The G2/M checkpoint is a critical regulatory mechanism ensuring proper DNA replication and repair before mitosis preventing genomic instability and aneuploidy. Its dysfunction is implicated in various cancers, including Malignant Pleural Mesothelioma (MPM), a rare and aggressive cancer with poor prognosis. The BUB gene family, particularly BUB1 and its interaction partners BUB1B and BUB3, plays a key role in both the G2/M checkpoint and Spindle Assembly Checkpoint (SAC), ensuring accurate chromosome segregation. Whole-genome CRISPR/Cas9 screening in MPM cell lines identified BUB1B and BUB3 as essential genes, suggesting their involvement in MPM progression. This study aims to investigate the molecular effects of BUB1B and BUB3 in MPM, assessing their role in cell proliferation and survival to explore their therapeutic potential.

Recently we conducted genome-wide CRISPR screening in H2052, H2452, and H28 MPM cell lines, identifying the BUB gene family (BUB1, BUB1B, and BUB3) as having a significant impact on MPM cell survival and proliferation. Following CRISPR/Cas9-mediated gene knockout, the dependency of MPM cells on BUB1B and BUB3 and their roles in aggressive phenotypes were assessed through functional assays such as BrdU incorporation, cell cycle, and 2D and 3D colony formation. Ongoing functional analyses aim to test the molecular and cellular implications of BUB1B and BUB3 in MPM.

Genetic silencing of BUB1B and BUB3 individually profoundly impaired MPM cell proliferation and survival. Cell cycle analysis revealed significant accumulation in the G2 phase. Elevated expression of BUB1B or BUB3 was strongly associated with poor overall survival, as predicted based on their expression levels in publicly available datasets. Critically, DepMap analyses conducted in MPM cell lines revealed a significant depletion of BUB1B and BUB3, supporting their impact on MPM survival. Interestingly, G2/M checkpoint molecules with high interaction scores in StringDB annotation have generally exhibited a depletion trend, highlighting that MPM cells are functionally dependent on proper G2/M modulation. Our ongoing studies aim to provide a deeper understanding of the functional and molecular effects of these genes on the G2/M checkpoint and their role in MPM pathobiology.

Findings highlight the critical role of BUB1B and BUB3 in the proliferation and survival of MPM cells, emphasizing their potential as both biomarkers and therapeutic targets. Silencing of these genes disrupted the cell cycle, particularly by inducing arrest in the G2 phase, pointing to their essential function in regulating the G2/M checkpoint. Further functional and molecular studies will be essential to fully understand the mechanisms by which BUB1B and BUB3 contribute to MPM pathogenesis. These investigations may offer valuable insights into novel therapeutic strategies targeting the G2/M checkpoint in MPM treatment.