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

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.

背景：瑞戈非尼（Regorafenib）与其他多激酶抑制剂可增强抗程序性细胞死亡蛋白1（anti-program cell death-1, anti-PD1）疗法在肝细胞癌（hepatocellular carcinoma, HCC）中的抗肿瘤效力，但其除抗血管生成作用之外的免疫调节效应尚未明确阐明。
方法：体内抗肿瘤效力测试在多种同源性肝癌模型中开展；采用小鼠骨髓来源巨噬细胞（murine bone marrow-derived macrophages, BMDMs）体外探究瑞戈非尼对巨噬细胞极化的调控作用，以及共培养T细胞的活化情况。通过定量反转录聚合酶链反应（quantitative reverse transcription PCR, RT-PCR）、精氨酸酶活性检测、流式细胞术与酶联免疫吸附实验（ELISA）测定M1/M2极化标志物。通过敲除上游丝裂原活化蛋白激酶14（MAPK14）激酶、使用p38激酶化学抑制剂以及染色质免疫沉淀实验，验证了p38激酶及下游Creb1/Klf4信号通路在巨噬细胞极化中的调控作用。
结果：瑞戈非尼（5 mg/kg/天，约为人类临床剂量的一半）在体内的抗肿瘤与抗血管生成效果与DC-101（抗VEGFR2抗体）相当，但可诱导更强的T细胞活化与M1型巨噬细胞极化；体外实验中，瑞戈非尼可提升M1/M2极化的骨髓来源巨噬细胞比例，并促进共培养T细胞的增殖与活化，提示其存在不依赖抗血管生成的免疫调节作用。瑞戈非尼可通过抑制骨髓来源巨噬细胞中p38激酶的磷酸化及其下游Creb1/Klf4活性，逆转M2型极化。瑞戈非尼可增强过继转移的抗原特异性T细胞的抗肿瘤效力。瑞戈非尼与抗PD1疗法的协同抗肿瘤效力与肿瘤微环境中的多条免疫相关通路密切相关。
结论：瑞戈非尼可通过调控巨噬细胞极化增强抗肿瘤免疫，这一作用不依赖其抗血管生成效应。优化瑞戈非尼给药剂量以合理设计联合治疗方案，或可提升临床治疗指数。