Introduction: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present significant therapeutic challenges in the metastatic setting due to their aggressive clinical course and limited treatment options. Recent integrative transcriptomic analyses have identified non-oncogene addiction (NOA) genes as potential therapeutic vulnerabilities in advanced PPGL. Among these, budding uninhibited by benzimidazoles 1 (BUB1), a mitotic checkpoint kinase, emerged as a leading candidate. BUB1 regulates chromosome alignment, spindle checkpoint fidelity, and chromatin modification through phosphorylation of histone H2A at threonine 120 (H2A-T120). Given its elevated expression in metastatic PPGL, BUB1 represents a promising therapeutic target. To interrogate this vulnerability pharmacologically, we employed BAY1816032, a selective first-in-class BUB1 kinase inhibitor. Here, we report its effects on PPGL-derived cells, with a focus on viability, colony formation, apoptosis, mitotic integrity, and metastatic behaviors such as migration and invasion.
Methods: The human PPGL-derived hPheo1 cell line, which expresses appreciable levels of BUB1, was treated with BAY1816032 across a range of concentrations (0–2 µM). Parallel assays were performed in BUB1-low cell lines (MTT and PC12) to assess specificity. Cell viability was measured by Sulforhodamine B (SRB) assays, while long-term proliferative capacity was quantified through colony formation assays. Apoptosis was assessed using a real-time Annexin V luminescence assay at 32 hours post-treatment. Cell cycle distribution was analyzed by propidium iodide staining and flow cytometry after 48 hours of treatment. Mitotic checkpoint fidelity was further examined by immunocytochemistry to detect micronuclei formation and by Western blotting for phosphorylation of histone H2A-T120. To evaluate metastatic potential, wound healing assays were conducted to monitor collective cell migration, and transwell assays were performed to quantify individual cell migration and invasion. Expression of epithelial-to-mesenchymal transition (EMT) markers N-cadherin and vimentin was assessed by Western blotting.
Results: Pharmacological inhibition of BUB1 with BAY1816032 demonstrated a strong antitumor effect in PPGL-derived cells. Treatment reduced cell viability and long-term proliferative capacity, while also inducing apoptosis and disrupting cell cycle progression. Evidence of impaired mitotic checkpoint signaling and chromosomal instability confirmed that the compound acts directly through BUB1 inhibition.
Beyond growth suppression, BAY1816032 significantly limited migratory and invasive behaviors, consistent with reduced expression of epithelial-to-mesenchymal transition markers. These findings establish that targeting BUB1 not only compromises tumor cell survival but also attenuates metastatic potential. Importantly, the similarity between pharmacological inhibition and siRNA-mediated silencing underscores the specificity of this approach.
Conclusion: Our study shows that BAY1816032, a selective BUB1 inhibitor, exerts strong antitumor activity in PPGL-derived cells. Treatment reduced cell growth and survival while also limiting invasive and migratory behaviors, highlighting BUB1’s role in both proliferation and metastasis. The overlap between genetic silencing and pharmacological inhibition confirms the specificity of this approach.
These findings provide the first evidence that targeting BUB1 may represent a promising therapeutic strategy for metastatic PPGL, supporting further exploration of BUB1 inhibitors in rare and aggressive neuroendocrine tumors.