结合ERAP1沉默和恩替诺特疗法,克服神经母细胞瘤对癌症免疫疗法的耐药性。

IF 11.4 1区 医学 Q1 ONCOLOGY Journal of Experimental & Clinical Cancer Research Pub Date : 2024-10-22 DOI:10.1186/s13046-024-03180-y
Patrizia Tempora, Silvia D'Amico, Paula Gragera, Verena Damiani, Kamila Krol, Valentina Scaldaferri, Kirti Pandey, Shanzou Chung, Valeria Lucarini, Ezio Giorda, Marco Scarsella, Gabriele Volpe, Marco Pezzullo, Cristiano De Stefanis, Valentina D'Oria, Lorenzo De Angelis, Roberto Giovannoni, Maria Antonietta De Ioris, Ombretta Melaiu, Anthony W Purcell, Franco Locatelli, Doriana Fruci
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引用次数: 0

摘要

背景:检查点免疫疗法可释放T细胞对肿瘤的控制作用,但对于非免疫原性肿瘤,如MHC I类低表达和新抗原负荷低的肿瘤,如神经母细胞瘤(NB),这种疗法就会受到破坏。内质网氨肽酶 1(ERAP1)是一种在肽载入 MHC I 类分子之前对其进行修饰的酶。抑制 ERAP1 会产生新的抗原,从而诱导有效的抗肿瘤免疫反应。在这里,我们发现了一种新型无毒组合策略,该策略基于对ERAP1的基因抑制和HDAC抑制剂(HDACi)恩替诺司他的施用,可增加NB的免疫原性,使其对PD-1疗法产生反应:方法:利用 CRISPR/Cas9 介导的基因编辑技术敲除(KO)来自 TH-MYCN 转基因小鼠自发性肿瘤的 9464D NB 细胞中的 ERAP1 基因。流式细胞术(FC)评估了 MHC I 类和 PD-L1 的表达。质谱法研究了这些细胞的免疫肽组。将携带 9464D 的小鼠脾脏细胞与肿瘤细胞共培养,可通过 FC 评估 ERAP1 抑制对炎症细胞因子分泌的影响,以及免疫细胞对 ERAP1 KO 细胞的激活和迁移。Caspase 3/7 检测法和流式细胞术分析评估了肿瘤细胞杀伤作用。通过FC、免疫组化和多重免疫荧光分析了抑制ERAP1对肿瘤免疫成分的影响:结果:我们发现,抑制ERAP1会增加CD4+和CD8+T细胞以及NK细胞的召回和活化,从而使9464D细胞更容易受到免疫细胞介导的杀伤。用恩替诺特治疗可诱导 9464D 细胞在体外和体内表达 MHC I 类和 PD-L1 分子。这不仅导致ERAP1抑制诱导的免疫肽组发生明显变化,还通过重塑肿瘤浸润T细胞区系抑制了体内ERAP1 KO肿瘤的生长。有趣的是,ERAP1缺失与恩替诺斯他和PD-1阻断联合使用,可克服对PD-1免疫疗法的耐药性,并提高宿主存活率:这些研究结果表明,ERAP1抑制与HDACi恩替诺司他治疗和PD-1阻断相结合,可重塑NB等非免疫原性肿瘤的免疫格局,使其对检查点免疫疗法产生反应。
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Combining ERAP1 silencing and entinostat therapy to overcome resistance to cancer immunotherapy in neuroblastoma.

Background: Checkpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy.

Methods: CRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence.

Results: We found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4+ and CD8+ T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival.

Conclusions: These findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy.

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来源期刊
CiteScore
18.20
自引率
1.80%
发文量
333
审稿时长
1 months
期刊介绍: The Journal of Experimental & Clinical Cancer Research is an esteemed peer-reviewed publication that focuses on cancer research, encompassing everything from fundamental discoveries to practical applications. We welcome submissions that showcase groundbreaking advancements in the field of cancer research, especially those that bridge the gap between laboratory findings and clinical implementation. Our goal is to foster a deeper understanding of cancer, improve prevention and detection strategies, facilitate accurate diagnosis, and enhance treatment options. We are particularly interested in manuscripts that shed light on the mechanisms behind the development and progression of cancer, including metastasis. Additionally, we encourage submissions that explore molecular alterations or biomarkers that can help predict the efficacy of different treatments or identify drug resistance. Translational research related to targeted therapies, personalized medicine, tumor immunotherapy, and innovative approaches applicable to clinical investigations are also of great interest to us. We provide a platform for the dissemination of large-scale molecular characterizations of human tumors and encourage researchers to share their insights, discoveries, and methodologies with the wider scientific community. By publishing high-quality research articles, reviews, and commentaries, the Journal of Experimental & Clinical Cancer Research strives to contribute to the continuous improvement of cancer care and make a meaningful impact on patients' lives.
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