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），细胞扩散 到脑脊液（CSF）空间以及脑和脊髓的膜覆盖物。之甚少 已知这种疾病的生物学，并且没有FDA批准的LMM疗法。这突出了一个 在知识和未满足的需要，在晚期黑色素瘤患者的临床护理的主要差距。我们的单细胞 黑色素瘤患者标本的RNAseq研究表明，LMM微环境是独特的， 免疫抑制，由非活性的功能失调的T细胞和交替激活的T细胞主导。 巨噬细胞LMM中的肿瘤细胞上调脂质代谢介质，包括相关酶 脂肪酸、脂类和肉毒碱合成。这是再加上增加外源性肉毒碱和脂质 与没有LMM的患者相比，在LMM患者的脑脊液中发现。虽然PD 1的表达 在LMM中的CD 8 + T细胞与在其他转移部位发现的相当，LMM中的肿瘤不 对PD 1抑制有反应，尽管在其他疾病部位有反应。我们将检验一个假设， 肿瘤中的脂质代谢和外源性脂肪酸、脂质和肉毒碱的上调， 浸润的巨噬细胞和CD 8 + T细胞的代谢有利于脂肪酸氧化和增加的氧化 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LMM患者和小鼠中代谢过程的综合图谱。 肿瘤中脂质代谢上调的免疫微环境。稳定同位素示踪， 将对患者巨噬细胞进行靶向代谢测定以及培养基补充和消耗 和CD 8 + T细胞的情况下，自体LMM衍生的CSF和原发性肿瘤培养物，以测量 外源性肉毒碱、脂肪酸和脂类对促进脂肪酸氧化和氧化还原的影响 磷酸化先前的研究表明脂质代谢在介导免疫应答中起着关键作用 并支持肿瘤在代谢应激条件下的存活。我们将确定是否针对脂质 使用脂肪酸合成酶或CPT 1a的抑制剂的代谢将促进营养不良中的肿瘤细胞死亡。 LMM微环境和恢复LMM中巨噬细胞和CD 8 + T细胞的抗肿瘤功能。我们将利用 与自体巨噬细胞和T细胞共培养的人LMM的原代培养物，以检查是否靶向 脂肪酸合成酶或CPT 1a将促进巨噬细胞的抗肿瘤极化和T细胞的活化。 我们将使用LMM的体内模型来测试脂肪酸合成酶或CPT 1a的抑制是否会减弱免疫- 抑制环境并使肿瘤对抗PD 1治疗敏感。总的来说，本提案将确定 促进LMM肿瘤微环境中促肿瘤发生相互作用的代谢驱动因素， 为LMM特异性疗法的开发提供药物靶点。

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.