Background: Current treatment of head and neck squamous cell carcinoma (HNSCC) consists of multi-modality therapy with surgery, radiation, and chemotherapy. Despite significant improvements in these modalities, there are limited treatment choices for recurrent/metastatic and platinum refractory HNSCCs, and overall survival remains very poor resulting in high morbidity. Therefore, novel therapeutic approaches are badly needed. Wee 1 is a tyrosine kinase that phosphorylates CDC2 at Tyr 15 and as such plays a pivotal role in the G2 DNA damage checkpoint. Recent clinical data show remarkable anti-tumor activity of the Wee1 kinase inhibitor AZD1775 in many cancer cells including HNSCC. This inhibitor acts to abrogate the G2 cell cycle checkpoint. HNSCC, especially those with TP53 mutations are reliant on repairing DNA damage during arrest at this cell cycle checkpoint. Tumor cells that are unable to rely on the G1 checkpoint are more sensitive to G2 checkpoint abrogation. High-level expression of Rad51, a key factor in homologous recombination, has been observed in a variety of human malignancies including HNSCC. During replication stress, Rad51 localizes with RPA32 to protect nascent DNA at stalled forks and mediates replication restart, thus allowing tumor cells to repair DNA damage. We and others have shown that, in addition to its effects on the cell cycle, inhibition of Wee1 impairs Rad51- mediated homologous recombination (HR) repair through forced activation of CDK1, leading to senescence and apoptosis in HNSCC cells. As with any targeted therapy, drug benefit could be achieved with novel therapeutic combinations. Therefore, we hypothesize that simultaneous targeting of Wee1 and Rad51 will result in greater cell killing in preclinical models of HNSCC.
Materials and Methods: Clonogenic survival assays, western blotting, and an orthotopic mouse model of oral cancer were used to examine the in vitro and in vivo sensitivity of mutant TP53 and HPV+ HNSCC cell lines to Rad51 inhibitor, B02, AZD1775 alone or in combination. Cell cycle analysis, DNA damage (γH2AX), 3D spheroid model and apoptosis assays (TUNEL staining and PI/Annexin V) were performed to dissect molecular mechanisms.
Results: Combination of B02 and AZD1775 resulted in synergistic effect in these cell lines. Mechanistically, these drugs interact synergistically to induce DNA damage, replication stress, and impaired Rad51-mediated HR through activation of CDK1 and decreased CHK1 phosphorylation in a time-dependent manner, culminating in mitosis associated with apoptotic cell death. Additionally, combination of B02 and AZD1775 is associated with an accumulation of cells in S-phase followed by an increase in Sub G1 fraction, indicative of replication stress induction and apoptosis respectively. Significant decrease in tumor growth was only found in vivo in HPV+ HNSCC tumor bearing mice following treatment with B02 and AZD1775 compared to controls and either drug alone, consistent with the in vitro findings.
Conclusions: Selective combined targeting of replication stress and Rad51 HR repair may represent an effective therapeutic approach for killing head and neck cancer.