Head and neck squamous cell carcinoma (HNSCC) is the sixth most common form of cancer. A mutant form of the tumor suppressor gene p53 is often found in HNSCC. p53 plays an important role in DNA repair and can initiate apoptosis in cells with irreparable DNA damage. In response to DNA damage, p53 can also hold cells at the G1/S regulation point to allow cells more time to fix DNA damage before proceeding to DNA replication. Cells with mutant p53 may pass through the G1/S checkpoint with DNA damage that may be repaired at the G2/M checkpoint before mitosis. Despite mutations in p53, HNSCC tumors can escape destruction by DNA damaging therapies. In order to repair damage, these cells likely rely on WEE1, a cell cycle kinase which can prevent entry of damaged cells into mitosis. The research group of the late Dr. Eduardo Mendez previously published studies showing that WEE1, a G2/M checkpoint regulator gene, is required for survival of p53 mutant HNSCC cells. Dr. Mendez led a phase I clinical trial showing that an inhibitor of WEE1, AZD1775, in combination with DNA damaging chemotherapy, reduced tumors in patients.
The laboratory of Dr. Mendez (Clinical Research Division) initiated a collaboration with the Sidorova lab at (University of Washington, Department of Pathology) to learn more about cellular responses to AZD1775. They published a study in Molecular Cancer Research in which they leverage this understanding to improve therapeutic options for HNSCC patients. This study, led by Post-Doctoral Research Fellow Dr. Ahmed Diab, followed HNSCC cell lines through multiple cell cycles after WEE1 inhibition. The researchers found that WEE1 inhibition resulted in some cells becoming phosphorylated on the histone H2AX (γH2AX), a marker of DNA damage. They next tested WEE1 inhibition in cells lines depleted of p53 and found a larger portion of these cells contained very high amounts of γH2AX. The authors confirmed the presence of DNA single- and double-strand breaks (SSB and DSB) in these cells through comet assays. Diab explained the results: "During a therapeutically relevant regimen of exposure to AZD1775 p53 status alters cellular responses in every phase of the cell cycle, and over consecutive cycles, resulting in a cytotoxic positive feedback loop. Not just the G1/S checkpoint, as was expected, but also the G2/M checkpoint response to WEE1 inhibition are attenuated in p53-deficient cells, and unexpectedly, the replication stress-related slowing of replication forks is exacerbated."
When both cell lines were treated with AZD1775, they found lower levels of 53BP1 bodies, an indicator of replication stress, in p53-deficient cells. In explaining the significance of the study Diab said, "While both in control and p53-null lines cells that replicated DNA and progressed through mitosis in AZD1775 can carry over residual DNA damage even if the drug is no longer present, only the p53-proficient line develops 53BP1 bodies, while p53-deficient cells instead mount a secondary γH2AX response. To my knowledge, a phenotypic interaction between p53 and 53BP1 in the context of the response to WEE1 inhibition has not been observed previously and its mechanism is as yet unknown."
While some forms of HNSCC are associated with heavy tobacco or alcohol use, others are thought to be caused by the human papillomavirus (HPV). HPV- HNSCC often have a mutant form of p53, while HPV in HPV+ HNSCC can inactivate p53. On the future of AZD1775 research Diab said, "Hypersensitivity of HPV+ HNSCC to AZD1775 may be determined by p53 inactivation plus additional, yet unknown, factors, and open up possibilities for much needed new therapies at the time when the incidence of HPV-positive oropharyngeal carcinoma in the US population is rising."
This work was funded by the National Cancer Institute, Seattle Translational Tumor Research, and the University of Washington Royalty Research Fund.
Fred Hutch/UW Cancer Consortium members Julia Sidorova (UW) and Eduardo Mendez (FH/UW) contributed to this work.
Diab A, Kao M, Kehrli K, Kim HY, Sidorova J, Méndez E. 2019. Multiple defects sensitize p53-deficient head and neck cancer cells to the WEE1 kinase inhibition. Mol Cancer Res. DOI: 10.1158/1541-7786.MCR-18-0860.