In Vivo Crispr Screening Identifies NF1/RASA1/TP53 CO-Mutations and Downstream MEK Signaling as a Common Key Mechanism of Sinonasal Tumorigenesis

Introduction: Sinonasal tumor histopathologic diversity is amongst the highest in the human body. This heterogeneity is in part due to a distinctive tissue composition including a unique olfactory neuroepithelium, respiratory mucosa, minor salivary glands, and others which directly interface with the external airborne environment. TP53 is the most frequently mutated gene amongst many sinonasal tumor types, but the identification of common molecular mechanisms governing sinonasal oncogenesis is lacking. In vivo screening approaches to identify genes driving sinonasal tumorigenesis have not been previously reported. We hypothesized that in vivo CRISPR screening would elucidate mechanisms of tumorigenesis across sinonasal cancer types and identify potential therapeutic targets.

Methods: We generated a pooled single-guide RNA (sgRNA) library targeting 167 genes selected from genes implicated in sinonasal and head and neck cancer tumorigenesis as well as prior in vivo CRISPR screening approaches in other organs. The sgRNA library was packaged in an adeno-associated virus (AAV) vector and instilled in the sinonasal cavity of Cas9 transgenic mice. Murine sinonasal tumor tissue was collected and tumor histology determined in 19 tumors for which sufficient tissue was available by immunohistochemical analysis. A custom capture library was applied followed by next-generation sequencing to identify inciting sgRNA for each tumor.

Results: Tumors were observed by micro-CT approximately 3-5 months after nasal instillation of the AAV sgRNA library ([Fig. 1]). A variety of sinonasal tumor types including the major sinonasal subtypes were identified namely sinonasal squamous cell carcinoma (SNSCC), adenocarcinoma, poorly differentiated sinonasal tumors similar to SNUC, and sinonasal neuroendocrine tumors characteristic of olfactory neuroblastoma (ONB) ([Fig. 2]). Surprisingly, rather than unique, distinct sgRNA mutational profiles across sinonasal tumor subtypes, common recurrent mutations were identified by next-generation sequencing in NF1 (78%), RASA1 (74%), and TP53 (68%) across these murine tumors. To support the in vivo CRISPR screen results through a second approach, we tested whether instillation of viral Cre recombinase vectors could incite sinonasal tumors in NF1/TP53 ^flox/flox mice. Sinonasal tumor development was observed approximately 12 months after AAV-Cre nasal instillation. NF1 and RASA1 have known Ras-GAP activity, thus loss of these genes may lead to increased Ras activity and downstream MEK signaling. MEK small molecule inhibitors including trametinib, mirdametinib, and selumetinib significantly reduced tumor cell proliferation in vitro in murine cell lines developed from these tumors (p < 0.05). MEK inhibitors significantly inhibited human tumor cell proliferation in a SNSCC cell line as well as newly developed human organoid models of sinonasal adenocarcinoma and ONB in vitro (p < 0.05).

Conclusion: Here, we performed the first sinonasal in vivo CRISPR screen in mice and common recurrent NF1/RASA1/TP53 co-mutations were observed across the major sinonasal tumor subtypes. We developed many of the first mouse sinonasal tumor cell lines and together with human cell line and organoid models elucidated the key function of downstream MEK signaling across sinonasal tumorigenesis. Collectively these results suggest that loss of NF1 and RASA1-mediated Ras-GAP activity leads to Ras activation and downstream MEK signaling which is a potential common target throughout major sinonasal tumor subtypes.