Biochemical and functional analyses of gp130 mutants unveil JAK1 as a novel therapeutic target in human inflammatory hepatocellular adenoma

Abstract

Inflammatory hepatocellular adenomas (IHCAs) are benign liver lesions that can be characterized histologically by the presence of an inflammatory infiltrate and at the molecular level by the overexpression of acute phase inflammatory response genes. Recurrent somatic mutations of the interleukin-6 (IL-6) signal transducer (IL6ST) locus, encoding the critical component of the IL-6 signal transduction machinery gp130, are present in 60% of IHCAs and in a subset (2%) of hepatocellular carcinoma (HCCs). By screening of 256 human hepatic adenoma specimens (the largest genetic analysis of IL6ST performed to date in this setting), we identified 24 distinct somatic IL6ST mutations among 66 mutant adenomas. The functional analysis of nine different gp130 mutants expressed in hepatic cancer cell lines consistently revealed the constitutive and IL-6-independent activation of the JAK/STAT signaling pathway. We further demonstrated that the signaling activity of mutant gp130 in IHCA remains responsive to suppressor of cytokine signaling 3 (SOCS3), a physiological gp130 inhibitor. Specifically, cells expressing a double mutant variant of gp130 with a disrupted SOCS3-binding site at residue 759 (Y186/Y759F) displayed a hyperactivation of signal transducer and activator of transcription 3 (STAT3) as compared with cells expressing the endogenous IHCA-associated Y186 gp130 mutant. Notably, we identified that constitutive signaling via gp130 in IHCA requires the Janus kinase family member JAK1, but not JAK2 or tyrosine kinase 2. In support of this notion, AG490, a tyrosine kinase inhibitor that selectively blocks JAK2, had no effect on gp130 activity. In stark contrast, we showed that ruxolitinib, a JAK1/JAK2-selective tyrosine kinase inhibitor used to treat patients with myelofibrosis, dramatically impaired JAK1-STAT signaling downstream of all IHCA-associated gp130 mutants. In conclusion, our findings provide a rationale for the use of JAK1 inhibitors for the treatment of HCAs expressing mutant gp130 as well as a subset of HCCs that bear similar mutations.

Keywords: IL6ST; JAK1; STAT3; ruxolitinib; targeted therapy.

Figure 1. Gain-of-function mutations of gp130 in IHCA. (A) Spectrum of somatic mutations affecting interleukin-6 (IL-6) signal transducer (IL6ST) in human inflammatory hepatocellular adenoma (IHCA) samples (n = 256). DNA sequencing of IL6ST of was performed to identify the resultant alterations in gp130, including in-frame deletions (in green), insertions or deletions (in pink) and amino acid substitutions (in yellow) occurring in the different domains of the protein (S, signal peptide; D1-D6, extracellular domains; TM, transmembrane domain). Right, occurrence of the different mutants with their official nomenclature. Mutants reproduced by site-directed mutagenesis (for functional analysis) are in blue. (B) Plasmids engineered to express either IHCA-associated gp130 mutants or wild-type (WT) gp130 were co-transfected into Hep3B cells (n = 3) along with a STAT3-driven luciferase (Luc) reporter. STAT3 activation (left) was measured by luciferase activity 6 h after serum starvation. Shown are the means ± SD luciferase activity. Quantitative PCR was also used to examine the effects of expressing mutant gp130 on SOCS3 (center) or CRP (right) mRNA expression levels in comparison to WT gp130. Shown is the mean ± SD of the normalized mRNA levels in mutants relative to WT gp130 controls (1-fold).

Figure 2. Signaling activity of IHCA gp130 mutants is attenuated by SOCS3. (A and B) Plasmids engineered to express either inflammatory hepatocellular adenoma (IHCA)-associated gp130 mutants (black bars) or wild-type (WT) gp130 were transfected into Hep3B cells (n = 3) together with a STAT3-driven luciferase (Luc) reporter construct (pSIEM-Luc). STAT3 activation was measured by luciferase activity 6 h after serum starvation. Shown are the means ± SD luciferase activity. (A) STAT3 activity following the co-transfection of Hep3B cells with the STAT3-luciferase reporter and a control empty plasmid (EP) or constructs coding withWT gp130, Y186 gp130 mutant, Y759F gp130 mutant, or Y186/Y759F gp130 double mutant. (B) Hep3B cells were co-transfected with the STAT3-luciferase reporter and a vector expressing the Y186 gp130 mutant or the corresponding EP along with a SOCS3 expression construct (+) or the corresponding EP (−). Data shown are the mean luciferase activities ± SD relative to pSIEM-Luc alone (EP control) following serum starvation (6 h). Where indicated, cells were treated for the final 3 h with 100 ng/mL interleukin-6 (IL-6). Statistical significance was determined by 2-tailed Student t test; ***P < 0.001.

Figure 3. Interaction of IHCA gp130 mutants with wild-type gp130 and OSMR. (A) MYC- or Flag-tagged constructs expressing either wild-type (WT) gp130 or the inflammatory hepatocellular adenoma (IHCA)-associated gp130 mutants A418 or Y186 were co-transfected (1:1) into Hep3B cells. Gp130 hetero- and homo-dimerization in the absence of interleukin-6 (IL-6) was assessed by immunoprecipitation using the anti-Flag antibodies followed by immunoblotting. (B and C) Hep3B cells (n = 3) were co-transfected with a control vector or a plasmid expressing the Y186 gp130 mutant and a control siRNA (−) or a siRNA specific for WT gp130 (+) and quantitative PCR was used to examine the effects on transcription of STAT3 target genes. Shown is the mean ± SD expression level of endogenous CRP (B) and SOCS3 (C) transcripts in cell expressing gp130 mutants relative to WT gp130 (1-fold). On hundred ng/mL IL-6 was added to the culture medium where indicated. (D) The dimerization potential of the oncostatin M receptor (OSMR) with WT or mutant gp130 in the absence of OSM was assessed in Hep3B cells expressing Flag-tagged WT or mutant gp130. Shown are the immunoblots of immunoprecipitates obtained with anti-OSMR antibodies in the presence or in the absence of 100 ng/mL OSM. (E) Hep3B cells were co-transfected with the STAT3-luciferase reporter and a control vector (EP) or plasmids encoding the Y186 gp130 mutant or OSMR, as indicated. One hundred ng/mL OSM was added when indicated. Shown are the mean ± SD luciferase activities determined from triplicate co-transfections. (F) The knockdown of OSMR in Hep3B cells (n = 3) impairs STAT3 activity a driven by the Y186 gp130 mutant or treatment with 100 ng/mL OSM. Shown are the mean luciferase activities ± SD relative to cells transfected with the EP only. Results were confirmed using a second siRNA (data not shown). Statistical significance was determined by 2-tailed Student t test; **P < 0.01; ***P < 0.001.

Figure 4. Activity of IHCA gp130 mutants is selectively dependent upon JAK1. (A) Two different siRNAs silencing JAK1 (siJAK1#1 and #2) and two different siRNAs silencing JAK2 (siJAK2#1 and #2) or TYK2 (siTYK2#1 and #2) were tested for their ability to interfere with STAT3 activity in HepG2 cells co-transfected with a STAT3-luciferase reporter and an empty plasmid (EP)- or a plasmid coding for the Y186 gp30 mutant (n = 3). Where indicated, cells were treated for 3 h with 100 ng/mL interleukin-6 (IL-6). Shown are the mean luciferase activity ± SD relative to cells receiving the EP and a non-targeting control siRNA (siCTL). (B) Hep3B cells transfected with a plasmid encoding the Y186 gp130 mutant (filled circles) or the corresponding EP and then treated with 100 ng/mL IL-6 were exposed for 16 h to increasing concentrations of the JAK1 inhibitor ruxolitinib (n = 3). The data shown are the mean luciferase activities ± SD relative to Hep3B cells not treated with the inhibitor. (C) Hep3B cells transfected with constructs coding for nine different IHCA gp130 mutants (K173, V184, Y186, S187, V189, E195, D215, P216 and A418) or the corresponding EP were treated with 100 ng/mL IL-6 and exposed for 18 h to increasing concentrations of ruxolitinib. Shown are the mean luciferase activities ± SD from triplicate assessments relative to transfected cells not treated with the inhibitor. Statistical significance was determined by 2-tailed Student t test; **P < 0.01; ***P < 0.001.