Malignant pleural mesothelioma (MPM) is a rare but highly aggressive malignancy with limited treatment options, a severe prognosis, and poor survival rates, necessitating the identification of novel therapeutic targets. BUB1, a mitotic checkpoint serine-threonine kinase, plays a pivotal role in the spindle assembly checkpoint (SAC) function and accurate chromosome segregation. Elevated BUB1 expression has been correlated with poor prognosis in multiple cancers. Here, we demonstrate the role of BUB1 as an emerging cancer-essential gene and a potential druggable vulnerability in MPM.

BUB1 was identified as a vulnerability for MPM cells by a whole-genome CRISPR screen and this role was validated by knockout and pharmacological inhibition (BAY-1816032). RNA sequencing was conducted to analyze transcriptomic profiles of BUB1-depleted MPM cells. Affected pathways and regulatory networks were defined by gene set enrichment analysis (GSEA). To explore BUB1’s functional role in mitotic regulation, localization of key mitotic checkpoint proteins and BUB1 interaction partners MAD1, MAD2, and Shugoshin (SGO1) was assessed by immunofluorescence (IF) staining and the expression of BUB1 local network genes (CDC20, Cyclin A, Cyclin B, and p21) was measured by western blot. Additionally, time-lapse live-cell imaging was conducted to track mitotic progression and cytokinesis dynamics in real time.

MPM cell survival and growth were significantly impaired by genetic depletion or pharmacological inhibition of BUB1, which also caused G2/M cell cycle arrest, cellular senescence, apoptosis, and a reduction in aggressive cancer cell traits. RNA-seq analysis of BUB1 knockout cells revealed widespread transcriptional changes associated with cell cycle regulation, G2/M checkpoint control, and mitotic reprogramming. Mechanistically, BUB1 is essential for the proper localization of MAD1, MAD2, and SGO1, ensuring the integrity of the spindle assembly checkpoint (SAC). The loss of BUB1 impaired SAC integrity, leading to mitotic errors. These defects were accompanied by significant downregulation of CDC20, Cyclin A, and Cyclin B, alongside an upregulation of p21, as well as G2/M cell cycle arrest reinforcing the role of BUB1 in G2/M checkpoint control. Live-cell imaging further confirmed that BUB1 KO cells exhibited prolonged mitosis, frequent cytokinesis failure, and multinucleation, underscoring the essential function of BUB1 in maintaining mitotic fidelity.

Our findings establish BUB1 as a critical regulator of mitosis by modulating the G2/M checkpoint in MPM. The observed mitotic defects and altered transcriptional programs reinforce the role of BUB1 in MPM pathophysiology. These results highlight BUB1 as a key dependency in MPM and provide a strong rationale for targeting BUB1 in preclinical models of MPM therapy.