Genome-wide CRISPR screening reveals a PKA-driven resistance mechanism to metformin for oral cancer prevention that can be exploited by combination with NSAIDs

Head and neck squamous cell carcinoma (HNSCC) is among the ten most common cancers worldwide and is associated with high morbidity and poor survival. Diminished HNSCC outcomes are often related to delayed diagnosis and treatment of occult progression of premalignant lesions, underscoring the need for effective and low risk chemoprevention strategies. In this regard, metformin has shown promising clinical activity for HNSCC prevention. Here, we performed a genome-wide CRISPR/Cas9 screen of metformin-treated HNSCC cells and identified activation of PKA signaling as the top resistance pathway. Overall design: LentiCas9-Blast plasmid was used to generate Cas9-expressing Cal33 HNSCC cells. The human Brunello whole-genome CRISPR pooled library DNA was transformed by electroporation using electrocompetent cells, and the amplified library DNA was then extracted and purified using the QIAGEN Midi Prep Kit. The high-titer VSV- G pseudotyped lentiviral vector was generated and purified by VectorBuilder. The screen was performed by culturing Cal33-Cas9 cells divided into two treatment arms: vehicle or 1mmol/L metformin treatment. The cells were cultured until the population doubling level reached 18. The entirety of isolated genomic DNA extracted from cells was used for subsequent amplification through PCR to ensure capturing the full representation of the libraries. PCR products were sequenced on a HiSeq4000 instrument.