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Several viruses are known to be associated with the development of certain cancers, including human papilloma virus (HPV), an established causative agent for a range of anogenital and head and neck cancers. However, the causality has been based on the presence of the virus, or its genetic material, in the sampled tumors. We have long wondered if viruses cause cancer via a “hit and run” mechanism such that they are no longer present in the resulting tumors. Here, we hypothesize that the absence of viral genes from the tumor does not necessarily exclude the viral aetiology. To test this, we used an HPV‐driven oropharyngeal cancer (OPC) tumor model and CRISPR to delete the viral oncogene, E7. Indeed, the genetic removal of HPV E7 oncogene eliminates tumors in vivo. Remarkably, E7 deleted tumors recurred over time and develop new mutations not previously seen in HPV+ OPC tumors. Importantly, a number of these new mutations are found to be already present in HPV− OPC tumors.

It has long been wondered if viruses cause cancer via a “hit-and-run” mechanism. We examined this in HPV-driven oropharyngeal cancer (OPC) using CRISPR to delete the viral oncogene, E7. Tumours regressed yet regrew over 3 months, despite the lack of E7, and had formed new mutations commonly found in HPV- OPC patient tumours. This is the first biological proof that HPV- OPCs can be formed via a hit-and-run HPV infection.

The major HPV oncogenes E6 and E7 are known for its notoriety in driving the carcinogenic process in human papilloma virus (HPV) driven cancers. It is well-established that the removal of E7 dampens HPV cancer cell growth and proliferation. This has made E7 one of the most attractive targets for HPV cancers. Seminal work from our laboratory employed in vivo CRISPR editing treatment to delete E7, resulting in the effective elimination of HPV positive (HPV+) cervical cancer tumours in vivo. We have also successfully delayed HPV+ tumour growth in vivo with aurora kinase (AURK) inhibitors, an effect which is strongly sensitized by the presence of E7 expression. Unlike our observations in cervical cancer cells, in vitro targeting of E6/E7 have only resulted in partial killing of HPV+ oropharyngeal cancer (OPC) cells. The effect of E7 removal on HPV+ OPC tumours in vivo have not been explored. In this study we investigated a staggered combination of aurora kinase inhibition, using alisertib, followed by CRISPR editing of E7, to determine if this would lead to better HPV+ OPC cell killing in vitro and in vivo. Remarkably, genetic deletion of E7 alone was sufficient to effectively eliminate established HPV+ OPC tumours in vivo suggesting that E7 is essential in the maintenance of HPV+ OPC cancers.