Regorafenib combined with PD1 blockade increases CD8 T-cell infiltration by inducing CXCL10 expression in hepatocellular carcinoma
Kohei Shigeta, Aya Matsui, Hiroto Kikuchi, Sebastian Klein, Emilie Mamessier, Ivy X Chen, Shuichi Aoki, Shuji Kitahara, Koetsu Inoue, Ayako Shigeta, Tai Hato, Rakesh R Ramjiawan, Daniel Staiculescu, Dieter Zopf, Lukas Fiebig, Gabriela S Hobbs, Alexander Quaas, Simona Dima, Irinel Popescu, Peigen Huang, Lance L Munn, Mark Cobbold, Lipika Goyal, Andrew X Zhu, Rakesh K Jain, Dan G Duda, Kohei Shigeta, Aya Matsui, Hiroto Kikuchi, Sebastian Klein, Emilie Mamessier, Ivy X Chen, Shuichi Aoki, Shuji Kitahara, Koetsu Inoue, Ayako Shigeta, Tai Hato, Rakesh R Ramjiawan, Daniel Staiculescu, Dieter Zopf, Lukas Fiebig, Gabriela S Hobbs, Alexander Quaas, Simona Dima, Irinel Popescu, Peigen Huang, Lance L Munn, Mark Cobbold, Lipika Goyal, Andrew X Zhu, Rakesh K Jain, Dan G Duda
Abstract
Background and purpose: Combining inhibitors of vascular endothelial growth factor and the programmed cell death protein 1 (PD1) pathway has shown efficacy in multiple cancers, but the disease-specific and agent-specific mechanisms of benefit remain unclear. We examined the efficacy and defined the mechanisms of benefit when combining regorafenib (a multikinase antivascular endothelial growth factor receptor inhibitor) with PD1 blockade in murine hepatocellular carcinoma (HCC) models.
Basic procedures: We used orthotopic models of HCC in mice with liver damage to test the effects of regorafenib-dosed orally at 5, 10 or 20 mg/kg daily-combined with anti-PD1 antibodies (10 mg/kg intraperitoneally thrice weekly). We evaluated the effects of therapy on tumor vasculature and immune microenvironment using immunofluorescence, flow cytometry, RNA-sequencing, ELISA and pharmacokinetic/pharmacodynamic studies in mice and in tissue and blood samples from patients with cancer.
Main findings: Regorafenib/anti-PD1 combination therapy increased survival compared with regofarenib or anti-PD1 alone in a regorafenib dose-dependent manner. Combination therapy increased regorafenib uptake into the tumor tissues by normalizing the HCC vasculature and increasing CD8 T-cell infiltration and activation at an intermediate regorafenib dose. The efficacy of regorafenib/anti-PD1 therapy was compromised in mice lacking functional T cells (Rag1-deficient mice). Regorafenib treatment increased the transcription and protein expression of CXCL10-a ligand for CXCR3 expressed on tumor-infiltrating lymphocytes-in murine HCC and in blood of patients with HCC. Using Cxcr3-deficient mice, we demonstrate that CXCR3 mediated the increased intratumoral CD8 T-cell infiltration and the added survival benefit when regorafenib was combined with anti-PD1 therapy.
Principal conclusions: Judicious regorafenib/anti-PD1 combination therapy can inhibit tumor growth and increase survival by normalizing tumor vasculature and increasing intratumoral CXCR3+CD8 T-cell infiltration through elevated CXCL10 expression in HCC cells.
Keywords: combination; drug therapy; liver neoplasms; programmed cell death 1 receptor.
Conflict of interest statement
Competing interests: LG reports personal fees from Agios Pharmaceuticals, Alentis Therapeutics, QED Therapeutics, H3 Biomedicine, Taiho Pharmaceuticals, Debiopharm, Incyte Corporation, SIRTEX and AstraZeneca. AXZ is a consultant/advisory board member for Bayer. RKJ received honorarium from Amgen and consultant fees from Chugai, Ophthotech, Merck, SPARC, SynDevRx and XTuit. Dr Jain owns equity in XTuit, Enlight, SPARC, SynDevRx and Accurius Therapeutics and serves as a paid member of the boards of trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund and Tekla World Healthcare Fund. He is a member of the scientific advisory board of Accurius Therapeutics. DZ, LF and the spouse of LLM are Bayer employees. MC is an AstraZeneca employee. DGD received consultant fees from Bayer, Simcere, Surface Oncology and Bristol Myers Squibb, and research grants from Bayer, Exelixis and Bristol Myers Squibb. No potential conflicts of interest were disclosed by other authors.
© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.
Figures
References
- Hernandez-Gea V, Toffanin S, Friedman SL, et al. . Role of the microenvironment in the pathogenesis and treatment of hepatocellular carcinoma. Gastroenterology 2013;144:512–27. 10.1053/j.gastro.2013.01.002
- Hoshida Y, Villanueva A, Kobayashi M, et al. . Gene expression in fixed tissues and outcome in hepatocellular carcinoma. N Engl J Med 2008;359:1995–2004. 10.1056/NEJMoa0804525
- Semela D, Dufour J-F. Angiogenesis and hepatocellular carcinoma. J Hepatol 2004;41:864–80. 10.1016/j.jhep.2004.09.006
- Llovet JM, Ricci S, Mazzaferro V, et al. . Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378–90. 10.1056/NEJMoa0708857
- Cheng A-L, Kang Y-K, Chen Z, et al. . Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol 2009;10:25–34. 10.1016/S1470-2045(08)70285-7
- Bruix J, Qin S, Merle P, et al. . Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 2017;389:56–66. 10.1016/S0140-6736(16)32453-9
- Abou-Alfa GK, Meyer T, Cheng A-L, et al. . Cabozantinib in patients with advanced and progressing hepatocellular carcinoma. N Engl J Med 2018;379:54–63. 10.1056/NEJMoa1717002
- Zhu AX, Kang Y-K, Yen C-J, et al. . Ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased α-fetoprotein concentrations (REACH-2): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol 2019;20:282–96. 10.1016/S1470-2045(18)30937-9
- Kudo M, Finn RS, Qin S, et al. . Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet 2018;391:1163–73. 10.1016/S0140-6736(18)30207-1
- Kudo M. Systemic therapy for hepatocellular carcinoma: 2017 update. Oncology 2017;93(Suppl 1):135–46. 10.1159/000481244
- Chen Y, Ramjiawan RR, Reiberger T, et al. . CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice. Hepatology 2015;61:1591–602. 10.1002/hep.27665
- Hato T, Goyal L, Greten TF, et al. . Immune checkpoint blockade in hepatocellular carcinoma: current progress and future directions. Hepatology 2014;60:1776–82. 10.1002/hep.27246
- Sia D, Jiao Y, Martinez-Quetglas I, et al. . Identification of an Immune-specific class of hepatocellular carcinoma, based on molecular features. Gastroenterology 2017;153:812–26. 10.1053/j.gastro.2017.06.007
- Zhu AX, Finn RS, Edeline J, et al. . Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial. Lancet Oncol 2018;19:940–52. 10.1016/S1470-2045(18)30351-6
- El-Khoueiry AB, Sangro B, Yau T, et al. . Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet 2017;389:2492–502. 10.1016/S0140-6736(17)31046-2
- Fukumura D, Kloepper J, Amoozgar Z, et al. . Enhancing cancer immunotherapy using antiangiogenics: opportunities and challenges. Nat Rev Clin Oncol 2018;15:325–40. 10.1038/nrclinonc.2018.29
- Hamzah J, Jugold M, Kiessling F, et al. . Vascular normalization in Rgs5-deficient tumours promotes immune destruction. Nature 2008;453:410–4. 10.1038/nature06868
- Jain RK. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Cancer Cell 2014;26:605–22. 10.1016/j.ccell.2014.10.006
- Huang Y, Yuan J, Righi E, et al. . Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Proc Natl Acad Sci U S A 2012;109:17561–6. 10.1073/pnas.1215397109
- Tian L, Goldstein A, Wang H, et al. . Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming. Nature 2017;544:250–4. 10.1038/nature21724
- Shigeta K, Datta M, Hato T, et al. . Dual programmed death receptor-1 and vascular endothelial growth factor receptor-2 blockade promotes vascular normalization and enhances antitumor immune responses in hepatocellular carcinoma. Hepatology 2020;71:1247–61. 10.1002/hep.30889
- Finn RS, Qin S, Ikeda M, et al. . Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma. N Engl J Med 2020;382:1894–905. 10.1056/NEJMoa1915745
- Fukuoka S, Hara H, Takahashi N, et al. . Regorafenib plus nivolumab in patients with advanced gastric or colorectal cancer: an open-label, dose-escalation, and Dose-Expansion phase Ib trial (REGONIVO, EPOC1603). J Clin Oncol 2020;38:2053–61. 10.1200/JCO.19.03296
- Tai W-T, Chu P-Y, Shiau C-W, et al. . STAT3 mediates regorafenib-induced apoptosis in hepatocellular carcinoma. Clin Cancer Res 2014;20:5768–76. 10.1158/1078-0432.CCR-14-0725
- Zopf D, Fichtner I, Bhargava A, et al. . Pharmacologic activity and pharmacokinetics of metabolites of regorafenib in preclinical models. Cancer Med 2016;5:3176–85. 10.1002/cam4.883
- Zender L, Xue W, Cordón-Cardo C, et al. . Generation and analysis of genetically defined liver carcinomas derived from bipotential liver progenitors. Cold Spring Harb Symp Quant Biol 2005;70:251–61. 10.1101/sqb.2005.70.059
- Chen Y, Huang Y, Reiberger T, et al. . Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice. Hepatology 2014;59:1435–47. 10.1002/hep.26790
- Tofilon PJ, Basic I, Milas L. Prediction of in vivo tumor response to chemotherapeutic agents by the in vitro sister chromatid exchange assay. Cancer Res 1985;45:2025–30.
- Reiberger T, Chen Y, Ramjiawan RR, et al. . An orthotopic mouse model of hepatocellular carcinoma with underlying liver cirrhosis. Nat Protoc 2015;10:1264–74. 10.1038/nprot.2015.080
- Zhou D, Conrad C, Xia F, et al. . Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 2009;16:425–38. 10.1016/j.ccr.2009.09.026
- Duda DG, Dima SO, Cucu D, et al. . Potential circulating biomarkers of recurrence after hepatic resection or liver transplantation in hepatocellular carcinoma patients. Cancers 2020;12:1275 10.3390/cancers12051275
- Wang F, Flanagan J, Su N, et al. . RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 2012;14:22–9. 10.1016/j.jmoldx.2011.08.002
- Hafner F-T, Werner D, Kaiser M. Determination of regorafenib (BAY 73-4506) and its major human metabolites BAY 75-7495 (M-2) and BAY 81-8752 (M-5) in human plasma by stable-isotope dilution liquid chromatography-tandem mass spectrometry. Bioanalysis 2014;6:1923–37. 10.4155/bio.14.52
- Wilhelm SM, Dumas J, Adnane L, et al. . Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int J Cancer 2011;129:245–55. 10.1002/ijc.25864
- Mpekris F, Voutouri C, Baish JW, et al. . Combining microenvironment normalization strategies to improve cancer immunotherapy. Proc Natl Acad Sci U S A 2020;117:3728–37. 10.1073/pnas.1919764117
- Jackson JD, Markert JM, Li L, et al. . Stat1 and NF-κB inhibitors diminish basal interferon-stimulated gene expression and improve the productive infection of oncolytic HSV in MPNST cells. Mol Cancer Res 2016;14:482–92. 10.1158/1541-7786.MCR-15-0427
- Regis G, Pensa S, Boselli D, et al. . Ups and downs: the STAT1:STAT3 seesaw of Interferon and gp130 receptor signalling. Semin Cell Dev Biol 2008;19:351–9. 10.1016/j.semcdb.2008.06.004
- Avalle L, Pensa S, Regis G, et al. . STAT1 and STAT3 in tumorigenesis: a matter of balance. JAKSTAT 2012;1:65–72. 10.4161/jkst.20045
- Avella DM, Li G, Schell TD, et al. . Regression of established hepatocellular carcinoma is induced by chemoimmunotherapy in an orthotopic murine model. Hepatology 2012;55:141–52. 10.1002/hep.24652
- Zheng C, Zheng L, Yoo J-K, et al. . Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 2017;169:e16:1342–56. 10.1016/j.cell.2017.05.035
- Chow MT, Ozga AJ, Servis RL, et al. . Intratumoral activity of the CXCR3 chemokine system is required for the efficacy of anti-PD-1 therapy. Immunity 2019;50:1498–512. 10.1016/j.immuni.2019.04.010
- Butera D, Marukian S, Iwamaye AE, et al. . Plasma chemokine levels correlate with the outcome of antiviral therapy in patients with hepatitis C. Blood 2005;106:1175–82. 10.1182/blood-2005-01-0126
Source: PubMed