PDK1 mediates Notch1-mutated head and neck squamous carcinoma vulnerability to therapeutic PI3K/mTOR inhibition

Purpose: Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/ mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function. Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression. Results: PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1^MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1^MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1^WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/ mTOR inhibition in NOTCH1^MUT but not NOTCH1^WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1^MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1^WT HNSCC but had little effect as single agents. Conclusions: Our findings suggest that NOTCH1^MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1^WT HNSCC to PI3K/ mTOR pathway inhibitors.