Regorafenib enhances antitumor immunity via inhibition of p38 kinase/Creb1/Klf4 axis in tumor-associated macrophages

J Immunother Cancer. 2021 Mar;9(3):e001657. doi: 10.1136/jitc-2020-001657.

Abstract

Background: Regorafenib 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.

Methods: In 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.

Results: Regorafenib (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.

Conclusion: Regorafenib 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.

Keywords: immunomodulation; immunotherapy; tumor microenvironment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiogenesis Inhibitors / pharmacology
  • Animals
  • Antineoplastic Agents / pharmacology*
  • Carcinoma, Hepatocellular / drug therapy*
  • Carcinoma, Hepatocellular / enzymology
  • Carcinoma, Hepatocellular / immunology
  • Cell Line, Tumor
  • Coculture Techniques
  • Cyclic AMP Response Element-Binding Protein / metabolism*
  • Kruppel-Like Factor 4 / metabolism
  • Liver Neoplasms / drug therapy*
  • Liver Neoplasms / enzymology
  • Liver Neoplasms / immunology
  • Lymphocyte Activation / drug effects
  • Lymphocytes, Tumor-Infiltrating / drug effects
  • Lymphocytes, Tumor-Infiltrating / enzymology
  • Lymphocytes, Tumor-Infiltrating / immunology
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Phenotype
  • Phenylurea Compounds / pharmacology*
  • Protein Kinase Inhibitors / pharmacology*
  • Pyridines / pharmacology*
  • Signal Transduction
  • Tumor Microenvironment
  • Tumor-Associated Macrophages / drug effects*
  • Tumor-Associated Macrophages / enzymology
  • Tumor-Associated Macrophages / immunology
  • p38 Mitogen-Activated Protein Kinases / metabolism*

Substances

  • Angiogenesis Inhibitors
  • Antineoplastic Agents
  • Creb1 protein, mouse
  • Cyclic AMP Response Element-Binding Protein
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Phenylurea Compounds
  • Protein Kinase Inhibitors
  • Pyridines
  • regorafenib
  • p38 Mitogen-Activated Protein Kinases