Data Sheet 1_Impact of MEK inhibition on T-cell infiltration and function after radiotherapy in KRAS-mutant lung cancer.pdf

IntroductionKi-ras2 (KRAS) mutation is a common driver of lung cancer, and KRAS-mutated tumors are relatively resistant to radiotherapy. Previously, we demonstrated that mitogen-activated protein kinase (MEK) inhibitors (MEKi) enhanced treatment efficacy by increasing the anti-tumor immune response after radiotherapy in KRAS-mutant tumors. In this study, we explored the potential mechanism underlying the MEKi-mediated increase in anti-tumor immune response.
Methods and resultRNA sequencing revealed that the MEKi+radiotherapy combination significantly activated the chemokine signaling pathway. Blocking the C-X-C motif chemokine ligand 10 (CXCL10) receptor reduced T-cell infiltration in vivo. The MEKi+radiotherapy combination increased CXCL10 expression and activated the cyclic GMP–AMP synthase-stimulator of interferon genes (cGAS-STING) pathway in KRAS-mutant lung cell lines. Using a STING inhibitor and cGAS-knockout LLC cells, we showed that CXCL10 production was mediated by the cGAS-STING pathway via nuclear factor kappa B activation. This combination also increased DNA damage and apoptosis in KRAS-mutant lung cancer cell lines, triggering the cGAS-STING pathway. Western blot analysis revealed that MEKi reduced checkpoint kinase 2 phosphorylation after radiotherapy, hindering DNA repair and increasing DNA damage. Flow cytometry revealed that MEKi combined with radiotherapy boosted tumor-infiltrating CD4+ and CD8+ T cells in vivo, enhancing their cytotoxic and secretory functions. In an LLC-bearing mouse model, combining MEKi with varying radiotherapy doses and extending drug holidays revealed that low-dose radiotherapy with MEKi effectively controlled tumor growth.
ConclusionOur findings suggest that MEKi activates the cGAS-STING-TANK-binding kinase 1-nuclear factor kappa B-CXCL10 axis post-radiotherapy in KRAS-mutant lung cancer, increasing T-cell infiltration and function, activating anti-tumor immunity, and inhibiting tumor growth. These results indicate the potential for clinical translation.

引言：Ki-ras2（KRAS）突变是肺癌常见的驱动因素，且KRAS突变肿瘤对放疗相对抗拒。此前本团队已证实，丝裂原活化蛋白激酶（mitogen-activated protein kinase, MEK）抑制剂（MEKi）可通过增强KRAS突变肿瘤放疗后的抗肿瘤免疫应答，提升治疗效果。本研究旨在探究MEKi介导的抗肿瘤免疫应答增强的潜在机制。
材料与方法及结果：RNA测序结果显示，MEKi联合放疗可显著激活趋化因子信号通路。阻断C-X-C基序趋化因子配体10（CXCL10）受体可降低体内T细胞浸润水平。MEKi联合放疗可上调KRAS突变肺细胞系中CXCL10的表达，并激活环状GMP-AMP合酶-干扰素基因刺激因子（cGAS-STING）通路。通过使用STING抑制剂及cGAS基因敲除的LLC细胞，本研究证实CXCL10的产生依赖于cGAS-STING通路经核因子κB（nuclear factor kappa B, NF-κB）的激活介导。该联合疗法还可增加KRAS突变肺癌细胞系的DNA损伤与细胞凋亡，进而触发cGAS-STING通路。蛋白质免疫印迹（Western blot）分析显示，MEKi可降低放疗后检查点激酶2的磷酸化水平，阻碍DNA修复并加剧DNA损伤。流式细胞术结果表明，MEKi联合放疗可在体内促进肿瘤浸润CD4+及CD8+T细胞的浸润，并增强其细胞毒功能与分泌功能。在LLC荷瘤小鼠模型中，通过调整MEKi联合放疗的剂量梯度及设置停药间歇期实验发现，低剂量放疗联合MEKi可有效控制肿瘤生长。
结论：本研究结果表明，在KRAS突变型肺癌中，MEKi可在放疗后激活cGAS-STING-TANK结合激酶1（TANK-binding kinase 1, TBK1）-核因子κB-CXCL10信号轴，促进T细胞浸润并增强其功能，激活抗肿瘤免疫并抑制肿瘤生长。上述结果提示该联合疗法具备临床转化潜力。