Repurposing Glucosylceramide Synthase to Promote Mitochondrial Lethality and Potentiate an Anti-Tumor Immune Response in Triple-Negative Breast Cancer

项目摘要 目前治疗三阴性乳腺癌（TNBC）的方法仍然不令人满意。那里 仍然需要建立新的替代疗法， 癌细胞以及刺激抗癌免疫反应，可能会提供额外的机会 与新兴的免疫疗法相结合。我们发现了TNBC中一种新的肿瘤代谢特征 其中癌细胞利用通过再利用可靶向的鞘磷脂脂质清除表型 选择性葡萄糖神经酰胺合成酶抑制剂eliglustat，一种FDA批准的治疗戈谢病的药物， 疾病我们的初步研究表明，eliglustat可促进有丝分裂毒性神经酰胺的蓄积， 结果从存活线粒体自噬转变为致死线粒体自噬和随后的癌细胞死亡。此外，我们显示 在TNBC荷瘤小鼠中，eliglustat以临床可达到的剂量抑制肿瘤生长， eliglustat的抗癌作用与肿瘤浸润性CD 4+和CD 8+显著增加相关 T细胞，表明eliglustat在增强抗肿瘤免疫应答中的额外作用。主 该提案的目的是建立eliglustat作为“免疫代谢佐剂”的新用途， 治疗TNBC，并定义eliglustat增强抗癌免疫的机制 反应为了验证这一点，我们将评估eliglustat在患者源性异种移植物（PDX）中的抗癌疗效。 从原发性初治TNBC患者中生成的模型，这些患者对 化疗（具体目标1a）。为了测试eliglustat+抗PD-L1的组合是否提高了产量， 与单独治疗相比，我们将使用BRCA 1co/co; MMTV-Cre; p53+/-和 TNBC的4 T1原位同基因小鼠模型（特异性目的1b）。体内关注的主要终点 研究将是总生存期和肿瘤生长;干预后对肿瘤免疫表型的影响 将使用多重免疫荧光板和单细胞转录组学进行单细胞水平评估 组织的表达谱。我们还旨在确定eliglustat诱导抗癌的机制， 免疫反应首先，我们将评估eliglustat对cGAS-STING信号传导蛋白的影响， TNBC细胞中的下游途径活性（特异性目标2a）。接下来，我们将使用先进的质谱仪 技术与细胞外囊泡（EV）的新分离方法相结合，以确定MHC-I结合 在eliglustat后TNBC细胞和TNBC衍生的循环和肿瘤内EV表面上的肽组 治疗将使用ELIspot和基于细胞毒性T细胞的肿瘤杀伤活细胞测定来测试功能性 EV对离体活化T细胞的影响（特异性目标2b）。如果成功，我们的潜在发现将提供关键的前- 使用eliglustat的临床证据和将eliglustat转入早期临床试验的理由。 鉴于eliglustat已被FDA批准用于另一种适应症，因此其很有可能易于转化 临床应用，并可能为癌症免疫治疗提供新的范例。

Project Summary Current approaches to treating triple-negative breast cancer (TNBC) remain unsatisfactory. There remains an unmet need to establish novel alternative therapies that can both exert targeted effects on cancer cells as well as stimulate an anti-cancer immune response that may render additional opportunity for combination with emerging immunotherapy. We have uncovered a novel onco-metabolic feature in TNBC wherein cancer cells exploit a sphingomyelin lipid scavenging phenotype that is targetable through repurposing of the selective glucosylceramide synthase inhibitor eliglustat, an FDA approved drug for treatment of Gaucher Disease. Our preliminary studies demonstrate that eliglustat promotes accumulation of mitotoxic ceramides with resultant shift from survival mitophagy to lethal mitophagy and subsequent cancer cell death. Moreover, we show that eliglustat suppresses tumor growth at clinically achievable doses in TNBC tumor-bearing mice and that the anti-cancer effects of eliglustat are associated with pronounced increases in tumor infiltrating CD4+ and CD8+ T-cells, suggesting an additional role of eliglustat in potentiating an anti-tumor immune response. The primary objectives of this proposal are to establish novel utility for eliglustat as an ‘immunometabolic adjuvant’ for the treatment of TNBC and to also define the mechanism(s) by which eliglustat potentiates an anti-cancer immune response. To test this, we will assess the anti-cancer efficacy of eliglustat in patient-derived xenograft (PDX) models generated from patients with primary treatment-naïve TNBC that did or did not go on to respond to chemotherapy (Specific Aim 1a). To test whether the combination of eliglustat plus anti-PD-L1 yields improved anti-cancer effects compared to either treatment alone, we will use the BRCA1co/co; MMTV-Cre; p53+/- and 4T1 orthotopic syngeneic mouse models of TNBC (Specific Aim 1b). Primary endpoints of interest for in vivo studies will be overall survival and tumor growth; effects on the tumor immunophenotype following intervention will be assess using multiplex immunofluorescence panels and single cell transcriptomics for single cell-level expression profiling of tissues. We also aim to define the mechanisms by which eliglustat induces an anti-cancer immune response. First, we will evaluate the effect of eliglustat on cGAS-STING signaling proteins and downstream pathway activities in TNBC cells (Specific Aim 2a). Next, we will use advanced mass spectrometry technologies coupled with novel isolation methods for extracellular vesicles (EVs) to define the MHC-I bound peptidome on surfaces of TNBC cells and TNBC-derived circulating and intra-tumor EVs following eliglustat treatment. ELIspot and Cytotoxic T Cell-based tumor killing live-cell assays will be used to test the functionality of EVs on activating T-cells ex vivo (Specific Aim 2b). If successful, our potential findings will provide key pre- clinical evidence for the use of eliglustat and justification for moving eliglustat into early phase clinical trials. Given that eliglustat is already FDA-approved for another indication, it has high potential to be readily translated into clinical use, and potentially providing a new paradigm for cancer immunotherapy.