Epigenetic reversal of lineage plasticity enhances responsiveness to anti-GD2 therapy in neuroblastoma [ATAC-seq SK-N-AS]

GD2 is a disialoganglioside that is highly expressed on the surface of neuroblastoma cells. Immunotherapy with anti-GD2 antibodies has revolutionized the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse and little is known about mechanisms of resistance to anti-GD2. Neuroblastomas harbor intrinsic transcriptional plasticity by co-opting divergent lineage-specific developmental programs between adrenergic and mesenchymal cell states. We found that reduced GD2 expression was significantly correlated with the adrenergic cell state in neuroblastoma and that an Adrenergic-to-Mesenchymal Transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Induced reprogramming of adrenergic cells with the master AMT regulator PRRX1 was sufficient to promote transcriptional rewiring in isogenic models and downregulate GD2 expression. Mechanistically, low-GD2 expressing cell lines demonstrate significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Primary neuroblastoma tumors enriched for mesenchymal features show demonstrably lower GD3 synthase expression as compared to adrenergic tumors. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to an anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma. SK-N-AS cells were treated for 14 days with vehicle (V) or 1 uM tazemetostat (T) in biological quadruplicate

GD2是一种在神经母细胞瘤细胞表面高表达的双唾液酸神经节苷脂（disialoganglioside）。抗GD2抗体免疫疗法彻底革新了高危儿童神经母细胞瘤的治疗方案，但仍有近半数患者出现复发，目前学界对抗GD2疗法的耐药机制尚不明晰。
神经母细胞瘤具备内在的转录可塑性，可通过共采纳肾上腺素能（adrenergic）与间质（mesenchymal）细胞状态间的不同谱系特异性发育程序实现表型转换。本研究发现，神经母细胞瘤中GD2表达下调与肾上腺素能细胞状态显著相关，而肾上腺素能向间质转化（Adrenergic-to-Mesenchymal Transition, AMT）可介导GD2表达下调及抗GD2抗体耐药性的产生。
借助AMT核心调控因子PRRX1诱导肾上腺素能细胞重编程，足以在同基因模型（isogenic models）中促成转录重编程并下调GD2的表达水平。从机制上看，低GD2表达的细胞系中，神经节苷脂合成酶ST8SIA1（GD3合酶）的表达水平显著降低，导致GD2合成通路出现瓶颈。相较于肾上腺素能表型的原发性神经母细胞瘤肿瘤，富含间质特征的肿瘤组织中GD3合酶的表达水平明显更低。
对EZH2进行药理学抑制，可使间质型神经母细胞瘤细胞发生表观遗传重编程（epigenetic rewiring）并重新表达ST8SIA1，恢复GD2的细胞表面表达，同时恢复其对抗GD2抗体的敏感性。
上述研究结果证实，发育谱系是决定抗GD2免疫疗法敏感性的关键因素，并验证了EZH2抑制剂可与抗GD2抗体联合开展临床测试，以改善儿童神经母细胞瘤患者的治疗预后。
SK-N-AS细胞以生物学四重重复的方式，分别用赋形剂（V）或1 μM他泽司他（T）处理14天。