Background:Sorafenib is the only drug approved for the treatment of hepatocellular carcinoma (HCC). The bioenergetic propensity of cancer cells has been correlated to anticancer drug resistance, but such correlation is unclear in sorafenib resistance of HCC.Methods:Six sorafenib-naive HCC cell lines and one sorafenib-resistant HCC cell line (Huh-7R; derived from sorafenib-sensitive Huh-7) were used. The bioenergetic propensity was calculated by measurement of lactate in the presence or absence of oligomycin. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, and siRNA of hexokinase 2 (HK2) were used to target relevant pathways of cancer metabolism. Cell viability, mitochondrial membrane potential, and sub-G1 fraction were measured for in vitro efficacy. Reactive oxygen species (ROS), adenosine triphosphate (ATP) and glucose uptake were also measured. A subcutaneous xenograft mouse model was used for in vivo efficacy.Results:The bioenergetic propensity for using glycolysis correlated with decreased sorafenib sensitivity (R2=0.9067, among sorafenib-naive cell lines; P=0.003, compared between Huh-7 and Huh-7 R). DCA reduced lactate production and increased ROS and ATP, indicating activation of oxidative phosphorylation (OXPHOS). DCA markedly sensitised sorafenib-resistant HCC cells to sorafenib-induced apoptosis (sub-G1 (combination vs sorafenib): Hep3B, 65.4±8.4% vs 13±2.9% Huh-7 R, 25.3± 5.7% vs 4.3±1.5% each P<0.0001), whereas siRNA of HK2 did not. Sorafenib (10 mg kg−1 per day) plus DCA (100 mg kg−1 per day) also resulted in superior tumour regression than sorafenib alone in mice (tumour size: −87% vs −36%, P<0.001).Conclusion:The bioenergetic propensity is a potentially useful predictive biomarker of sorafenib sensitivity, and activation of OXPHOS by PDK inhibitors may overcome sorafenib resistance of HCC.

Markers that could accurately predict responses to the general kinase inhibitor sorafenib are needed to better leverage its clinical applications. In this study, we examined a hypothesized role in the drug response for the growth arrest DNA damage-inducible gene 45β (GADD45β), which is commonly underexpressed in hepatocellular carcinoma (HCC) where sorafenib may offer an important new therapeutic option. The anticancer activity of sorafenib-induced GADD45β expression was tested in a panel of HCC cell lines and xenograft models. We found that GADD45β mRNA and protein expression were induced relatively more prominently in HCC cells that were biologically sensitive to sorafenib treatment. GADD45β induction was not found after treatment with either the mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 or the Raf inhibitor ZM336372, suggesting that GADD45β induction by sorafenib was independent of Raf/MEK/ERK signaling activity. However, c-Jun NH 2 -terminal kinase (JNK) kinase activation occurred preferentially in sorafenib-sensitive cells. Small interfering RNA-mediated knockdown of GADD45βor JNK kinase limited the proapoptotic effects of sorafenib in sorafenib-sensitive cells. We defined the −339/−267 region in the GADD45β promoter containing activator protein-1 and SP1-binding sites as a crucial region for GADD45β induction by sorafenib. Together, our findings suggest that GADD45β induction contributes to sorafenib-induced apoptosis in HCC cells, prompting further studies to validate its potential value in predicting sorafenib efficacy. Cancer Res; 70(22); 9309-18. ©2010 AACR.

<b><i>Background:</i></b> Reversal of CD8 T-cell exhaustion was considered a major antitumor mechanism of anti-programmed cell death-1 (PD-1)/ anti-programmed death ligand-1 (PD-L1)-based immune checkpoint inhibitor (ICI) therapy. <b><i>Objectives:</i></b> The aim of this study was to identify markers of T-cell exhaustion that is best associated with ICI treatment efficacy for advanced hepatocellular carcinoma (HCC). <b><i>Methods:</i></b> Immune cell composition of archival tumor samples was analyzed by transcriptomic analysis and multiplex immunofluorescence staining. <b><i>Results:</i></b> HCC patients with objective response after anti-PD-1/anti-PD-L1-based ICI therapy (<i>n</i> = 42) had higher expression of genes related to T-cell exhaustion. A 9-gene signature (LAG3, CD244, CCL5, CXCL9, CXCL13, MSR1, CSF3R, CYBB, and KLRK1) was defined, whose expression was higher in patients with response to ICI therapy, correlated with density of CD8⁺LAG3⁺ cells in tumor microenvironment, and independently predicted better progression-free and overall survival. This 9-gene signature had similar predictive values for patients who received single-agent or combination ICI therapy and was not associated with prognosis in HCC patients who received surgery, suggesting that it may outperform other T-cell signatures for predicting efficacy of ICI therapy for HCC. For HCC patients who underwent surgery for both the primary liver and metastatic lung tumors (<i>n</i> = 31), lung metastatic HCC was associated with a higher exhausted CD8 T-cell signature, consistent with prior observation that patients with lung metastatic HCC may have higher probability of response to ICI therapy. <b><i>Conclusions:</i></b> CD8 T-cell exhaustion in tumor microenvironment may predict better efficacy of ICI therapy for HCC.

Multikinase inhibitors with antiangiogenic properties used to be standard therapy for patients with advanced hepatocellular carcinoma (HCC). Recently, several antiangiogenic agents (lenvatinib, cabozantinib, and ramucirumab) have demonstrated antitumor activity for advanced HCC in randomized controlled trials. However, the landscape of drug development for HCC may change dramatically with the advent of immune checkpoint inhibitor therapy, particularly the anti–programmed cell death-1 (anti-PD1) agents. In addition, early-phase clinical trials of combination of anti–PD-1 and antiangiogenic agents have shown very promising anti-tumor activity in patients with advanced HCC. Therefore, the critical research questions at present are whether this combination strategy will be the next generation of standard therapy and which antiangiogenic agents will be the optimal partner for the combination. All of the 4 multikinase inhibitors for HCC (sorafenib, regorafenib, lenvatinib, and cabozantinib) have been reported to have immune modulatory effects. The authors systematically reviewed the pre-clinical evidence of their immune modulatory effects to explore whether these effects were mediated by angiogenesis inhibition or by other “off-target” effects on the tumor microenvironment. Studies of sorafenib comprised the majority (58 of the 71) of the research articles reviewed. Potentially beneficial effects on anti-tumor immunity may result from increased M1 polarization of macrophages and stimulation of CD8 T cell function. On the other hand, high dosage of the kinase inhibitors in pre-clinical models and hypoxia associated with angiogenesis may contribute to immune suppression in the tumor microenvironment. Sorafenib and other multikinase inhibitors may promote anti-tumor immunity through modulation of multiple immune cell types as well as the tumor microenvironment. The optimal immune modulatory dosage should be defined to facilitate design of future combination regimens.

BackgroundRegorafenib and other multikinase inhibitors may enhance antitumor efficacy of anti-program cell death-1 (anti-PD1) therapy in hepatocellular carcinoma (HCC). Its immunomodulatory effects, besides anti-angiogenesis, were not clearly defined.MethodsIn vivo antitumor efficacy was tested in multiple syngeneic liver cancer models. Murine bone marrow–derived macrophages (BMDMs) were tested in vitro for modulation of polarization by regorafenib and activation of cocultured T cells. Markers of M1/M2 polarization were measured by quantitative reverse transcription PCR (RT-PCR), arginase activity, flow cytometry, and ELISA. Knockdown of p38 kinase and downstream Creb1/Klf4 signaling on macrophage polarization were confirmed by using knockdown of the upstream MAPK14 kinase, chemical p38 kinase inhibitor, and chromatin immunoprecipitation.ResultsRegorafenib (5 mg/kg/day, corresponding to about half of human clinical dosage) inhibited tumor growth and angiogenesis in vivo similarly to DC-101 (anti-VEGFR2 antibody) but produced higher T cell activation and M1 macrophage polarization, increased the ratio of M1/M2 polarized BMDMs and proliferation/activation of cocultured T cells in vitro, indicating angiogenesis-independent immunomodulatory effects. Suppression of p38 kinase phosphorylation and downstream Creb1/Klf4 activity in BMDMs by regorafenib reversed M2 polarization. Regorafenib enhanced antitumor efficacy of adoptively transferred antigen-specific T cells. Synergistic antitumor efficacy between regorafenib and anti-PD1 was associated with multiple immune-related pathways in the tumor microenvironment.ConclusionRegorafenib may enhance antitumor immunity through modulation of macrophage polarization, independent of its anti-angiogenic effects. Optimization of regorafenib dosage for rational design of combination therapy regimen may improve the therapeutic index in the clinic.