Docking protein-1 in macrophage immunotherapy of gastric cancer

胃癌（GC）患者通常处于晚期，无法进行有效治疗，导致临床结局不佳。尽管GC在中国和世界范围内的发病率和患病率正在下降，但在老龄化人口中，GC的死亡率仍然很高，是一个具有挑战性的健康问题。新的治疗方法被设计为促进宿主免疫应答朝向肿瘤攻击模式，防止免疫逃避和宿主无能。然而，在GC中保证免疫疗法（例如针对免疫检查点（PD 1/PD-L1）的抗体）的临床功效的潜在机制是未知的。对接蛋白-1（DOK 1）是一种细胞内的衔接子，它抑制致癌Ras信号传导并控制免疫受体活性。DOK 1由胰岛素增敏剂（格列酮）诱导，并通过与过氧化物酶体增殖物激活受体-γ（PPARg）相互作用而稳定，导致体外和体内GC生长减弱。我们最近发现DOK 1抑制PD-L1表达，并促进人巨噬细胞对肿瘤攻击、促炎M1表型的极化和效应功能。DOK 1和M1（例如toll样受体）基因的改变导致GC患者预后不良，并且DOK 1 mRNA/蛋白在肿瘤进展期间在肿瘤和免疫细胞区室中受到差异调节。因此，本提案研究将探索DOK 1的可药用性是否可用于与临床使用的免疫抑制抗体协同作用（针对PD 1/PD-L1）或新型吞噬刺激剂我们的翻译方法包括三个具体目标：1）使用生物素捕获测定鉴定DOK 1结合的巨噬细胞特异性免疫受体; 2）在人巨噬细胞系和原代细胞中使用功能获得和CRISPR/Cas9敲除来研究DOK 1的免疫调节作用的细胞和分子机制; 3）研究DOK 1表达和巨噬细胞（骨髓谱系）浸润在GC小鼠模型和治疗中的患者组织中的（临床前）意义。这些结果有望为DOK 1在GC巨噬细胞中的功能提供机制性见解，加强我们对癌症免疫治疗的认识，并促进GC患者未来有效的治疗策略。

Gastric cancer (GC) patients usually present at an advanced stage, where effective treatment is impossible, resulting in poor clinical outcome. Although the incidence and prevalence of GC in China and world-wide is declining, the mortality from GC in an ageing population is still high and a challenging health problem. Novel therapies were designed to boost the host immune response towards a tumor-attacking mode, preventing immune evasion and host anergy. However, the underlying mechanisms that warrant clinical efficacy of immunotherapies, e.g. such as antibodies against immune checkpoints (PD1/PD-L1), in GC are unknown. Docking protein-1 (DOK1) is an intracellular adapter which inhibits oncogenic Ras signaling and controls immune receptor activities. DOK1 is induced by insulin sensitizers (glitazones) and stabilized by interaction with peroxisome proliferator-activated receptor-gamma (PPARg), resulting in attenuated GC growth in vitro and in vivo. We have recently shown that DOK1 inhibits PD-L1 expression and promotes polarization and effector functions of human macrophages towards the tumor-attacking, pro-inflammatory M1 phenotype. Alterations in DOK1 and M1 (e.g. toll-like receptors) genes conferred poor prognosis in GC patients, and DOK1 mRNA/protein was differentially regulated during tumor progression in tumor and immune cell compartments. The present proposal study shall therefore explore whether the drugability of DOK1 can be exploited to synergize with clinically-in-use immunotherapeutic antibodies (against PD1/PD-L1) or novel phagocytosis stimulating agents (against CD47) to enforce a tumor-attacking macrophage response.Our translational approach comprises three specific aims: 1) to identify macrophage-specific immune receptor(s) bound by DOK1 using biotin capture assay; 2) to investigate the cellular and molecular mechanisms underlying the immune-regulatory role of DOK1 using gain-of-function and CRISPR/Cas9-knock-out in human macrophage lines and primary cells; 3) to study the (pre)clinical significance of DOK1 expression and macrophage (myeloid lineage) infiltration in GC mouse models and patients’ tissues under therapy. Results are expected to deliver mechanistic insight into the function of DOK1 in macrophages of GC, strengthen our knowledge of cancer immunotherapy and facilitate future effective therapeutic strategies for GC patients.