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 建立新的替代疗法的需求仍然没有得到满足，这种疗法既可以对 以及刺激抗癌免疫反应，这可能会提供额外的机会 与新兴的免疫疗法相结合。我们在TNBC中发现了一种新的肿瘤代谢特征 其中癌细胞利用鞘磷脂清除脂质的表型，该表型可通过重新调整用途来靶向 FDA批准的治疗高雪症的药物--选择性葡萄糖神经酰胺合成酶抑制剂利格卢斯特 疾病。我们的初步研究表明，伊格列斯特通过以下途径促进丝裂毒性神经酰胺的积累 结果是从存活的有丝分裂转变为致命的有丝分裂和随后的癌细胞死亡。此外，我们还展示了 力格列斯特在临床可达到的剂量下抑制TNBC荷瘤小鼠的肿瘤生长，并且 伊立格列他的抗癌作用与肿瘤浸润性CD4和CD8的显著增加有关 T细胞，这表明利格列斯特在增强抗肿瘤免疫反应方面具有额外的作用。初级阶段 这项建议的目的是建立新的用途，使伊立伐他作为一种免疫代谢佐剂，用于 治疗肿瘤细胞癌并明确其增强抗癌免疫的机制(S) 回应。为了测试这一点，我们将评估伊立格列他在患者来源的异种移植(Pdx)中的抗癌效果。 从接受初级治疗的患者产生的模型-幼稚的TNBC对 化疗(具体目标1a)。为了测试联合使用伊立格列他加抗PD-L1抗体的效果是否有所改善 抗癌效果与单独治疗相比，我们将使用BRCA1co/co；MMTV-CRE；P53/-和 4T1原位同基因小鼠TNBC模型(特异性靶点1b)。体内感兴趣的主要终点 研究将包括总体存活率和肿瘤生长；干预后对肿瘤免疫表型的影响 将使用多重免疫荧光面板和单细胞转录切割法对单细胞水平进行评估 组织的表达图谱。我们的目标也是确定利格卢斯特诱导抗癌的机制。 免疫反应。首先，我们将评估利格列斯特对cGAS-STING信号蛋白和 下游通路在TNBC细胞中的活性(特异性靶2a)。接下来，我们将使用先进的质谱学 结合新的细胞外小泡分离方法确定MHC-I结合的技术 伊立伐他汀对TNBC细胞及肿瘤细胞来源的循环和瘤内EV表面的影响 治疗。ELISPOT和基于细胞毒T细胞的肿瘤杀伤活细胞检测将用于测试该功能 EVS对体外激活T细胞的影响(特异靶2b)。如果成功，我们的潜在发现将提供关键的预 使用伊立格列他的临床证据和将伊立格列他转移到早期临床试验的理由。 鉴于iglustat已经被FDA批准用于另一种适应症，它有很大的潜力很容易被翻译 进入临床应用，并有可能为癌症免疫治疗提供一个新的范例。

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.