Abstract A047: PML nuclear bodies under siege: How a single phospho-switch fuels neuroblastoma aggression

Abstract Promyelocytic leukemia protein (PML) is a multifunctional nuclear scaffold that are critical transcriptional regulators through chromatin remodeling, DNA repair and signal transduction. In neuroblastoma (NBL), an aggressive pediatric cancer driven by profound transcriptional plasticity and lineage infidelity, PML nuclear bodies (PML-NBs) govern the dynamic equilibrium between differentiated and dedifferentiated cell states. Nonetheless, this regulatory framework is intricately controlled by synchronized post-translational modifications, particularly phosphorylation, which leads to the destabilization of PML-NBs. Herein, we investigated serine 518 (S518) phosphorylation driven destabilization of PML-NBs that rewires miRNome to promote NB evolution. Site-directed mutagenesis was employed to dissect the role of phosphorylation of PML in NBL reprogramming. In cells from patients with progressive disease unresponsive to intensive multimodal therapy, S518 of PML was substituted with alanine (A518) to generate a phospho-dead mutant. In contrast, cells obtained at diagnosis were engineered to express a phospho-mimetic variant by replacing S518 with glutamic acid (E518). All mutations were stably introduced and confirmed through sequencing. Co-immunoprecipitation assays of S518 mutant cells unveiled phosphorylation mediated destabilization of PML was independent of the canonical PML-RARα signaling. Notably, sustained S518 phosphorylation disrupts PML-RARα interaction. miRNome modifications independently driven by phosphorylation at S518 were assessed using Nanostring ncounter miRNome expression assay and analyzed with nSolver software. Differential expression analysis coupled with stringent criteria (32-fold up/down modulation) calculation identified distinctive dysregulation of 76 miRs on phosphorylation of PML. miRNet 2.0 bioinformatic analysis unveiled that these miRs regulate key molecular pathways including TGFβ, Hippo, WNT, NOTCH, NFκB, JAK-STAT, FOXO, and mTOR that are critically involved in regulation of epithelial–mesenchymal transition (EMT), cancer stem cell (CSC) maintenance, self-renewal, therapy resistance, dedifferentiation, and metastasis. Together, our findings uncover a previously unrecognized role of PML phosphorylation at S518 in destabilizing PML nuclear bodies independently of canonical PML–RARα signaling, that reprograms the miRNA landscape, converging on key oncogenic pathways that drives cellular plasticity, stemness, therapy resistance, and metastatic progression in neuroblastoma. These insights highlight S518 phosphorylation as a critical regulatory node in neuroblastoma plasticity and a potential therapeutic target for disrupting tumor evolution. Funding: This work was partially or in full, funded by Department of Defense CA-210339; OCAST-HR19-04; NIH-P20GM103639. Citation Format: Sreenidhi Mohanvelu, Poorvi Subramanian, Natarajan Aravindan. PML nuclear bodies under siege: How a single phospho-switch fuels neuroblastoma aggression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A047.