Shokraie Fatemeh, Lechermeier Larissa, Bordihn Pia, Kaps Philipp, Möller Steffen, Schulz Anna Sophie, Schneider Björn, Koczan Dirk, Roshan Samira Khanipour, Lode Holger N, Classen Carl-Friedrich, Hahn Olga, Troschke-Meurer Sascha, Maletzki Claudia
University Children's Hospital, Rostock University Medical Center, University of Rostock, Rostock, Germany.
Institute of Clinical Chemistry and Laboratory Medicine, Rostock University Medical Center, University of Rostock, Rostock, Germany.
Cell Death Discov. 2025 Aug 2;11(1):363. doi: 10.1038/s41420-025-02637-z.
The rarity of recurrent somatic mutations poses a challenge for the targeted treatment of neuroblastoma (NB). Differentiation therapy is an encouraging prospect, with cyclin-dependent kinase inhibitors (CDKis) representing a promising avenue for promoting NB differentiation. This study investigated three CDKis (abemaciclib, fadraciclib, and dinaciclib) alone or combined with retinoic acid (RA) to assess the effects on morphology, growth, gene expression, and the induction of immunogenic cell death in NB cell lines with (LAN-1 and CHLA-90) and without (CHLA-172) MYCN amplification. All cell lines demonstrated sensitivity to CDK inhibition. Notably, low-dose abemaciclib promoted cellular differentiation, as evidenced by the emergence of stromal-like morphological features and upregulation of the differentiation markers STMN4 and ROBO2. Treatment with abemaciclib or fadraciclib led to the upregulation of calnexin and holocytochrome C, which are part of the global stress response, along with the protein p27, which arrests the cell cycle. Molecularly, CDKis sensitivity correlated with an increased CDK4-specific copy number, along with a partial deletion of CDKN2a in two cases (LAN-1, CHLA-172). The addition of RA augmented the effects of the monotherapy, particularly in LAN-1 cells, in both 2D and 3D culture, and both treatments triggered immunogenic cell death, evidenced by calreticulin translocation. Transcriptomic analysis of LAN-1 and CHLA-90 cells revealed that genes deregulated by monotherapy (fadraciclib or RA) were re-regulated in the presence of the second drug. Combination therapy significantly downregulated CRABP2 and CYP26B1, both of which are involved in RA metabolism and its degradation. Furthermore, CCNE2, MYBL2, and MCM4 were strongly suppressed in the fadraciclib/RA combination, confirming the induction of cell cycle arrest. CDKi treatments promote NB differentiation via ER stress, with cytotoxicity enhanced by RA co-treatment. This may increase NB immunogenicity and support immunotherapy eligibility.
复发性体细胞突变的罕见性给神经母细胞瘤（NB）的靶向治疗带来了挑战。分化疗法是一个令人鼓舞的前景，细胞周期蛋白依赖性激酶抑制剂（CDKis）是促进NB分化的一个有前景的途径。本研究调查了三种CDKis（阿贝西利、法曲利布和地西利布）单独使用或与视黄酸（RA）联合使用，以评估其对具有（LAN-1和CHLA-90）和不具有（CHLA-172）MYCN扩增的NB细胞系的形态、生长、基因表达以及免疫原性细胞死亡诱导的影响。所有细胞系均表现出对CDK抑制的敏感性。值得注意的是，低剂量阿贝西利促进了细胞分化，这表现为基质样形态特征的出现以及分化标志物STMN4和ROBO2的上调。用阿贝西利或法曲利布处理导致钙网蛋白和全细胞色素C上调，它们是全局应激反应的一部分，同时蛋白p27上调，p27可使细胞周期停滞。在分子水平上，CDKis敏感性与CDK4特异性拷贝数增加相关，在两例（LAN-1、CHLA-172）中还伴有CDKN2a的部分缺失。在二维和三维培养中，添加RA增强了单药治疗的效果，特别是在LAN-1细胞中，两种治疗均引发了免疫原性细胞死亡，这通过钙网蛋白易位得以证明。对LAN-1和CHLA-90细胞的转录组分析表明，单药治疗（法曲利布或RA）失调的基因在第二种药物存在时会重新调节。联合治疗显著下调了CRABP2和CYP26B1，这两者均参与RA代谢及其降解。此外，在法曲利布/RA联合治疗中，CCNE2、MYBL2和MCM4被强烈抑制，证实了细胞周期停滞的诱导。CDKi治疗通过内质网应激促进NB分化，RA联合治疗增强了细胞毒性。这可能会增加NB的免疫原性并支持免疫治疗的适用性。
