Glutamine antagonist JHU083 reprograms immunosuppressive tumor-associated macrophages to drive tumor immunity in urologic cancers, bulk RNA-seq

Glutamine metabolism in the tumor microenvironment is emerging as a critical regulator of immune-mediated anti-tumor responses. We report potent tumor growth inhibition by the glutamine antagonist prodrug JHU083 in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes (TIMs). Using orthogonal approaches, we show that JHU083-mediated glutamine antagonism in the tumor microenvironment induces TNF, inflammatory, and mTORC1 signaling in different intra-tumoral TAM clusters. Additionally, we report that JHU083 increases proliferation in tissue-resident macrophages intratumorally and in different TAM sub-clusters. Functionally, we report that JHU083-reprogrammed TAMs have increased tumor cell phagocytosis and diminished pro-angiogenic capacities. In vivo inhibition of glutamine consumption in TAMs results in increased glycolysis, broken TCA cycle, and disruption in purine metabolism. Although the effect of glutamine antagonism was less profound on tumor-infiltrating T cells for their anti-tumor activity, it promoted a stem cell-like phenotype in CD8+ T cells and decreased the CD4+ Treg abundance. Additionally, we report that JHU083 causes a global shutdown in glutamine utilizing metabolic pathways in tumor cells, leading to reduced HIF-1?, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key anti-tumoral features. Overall design: Subcutaneous B6CaP tumors were harvested from mice treated with JHU083 or control. Tumors were dissociated and macrophages were fluorescence-activated cell sorted (FACS) by CD45+CD3-Ly6G-CD11b-F4/80+ and submitted for bulk RNA-sequencing.

肿瘤微环境中的谷氨酰胺代谢，正逐渐成为免疫介导抗肿瘤应答的关键调控因子。本研究证实，谷氨酰胺拮抗剂前药JHU083可通过重编程肿瘤相关巨噬细胞（TAMs）与肿瘤浸润单核细胞（TIMs），对泌尿系统肿瘤产生强效生长抑制效应。采用正交实验策略，我们证实肿瘤微环境中JHU083介导的谷氨酰胺拮抗作用，可在不同肿瘤内TAM亚群中激活TNF信号、炎症信号以及mTORC1信号通路。此外，本研究发现JHU083可促进肿瘤内组织驻留巨噬细胞以及不同TAM亚群的增殖活性。功能层面上，经JHU083重编程的TAMs可增强肿瘤细胞吞噬能力，并削弱其促血管生成特性。在体内抑制TAMs的谷氨酰胺消耗，可引发糖酵解增强、三羧酸（TCA）循环紊乱以及嘌呤代谢失衡。尽管谷氨酰胺拮抗作用对肿瘤浸润T细胞的抗肿瘤活性影响较弱，但可促使CD8+ T细胞获得干细胞样表型，并降低CD4+调节性T细胞（Treg）的丰度。此外，本研究证实JHU083可全面阻断肿瘤细胞内谷氨酰胺依赖的代谢通路，进而降低HIF-1α、c-MYC的磷酸化水平，并诱导肿瘤细胞凋亡——上述均为关键的抗肿瘤特性。实验整体设计：将经JHU083或对照试剂处理的小鼠皮下移植B6CaP肿瘤组织取材分离；将肿瘤组织解离后，通过CD45+CD3-Ly6G-CD11b-F4/80+标记，采用荧光激活细胞分选术（FACS）分离巨噬细胞，随后进行批量RNA测序（bulk RNA-sequencing）。