RBM39降解剂对神经母细胞瘤的抗癌活性：CDKN2A/B缺失作为一种生物标志物

Robust anticancer effects of molecular glue RBM39 degraders have been reported in human neuroblastoma models (DOI: 10.1126/sciadv.abj5405 and 10.1038/s41467-022-28907-3). In the present study we further explored the MOA underlying the potent activity of RBM39 degraders in neuroblastoma, utilizing SEED Therapeutics' optimized RBM39 degrader ST-00937, and its deuterium derivative ST-01156 that was recently cleared by the FDA for clinical testing (NCT07197554 ) . Method In vitro anticancer activity was profiled in 3D clonogenic assays utilizing tissue derived from 4 neuroblastoma patients and 6 human neuroblastoma tumor cell lines. In vivo anticancer efficacy was evaluated in nude mice bearing subcutaneous tumors established with SH-SY5Y human neuroblastoma cells. Cell lysates generated from 2D culture of neuroblastoma cell lines treated with ST-01156 were evaluated by TMT labeling mass spectrometry and Western Blot. Result ST-00937 treatment showed differential anticancer activity in 6 neuroblastoma cell lines and 4 patient-derived models. For the 6 neuroblastoma cell lines, the IC 50 values ranged from 0.04 to 0.3µM among 5 sensitive cell lines, while SH-EP cells did not respond. The IC 50 for colony formation ranged from 0.03 to 2.3µm in 4 patient-derived models. Notably, the most insensitive patient-derived model NB0277 has a homozygous CDKN2A/B deletion. In vitro activity was extended to in vivo activity utilizing SH-SY5Y cells. In vivo, complete tumor regressions were observed with 5-Days On/2-Days Off, twice daily oral treatment with ST-01156, without body weight loss. Proteomic analysis in vitro showed that ST-01156 decreased proteins involved in DNA damage and cell cycle control, and increased p53 signaling after 16h treatment. The divergent anticancer responses of SH-SY5Y and SH-EP subclones (both derived from SK-N-SH) warranted further investigation. Although both lines expressed similar levels of RBM39 and exhibited complete RBM39 degradation after treatment, distinct downstream effects were observed. In SH-SY5Y cells (adrenergic CDKN2A wild-type, cMYC amplified), cMYC expression was downregulated and the p21/p53 pathway was upregulated by ST-01156. In contrast, these changes were not detected in SH-EP cells (mesenchymal, CDKN2A/B deletion). This differential regulation may explain why cleaved caspase-3 (apoptosis) was not observed in ST-01156 treated SH-EP CDKN2A/B deleted cells, although DNA damage indicated by gammaH2AX was increased in both cell lines. Conclusion RBM39 degraders demonstrate differential potency in 6 neuroblastoma cell lines and 4 patient-derived models in vitro, and total tumor regression in SH-SY5Y in vivo. DNA damage and increased p53-mediated apoptosis are confirmed in the sensitive neuroblastoma model SH-SY5Y, while resistance in CDKN2A/B-deleted lines suggests CDKN2A loss may serve as a predictive biomarker of resistance to RBM39-targed therapy.