低剂量去甲基化药物通过激活Th1免疫应答逆转结直肠癌PD-1抑制剂抵抗的机制研究

Colrectal Carcinoma （CRC）is one of the most common tumor types in China. Immune checkpoint blockade has become an important approach for tumor treatment. However, owing to PD-1 blockade resistance, most of CRC patients cannot benefit from PD-1 blockade. Previous sudies have demonstrated that PD-1 blockade resistance results in Th1 anergy. Emerging evidence indicates that DNA methylation can regulate Th1 activation, providing the theoretical basis for the combination of demethylating agent and PD-1 blockade. Our preliminary results found that the combination of low-dose demethylating agent and PD-1 blockade markedly inhibits disease progression in 9/11 patients with PD-1 blockade resistance, suggesting that low-dose demethylating agent could reverse PD-1 blockade resistance. Therefore, we aim to investigate the molecular mechnism of Th1 activation in CRC pateints with PD-1 blockade by the combined treatment of low-dose demethylating agent and PD-1 blockade, and to establish optimized regimen of low-dose demethylating agent combined with PD-1 blockade in CRC patients PD-1 blockade resistance.

结直肠癌是我国最常见恶性肿瘤之一，以PD-1抑制剂为代表的免疫检验点阻断治疗已成为肿瘤治疗的重要手段，但耐药性导致多数肿瘤患者无法获益。研究表明PD-1抑制剂抵抗导致Th1失能，而Th1活性受甲基化调控，为研究去甲基化药物通过激活Th1免疫应答逆转PD-1抑制剂抵抗提供理论基础。前期临床试验显示低剂量去甲基化药物联合PD-1抑制剂显著抑制9/11例PD-1抑制剂抵抗肿瘤患者的疾病进展，提示低剂量去甲基化药物可逆转PD-1抑制剂抵抗。我们拟从分子、细胞、动物模型及临床样本四个层面解析低剂量去甲基化药物通过激活Th1免疫应答逆转结直肠癌PD-1抑制剂抵抗的分子机制，建立优化的低剂量去甲基化药物联合PD-1抑制剂治疗结直肠癌的预后评估体系。

虽然PD-1阻断治疗已成为肿瘤免疫治疗的重要手段，但由于治疗抵抗导致大多数肿瘤患者无法获益。CD4+ T细胞在肿瘤免疫治疗中具有重要作用。DNA甲基化介导的CD4+ T细胞失能或耗竭减弱PD-1阻断治疗的反应性，导致肿瘤免疫逃逸。本研究中，我们探索低剂量DNA去甲基化药物地西他滨能否增强CD4+ T细胞介导的抗肿瘤免疫应答。本研究主要包含两个部分：（1）体外实验中，我们使用低剂量地西他滨处理分选的CD4+ T细胞。结果显示，低剂量地西他滨促进CD4+ T细胞的活化、增殖，增加IFN-γ+CD4+ T（Th1）亚群比例，增强CD4+ T细胞的细胞溶解能力并抑制小鼠结直肠癌MC38肿瘤生长。其机制是低剂量地西他滨通过上调E3泛素连接酶β-TrCP表达促进IκBα泛素化与降解，增强NF-κB活化进而促进CD4+ T细胞介导的抗肿瘤免疫应答。（2）使用低剂量地西他滨联合治疗小鼠结直肠癌模型。结果显示，低剂量地西他滨联合PD-1抑制剂增强对MC38肿瘤生长的抑制，改善PD-1抑制剂抵抗。流式细胞结果显示，低剂量地西他滨联合PD-1抑制剂增强肿瘤浸润的CD4+ T细胞活性、抑制CD4+ T细胞终末耗竭、增加CD4+耗竭前体细胞。总体上，本研究证实低剂量地西他滨增强CD4+ T细胞介导的抗肿瘤免疫应答，为改善肿瘤免疫治疗临床疗效提供理论基础。