Defining and targeting the immune-suppressive metabolic microenvironment of leptomeningeal melanoma metastases

晚期黑色素瘤最致命的并发症是软脑膜转移(Lmm)，在那里细胞扩散。 进入脑脊液(CSF)间隙以及大脑和脊髓的膜覆盖物。很少有 了解这种疾病的生物学，而且还没有FDA批准的治疗LMM的方法。这突出了一个 在晚期黑色素瘤患者的临床护理中，知识上的重大差距和未得到满足的需求。我们的单细胞 对黑色素瘤患者样本的RNAseq研究表明，LMM微环境是独特的 免疫抑制，主要由不活跃、功能失调的T细胞主导，也可被激活 巨噬细胞。LMM中的肿瘤细胞上调脂代谢的介质，包括涉及的酶 在脂肪酸、脂肪和肉碱的合成中。这与外源性肉碱和脂类的增加有关。 在LMM患者与非LMM患者的脑脊液中发现。虽然PD1的表达 在CD8T细胞上，LMM与其他转移部位的T细胞相似，而LMM的肿瘤则不是 对PD1抑制有反应，尽管在其他疾病部位有反应。我们将检验增加的假设 肿瘤中的脂代谢以及外源性脂肪酸、脂类和肉碱的上调推动了肿瘤细胞的转变 浸润性巨噬细胞和CD8 T细胞的代谢有利于脂肪酸氧化和氧化增加 磷酸化，支持软脑膜的免疫功能障碍。我们将利用我们广泛的单细胞 来自LMM患者和小鼠样本的RNAseq数据，以创建一张全面的代谢过程图 肿瘤中脂质代谢上调周围的免疫微环境。稳定的同位素追踪， 将对患者巨噬细胞进行有针对性的代谢分析和介质补充和耗竭 和CD8 T细胞在自体LMM来源的脑脊液和原代肿瘤培养的背景下测量 外源性肉碱、脂肪酸和脂肪促进脂肪酸氧化和氧化的作用 磷酸化。先前的研究表明，脂代谢在调节免疫反应中起着关键作用。 并支持在代谢应激条件下的肿瘤存活。我们将确定是否以脂质为靶点 使用脂肪酸合酶抑制剂或CPT1a的代谢会促进营养贫乏者的肿瘤细胞死亡 LMM的微环境和恢复巨噬细胞和CD8T细胞的抗肿瘤功能。我们将利用 人LMM与自体巨噬细胞和T细胞共培养的原代培养 脂肪酸合酶或CPT1A可促进巨噬细胞的抗肿瘤极化和T细胞的激活。 我们将使用LMM的体内模型来测试抑制脂肪酸合成酶或CPT1A是否会减弱免疫- 抑制环境，并使肿瘤对抗PD1治疗敏感。总体而言，该提案将确定 促进LMM和Will肿瘤微环境中促肿瘤相互作用的代谢驱动因素 为LMM特异性疗法的发展提供可用药靶点。

The deadliest complication of late-stage melanoma is leptomeningeal metastasis (LMM), where the cells spread to the cerebrospinal fluid (CSF) space and the membrane coverings of the brain and spinal cord. Very little is known about the biology of this disease and there are no FDA-approved therapies for LMM. This highlights a major gap in knowledge and an unmet need in the clinical care of late-stage melanoma patients. Our single-cell RNAseq studies of melanoma patient specimens demonstrate that the LMM microenvironment is uniquely immune-suppressed and is dominated by inactive, dysfunctional T cells and alternatively activated macrophages. The tumor cells in the LMM upregulate mediators of lipid metabolism, including enzymes involved in fatty acid, lipid and carnitine synthesis. This is coupled with an increase in exogenous carnitines and lipids found in the cerebrospinal fluid of patients with LMM compared to those without. Although the expression of PD1 on CD8+ T cells in the LMM are comparable to those found at other sites of metastasis, tumors at LMM do not respond to PD1 inhibition despite responding at other sites of disease. We will test the hypothesis that increased lipid metabolism in the tumor and upregulation of exogenous fatty acids, lipids and carnitines fuel a shift in the metabolism of infiltrating macrophages and CD8+ T cells to favor fatty acid oxidation and increased oxidative phosphorylation, supporting immune dysfunction at the leptomeninges. We will utilize our extensive single-cell RNAseq data from LMM patient and mouse samples to create a comprehensive map of metabolic processes in the immune microenvironment surrounding the upregulated lipid metabolism in the tumor. Stable isotope tracing, targeted metabolic assays and media supplementation and depletion will be performed on patient macrophage and CD8+ T cells in the context of autologous LMM-derived CSF and primary tumor cultures to measure the effects of exogenous carnitines, fatty acids and lipids on promoting fatty acid oxidation and oxidative phosphorylation. Previous studies suggest lipid metabolism plays a critical role in mediating immune responses in cancer and support tumor survival under conditions of metabolic stress. We will determine if targeting the lipid metabolism using inhibitors of fatty acid synthase or CPT1a would promote tumor cell death in the nutrient poor LMM microenvironment and restore anti-tumor function of macrophages and CD8+ T cells in LMM. We will utilize primary cultures of human LMM in co-culture with autologous macrophages and T cells to examine if targeting fatty acid synthase or CPT1a would promote anti-tumor polarization of macrophages and activation of T cells. We will use in vivo models of LMM to test if inhibition of fatty acid synthase or CPT1a will attenuate the immune- suppressive environment and sensitize the tumors to anti-PD1 therapy. Overall, this proposal will identify the metabolic drivers promoting the pro-tumorigenic interactions in the tumor microenvironment of LMM and will provide druggable targets for the development of LMM-specific therapies.