically target Notch1, Notch2, Notch3, or jagged1 (Jag1) in a mouse model of primary liver cancer driven by v-akt murine thymoma viral oncogene homolog and neuroblastoma RAS viral oncogene homolog (NRas). We show that inhibition of Notch2 reduces tumor burden by eliminating highly malignant HCC- and CCA-like tumors. Inhibition of the Notch ligand, Jag1, had a similar effect, consistent with Jag1 acting in cooperation with Notch2. This effect was specific to Notch2, because Notch3 inhibition did not decrease tumor burden. Unexpectedly, Notch1 inhibition altered the relative proportion of tumor types, reducing HCC-like tumors but dramatically increasing CC-like tumors. Finally, we show that Notch2 and Jag1 are expressed in, and Notch2 signaling is activated in, a subset of human HCC samples.ConclusionsThese findings underscore the distinct roles of different Notch receptors in the liver and suggest that inhibition of Notch2 signaling represents a novel therapeutic option in the treatment of liver cancer

Differential effects of targeting Notch receptors in a mouse model of liver cancer

Primary liver cancer encompasses both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The Notch signaling pathway, known to be important for the proper development of liver architecture, is also a potential driver of primary liver cancer. However, with four known Notch receptors and several Notch ligands, it is not clear which Notch pathway members play the predominant role in liver cancer. To address this question we utilized antibodies to specifically target Notch1, Notch2, Notch3 or Jag1 in a mouse model of primary liver cancer driven by AKT and NRas. We show that inhibition of Notch2 reduces tumor burden by eliminating highly malignant hepatocellular carcinoma- and cholangiocarcinoma-like tumors. Inhibition of the Notch ligand Jag 1 had a similar effect, consistent with Jag1 acting in cooperation with Notch2. This effect was specific to Notch2, as Notch3 inhibition did not decrease tumor burden. Unexpectedly, Notch1 inhibition altered the relative proportion of tumor types, reducing HCC-like tumors but dramatically increasing CC-like tumors. Finally, we show that Notch2 and Jag1 are expressed in, and Notch2 signaling is activated in, a subset of human HCC samples. Conclusions: These findings underscore the distinct roles of different Notch receptors in the liver and suggest that inhibition of Notch2 signaling represents a novel therapeutic option in the treatment of liver cancer

Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs

In vivo efficacy of LD1.

<p><i>A</i>, LD1 inhibits FGF19-regulated <i>FOS</i> expression in mouse liver. The results are represented as fold expression relative to <i>FOS</i> levels in the livers of non-treated mice. <i>B</i>, LD1 (30 mg/kg; once weekly) inhibits HUH7 xenograft tumor growth in vivo. <i>C</i>, Effects of LD1 on the mRNA expression of <i>FGFR4</i>, <i>CYP7A1</i>, <i>FOS</i>, and <i>EGR1</i> in HUH7 xenograft tumors from Fig. 5B. <i>D</i>, Multiple, large, raised tumors (arrows) protruding from the hepatic surface of a DEN-accelerated FGF19-TG:FGFR4-WT mouse treated with a control antibody (upper panel). Liver of DEN-accelerated FGF19-TG:FGFR4-WT mouse treated with LD1 (lower panel). <i>E</i>, Liver weights of DEN–accelerated FGF19-TG:FGFR4-WT mice treated with control antibody, LD1, or 1A6 (anti-FGF19 antibody).</p

LD1 binds to FGFR4.

<p><i>A</i>, LD1 binds to human (h), mouse (m), and cynomolgus monkey (c) FGFR4, but does not bind to hFGFR1, hFGFR2, or hFGFR3. The binding of LD1 to immobilized FGFR-Fc chimeric proteins was determined by solid phase binding assay. <i>B</i>, Affinity of LD1 binding to mouse, cynomolgus monkey, and human FGFR4 as determined by surface plasmon resonance. <i>C</i>, Binding of LD1 to hFGFR4 expressed at the cell surface of stably transfected HEK293 cells as measured by FACS (RFU  =  Relative Fluorescence Unit). <i>D</i>, The binding of LD1 to immobilized hFGFR4-Flag chimeric proteins bearing point mutations as measured by a solid phase binding assay. <i>E</i>, The binding of LD1 to hFGFR4-Flag chimeric proteins bearing point mutations as evaluated by Western blot. Mutated proteins were electrophoresed and sequentially immunoblotted using LD1, an anti-FGFR4 (8G11), and an anti-Flag antibody. <i>F</i>, Dimer model illustrating the position of G165 (blue) on FGFR4 (red and yellow) bound to FGF19 (green).</p

LD1 inhibits FGFR4 activities.

<p><i>A</i>, LD1 inhibits FGFR4 binding to FGF1 and FGF19 as determined by solid phase binding assay. <i>B</i>, LD1 inhibits FGF1-stimulated proliferation of BaF3 cells stably expressing FGFR4/R1. <i>C</i>, LD1 inhibits FGFR4 signaling in L6 cells stably expressing FGFR4. <i>D</i>, Cell surface expression of FGFR4 protein in a subset of liver tumor cell lines as determined by FACS analysis using LD1.</p

LD1 inhibits FGFR4 biological activities in liver cancer cell lines.

<p><i>A</i>, LD1 inhibits FGFR4 signaling in HEP3B cells as evaluated by Western blot. <i>B</i>, LD1 inhibits the FGFR4-regulated <i>CYP7A1</i> repression in HEP3B cells. <i>CYP7A1</i> levels are represented as fold expression relative to the level in untreated cells. <i>C</i>, LD1 inhibits FGFR4-regulated <i>FOS</i> expression in a panel of liver cancer cell lines. The results are represented as fold expression relative to the <i>FOS</i> level in untreated cells. <i>D</i>, Inhibition of colony formation by repression of FGFR4 expression in JHH5 cells stably transfected with an FGFR4 shRNA doxycycline-inducible vector. <i>E</i>, Enumeration of LD1-inhibited liver cancer cell line colony formation. The values are represented as percent of the number of colonies enumerated in the absence of added LD1. <i>F</i>, LD1 inhibits HCC cell line colony formation.</p