E2F3 amplification primes bladder cancer cells for premature mitosis.

The E2F-RB pathway is essential to enforce the G1/S checkpoint. Hence, many tumors contain mutations that override this checkpoint, such as RB1 loss or CDKN2A loss, to activate E2F dependent transcription, which facilitates cell cycle entry. However, these mutations do not necessarily result in sustained E2F-dependent transcription throughout the cell cycle. This is different in tumors with E2F3 amplification, because the resulting E2F3 overexpression persists through S- and G2-phase to cause excessive and unscheduled E2F-dependent transcription. E2F3 is a bona fide oncogene, which is frequently amplified in bladder cancer. Here we investigated how E2F3 amplification impacts the cancer cell cycle. Using isogenic bladder cancer cell lines and patient data we found that E2F3 amplification causes hyperactivation of the FOXM1-dependent mitotic gene expression program, including cyclin B1. This primes cells to undergo unscheduled and catastrophic mitosis when the mitotic kinase CDK1 was hyperactivated by treating cells with the PKMYT1 inhibitor RP-6306, thus uncovering a promising synthetic lethal interaction. Bladder cancer cells with E2F3 amplification developed resistance to RP-6306 by reducing cyclin B1 expression, thereby mitigating premature mitotic entry. This resistance could be overcome by adding a low dose of WEE1 inhibitor to PKMYT1 inhibition. Together, these data strongly suggest that PKMYT1-dependent inactivation of CDK1 is essential to prevent premature mitosis in bladder cancer cells harboring E2F3 amplifications. This suggests an opportunity for precision medicine strategies in bladder cancer patients with E2F3 amplification and/or excessive cyclin B1 expression. SignificanceE2F3 gene amplification is a recurrent genetic alteration in muscle-invasive bladder cancer, creating a synthetic lethal dependency on PKMYT1. This vulnerability provides a rationale for development of personalized therapies targeting PKMYT1.