Infection with high-risk strains of human papillomavirus (HPV) is one of the major risk factors for developing head and neck squamous cell carcinoma (HNSCC), a major cause of cancer deaths worldwide. In the United States, the incidence of HPV-positive HNSCC is increasing, fueling the efforts to identify therapeutic targets in this type of cancer. New research from the Clurman lab in the Human Biology Division, led by postdoctoral fellow Dr. Ahmed Diab, shows that a critical vulnerability in HPV-positive HNSCC tumors renders them sensitive to AZD1775, a WEE1 inhibitor (WEE1i), currently in clinical trials for a variety of cancers. WEE1 is a kinase that inhibits cell cycle regulators CDK1 and CDK2; inhibition of WEE1 forces cells through cell cycle checkpoints resulting in massive DNA damage and, eventually, cell death. Diab explained the initial observation that lay the ground for his discovery, “the work started by an observation made by the late Eddie Méndez and his team in Phase 1 clinical trial for AZD1775, a small molecule WEE1 inhibitor. They noticed that patients with HPV+ tumors responded remarkably to the drug. So contrary to the bench-to-bedside paradigm, we took back to the bench from bedside, and attempted to understand how HPV predisposes cancer cells to WEE1i-induced DNA damage.”
To address this question, the investigators first determined the effect of WEEi treatment in HPV-negative and HPV-positive HNSCC cell lines by following the expression of proliferation marker pHH3 and DNA damage marker gH2AX. In HPV-positive HNSCC cell lines, WEE1i treatment increased the fraction pHH3+/gH2AX+ cells, demonstrating that HPV-positive cells enter mitosis before completing DNA replication faster than HPV-negative cells, an event referred to as “premature mitosis”. These findings suggested that HPV-mediated cell cycle dysregulation accelerates the premature mitosis induced by WEE1i treatment. Typically, HPV oncogenes E6 and E7 mediate cell-cycle dysregulation by inactivating tumor suppressor proteins p53 and pRb, respectively. To determine the role of E6/E7 in WEE1i-induced premature mitosis in HPV-positive cells, the investigators introduced the HPV oncogenes E6 and/or E7 to HPV-negative HNSCC cell lines and found that WEEi-treated E6 and E6/E7 cell lines but not E7 or control cell lines entered mitosis prematurely.
In experiments to further characterize the timing of mitosis entry, the investigators quantified pCHK1-positive cells, a marker for cells that have entered mitosis with unresolved replication stress, and pHH3-positive cells by flow cytometry and immunoblotting. They compared the abundance of these markers to negative controls, including a cell line with a non-HPV mediated p53 knockdown (p53KD), and reported that pCHK1+/pHH3+ cells were found in WEE1i-treated E6 and E6/E7 cells, but not in E7 or p53KD. Taken together, these results demonstrated that HPV oncogenes, particularly E6, confer sensitivity of HNSCC cells to WEE1i-induced premature mitosis. To determine how E6/E7 affects CDK dynamics in real time after WEE1i treatment, the investigators used live-cell imaging that followed CDK1/2 biosensors. In controls cells, WEE1i treatment led to increase CDK activity for up to 8 hours, as expected, followed by a period of prolonged CDK suppression. In contrast, WEE1i treatment in E6/E7 cells resulted in sustained CDK activity throughout treatment. Using CDK-associated kinase assays, the investigators demonstrated that CDK suppression in controls cells is mediated by a p53-dependent response, which is abrogated by E6-mediated p53 degradation in E6/E7 cells. Thus, HPV oncogenes E6/E7 bypass the regulation mediated by p53 to suppress CDK hyperactivity, making HPV-positive cells more sensitive to WEE1i treatment.
Finally, using an 84-gene array in WEE1i-treated E6/E7 and control oral keratinocytes, the investigators identified 25 genes highly expressed in E6/E7 WEE1i-treated cells. Motif analyses in this subset of genes revealed binding sites for FOXM1, a master transcriptional regulator of mitotic genes, and a target for CDK1 phosphorylation. Acting on a circuit, FOXM1 phosphorylation in turn increases CDK1 activity by recruiting FOXM1 to promoters of its target genes. The investigators then hypothesized that CDK1-driven FOXM1 hyperactivity contributes to WEE1i-induced premature mitosis in HPV-positive HNSCC cells. Consistent with this hypothesis, the investigators found high levels of FOXM1 in serial biopsies obtained from patients with HPV-positive HNSCC relative to HPV-negative HNSCC. Gene set enrichment analyses in these samples demonstrated that a signature composed of FOXM1-target genes was highly enriched in HPV-positive tumors compared with HPV-negative tumors. In summary, this work elucidates an E6/E7-driven mechanism acting on a FOXM1-CDK1 circuit, which underlies the WEE1i hypersensitivity in HPV-positive HNSCC.
Diab A, Gem H, Swanger J, Kim HY, Smith K, Zou G, Raju S, Kao M, Fitzgibbon M, Loeb KR, Rodriguez CP, Méndez E, Galloway DA, Sidorova JM, & Clurman BE. (2020). FOXM1 drives HPV+ HNSCC sensitivity to WEE1 inhibition. Proceedings of the National Academy of Sciences of the United States of America, 202013921. Advance online publication. https://doi.org/10.1073/pnas.2013921117
This work was supported by grants from the NIH/National Cancer Institute, the Seattle Translational Tumor Research programmatic investment grant, and the Shared Resources of the Fred Hutch/University of Washington Cancer Consortium Grant.
Fred Hutch/UW Cancer Consortium members Denise Galloway, Julia Sirodova, Cristina Rodriguez, Keith Loeb, the late Eddie Mendez and Bruce Clurman contributed to this research.