E2F3 amplification primes bladder cancer cells for premature mitosis

The E2F-RB pathway controls the G1/S checkpoint, and tumors often bypass it through RB1 or CDKN2A loss. Unlike these lesions, E2F3 amplification drives persistent excessive E2F-dependent transcription through S and G2 phases. This oncogene is frequently amplified in for example bladder cancer, but its impact on the cancer cell cycle remains unclear. Using isogenic bladder cancer models and patient data, we show that E2F3 amplification hyperactivates a mitotic gene expression program, including cyclin B1. This predisposes cells to unscheduled mitosis when the G2/M checkpoint is inhibited using the PKMYT1 inhibitor lunresertib, alone or in combination with low dosages of the WEE1 inhibitor zederosertib. E2F3-amplified cells acquired resistance to lunresertib by permanently reducing cyclin B1 expression, thereby preventing premature mitotic entry. Importantly, this resistance was reversed by co-treatment with a low dose of WEE1 inhibitor. These findings identify PKMYT1-dependent CDK1 inhibition as a critical safeguard against premature mitosis in E2F3-amplified bladder cancer. Thus, we uncover an opportunity for precision medicine strategies aimed at G2/M checkpoint inhibition to promote catastrophic mitosis in bladder cancer patients with E2F3 amplification and excessive cyclin B1 expression.