定量磷酸化蛋白质组学揭示CX-4945对神经母细胞瘤翻译调控和分化的影响

Introduction: Neuroblastoma is a third most prevalent pediatric cancer of the sympathetic nervous system, which is often diagnosed at a clinically advanced stage with primary or acquired resistance to conventional chemotherapy. Protein Kinase CSNK2A1 (CK2) is an intrinsically active, ubiquitously present serine-threonine kinase overactive in various pediatric solid cancers, including neuroblastoma (NBL) and Ewing sarcoma (EWS). Recurrent genetic alterations in CK2 were not observed in NBL, indicating that its oncogenic role is driven by dysregulated activity rather than mutation. CX-4945 (Silmitasertib) is a potent, ATP-competitive CK2 inhibitor with low-nanomolar Ki values and high selectivity, targeting 49 of 250 tested kinases. CX-4945 has demonstrated potent in vivo antitumor activity in solid tumors and leukemia, with favorable pharmacokinetics and good tolerability in human studies. It is currently under Phase I clinical evaluation for pediatric solid tumors. Here, we report the quantitative phospho-proteomic profiling of CX-4945 treated NBL cells Methods: The susceptibility of NBL cell lines with and without MYCN amplification was tested against CX-4945. We performed label-free quantitative Phosphoproteomic profiling of 2 NBL cell lines (SMS-KCNR and SHSY5Y) following in vitro treatment with CX-4945 for 24 h. Trypsin digested peptides were enriched for phospho-peptides by using Titanium Ion-Metal Affinity Chromatography. Differentially expressed phospho-peptides and their corresponding phosphoproteins were identified using high-resolution LC-MS/MS, and functional enrichment analysis was conducted to map the affected signaling networks. We also performed transcriptomic analysis for comparing the commonly regulated proteins at mRNA and Protein level. For validation studies, WB and IHC-based quantification of selected phosphoproteins, and their associated pathways, was tested in pre-clinical model-derived tissue treated with CX-4945. Results: Quantitative phospho-proteomics analysis revealed 4064 and 3131 downregulated phospho-proteins in CX-4945-treated NBL cell lines, SMS-KCNR and SHSY5Y, respectively. GPCR5C/IRS4-PI3K-AKT/MYC axis and EIF4A1/MYC axis are found to be downregulated in multi-omics approach. Network analysis highlighted the disruption of translation initiation, proliferation, and neuronal differentiation signaling, suggesting a mechanistic link between CK2 inhibition and altered signaling pathways. CK2 mediated cMYC signaling inhibition was validated using WB and IHC studies. Conclusion: Protein kinase CK2 is intrinsically active and negatively impacts survival in HR-NBL patients. Phospho-proteomic analysis reveals, changes in multiple pathways which leads to reduced proliferation and increased neuronal differentiation.