CYP4X1抑制剂调控肿瘤相关巨噬细胞PD-L1抗肿瘤作用和机制研究

PD-1/PD-L1 inhibitors have demonstrated remarkable efficacy in a wide range of advanced cancers, but their overall response rates are low and some patients can be undone by their development of resistance. Thus, identification of such mechanisms and development of novel target, biomarkers and appropriate combination strategies are highly prudent. Our previous work demonstrated that cytochrome P450 (CYP) 4X1 is over-expressed in tumor-associated macrophages (TAMs) from human cancer tissues and positive correlated with PD-L1. We first dynamically investigate the effects of CYP4X1 knockout or the small molecule inhibitors (CH625 and 17-ODYA) on the growth and metastasis of Lewis lung cancer and B16F10 melanoma, metabolism of the endocannabinoid anandamide (AEA) and PD-L1 expression in TAMs as well as infiltration and activation of CD8+T cells. We then study whether the CYP4X1 inhibitors down-regulate CB2 and EGFR signaling-mediated PD-L1 in TAMs, and thereby enhance the anti-tumor activity of CD8+T cells in the CB2 or EGFR knockout mice models, co-implantation models of wild type or CYP4X1-overexpressed macrophages with tumor cells, or in vitro co-culture of TAMs with CD8+T cells. Finally, we investigate the chemical structural modification of CH625, and confirm the synergy of the novel CYP4X1 inhibitors and anti-PD-1/PD-L1 antibody. Together, the CYP4X1 inhibitors attenuate tumor immune escape mediated by PD-L1 in TAMs through the regulation of metabolism of the endocannabinoid AEA as well as CB2 and EGFR-STAT3 axis, and CYP4X1 may serve as a potential therapeutic target and biomarker in human cancer, and thus have important theoretical significance and clinical value.

PD-1/PD-L1抑制剂对肿瘤疗效显著，但总体应答率偏低。因此，研究新的靶点、标志物和联合治疗策略是当前热点。本项目是在前期发现CYP4X1在人癌组织中高表达并与肿瘤相关巨噬细胞PD-L1表达显著正相关的基础上，首先研究CYP4X1敲除或CH625等抑制剂抗小鼠Lewis肺癌和B16F10黑色素瘤生长和转移及其与巨噬细胞内源性大麻素代谢、PD-L1表达和CD8+T细胞浸润和活化的关系。随后采用CB2或EGFR敲除、巨噬细胞与肿瘤细胞混合接种或与T细胞共培养模型，研究CYP4X1抑制剂通过CB2和EGFR信号下调巨噬细胞PD-L1提高CD8+T细胞的抗肿瘤免疫反应；最后结构修饰CH625并验证与PD-1/PD-L1单抗联用的治疗效能。阐明CYP4X1抑制剂调控肿瘤微环境内源性大麻素代谢抗PD-L1介导免疫逃逸的作用及机理，确定潜在免疫治疗靶点、候选药物和联合疗法具有重要理论意义和临床价值。

PD-1/PD-L1抑制剂对肿瘤疗效显著，但总体应答率偏低。因此，研究新的靶点、标志物和联合治疗策略是当前热点。本项目首先通过生物信息学及临床标本分析发现CYP4X1在人结肠癌组织中高表达，且巨噬细胞中CYP4X1高表达与总存活期和和T细胞免疫功能负相关。随后，采用Cyp4x1-/-小鼠研究CYP4X1敲除抗小鼠Lewis肺癌、B16F10黑色素瘤、MC38结肠癌生长和转移及其与巨噬细胞内源性大麻素代谢、PD-L1表达和CD8+ T细胞浸润和活化的关系。结果，CYP4X1敲除显著抑制B16F10黑色素瘤和MC38结肠癌移植瘤生长，但促进Lewis肺癌生长。在小鼠结肠癌脾脏注射实验性肝转移动物模型中，CYP4X1敲除可显著抑制CT26结肠癌细胞肝转移。在巨噬细胞与肿瘤细胞混合接种模型中，敲除CYP4X1.或CYP4X1抑制剂17-ODYA处理可显著抑制4T1乳腺癌和MC38结肠癌生长并促进CD8+ T细胞浸润及功能，抑制巨噬细胞浸润及其M2标志（Arginase-1和CD206）和PD-L1表达。在PD-1/PD-L1抑制剂BMS-1诱导的心肌毒性模型中发现，敲除CYP4X1促进PD-1/PD-L1抑制剂诱导的心肌毒性但增加其抗黑色素瘤的疗效。在DOX诱导的肾损伤模型中，敲除CYP4X1加重DOX诱导的巨噬细胞M1样极化和焦亡，从而加重DOX诱导的足细胞损伤。综上所述，本项目在国内外首次从巨噬细胞CYP4X1过表达→内源性大麻素AEA代谢生成14,15-EET-EA↑→CB2信号灭活/EGFR信号激活↑→PD-L1表达↑→CD8+ T细胞凋亡、无能和耗竭↑→肿瘤生长和转移↑角度阐明CYP4X1抑制剂调控肿瘤微环境内源性大麻素代谢抗PD-L1介导免疫逃逸的作用及机理，为肿瘤治疗提供了新的靶点和候选药物，因而具有重要的理论意义和潜在临床应用价值。