Abstract 3913: PDK1 drives susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition
Authors: Sambandam Vaishnavi , Shen Li , Peng Shaohua , Mazumdar Tuhina , Pickering Curtis , Myers Jeffrey , Wang Jing , Frederick Mitchell , Johnson Faye 
Published: August 2019

Abstract
Head and neck squamous cell carcinoma (HNSCC) is primarily driven by the loss of tumor suppressor genes. Although approaches to target driver oncogenes have been clinically successful, targeting tumor suppressors has been challenging. To address this translational gap, we previously demonstrated that HNSCC cell lines with loss of function (LOF) NOTCH1 mutations (NOTCH1MUT n=14) were more sensitive to 6 PI3K pathway inhibitors (Omipalisib, Alpelisib, Bimiralisib, Dactolisib, Copanlisib, Apitolisib) than NOTCH1WT cells (n=45). In contrast to PIK3CAMUT cell lines, NOTCH1MUT lines underwent significant apoptosis after PI3K/mTOR pathway inhibition. NOTCH1MUT lines also showed significantly reduced clonogenic growth and significant tumor growth inhibition in both oral orthotopic and subcutaneous xenograft models. We employed CRISPR-Cas9 gene editing technology to knock out NOTCH1 gene in two NOTCH1WT lines, PJ34 and UMSCC49. After Omipalisib treatment, NOTCH1-KO lines showed decreased cell viability compared to parental lines. The NOTCH1 knock out lines showed significantly increased apoptosis after Omipalisib treatment compared to parental lines (PJ34 KO: 1.62-fld, P&lt;0.003; UM49 KO: 1.37-fld, P=0.002). Both NOTCH1 KO lines also showed increased cleaved PARP and cleaved caspase 3 by western blot analysis. With NOTCH1 knock out lines, treatment significantly decreased number of colonies (PJ34 KO: 1.35-fld, P&lt;0.005; UM49 KO: 1.62-fld, P&lt;0.0001). As no canonical pathways account for the underlying mechanism of sensitivity, we measured 301 proteins by reverse phase protein array (RPPA) in NOTCH1MUT and NOTCH1WT lines after Omipalisib treatment. Several proteins were differentially regulated in NOTCH1MUT cells compared with NOTCH1WT lines including PDK1, FoxM1 and p-ERK. We then investigated PDK1 because it is activated by PI3K, and can regulate FOXM1 and ERK which were also inhibited more robustly in NOTCH1MUT HNSCC. To test the role of PDK1 in PI3K/mTOR inhibition, we over expressed PDK1 in NOTCH1MUT cells and it successfully led to decreased apoptosis after Omipalisib treatment as compared to the parental lines. The combination of AKT and PDK1 inhibition led to decreased cell viability in all four NOTCH1WT lines tested as compared to single agents. The combination also led to significantly increased apoptosis in all NOTCH1WT cell lines tested as demonstrated by cleaved PARP and Caspase 3 levels by western blot analysis. Our research is the first to establish a therapeutic vulnerability of NOTCH1MUT HNSCC to any class of drugs and may inform the development of the first biomarker-driven targeted therapy for HNSCC. Also, we may uncover combination therapies and define a heretofore-unknown mechanism of PDK1 regulation. Because NOTCH1 LOF mutations are common in other squamous carcinomas, including skin, esophagus and lung, our findings will likely have implications beyond HNSCC.
Citation Format: Vaishnavi Sambandam, Li Shen, Shaohua Peng, Tuhina Mazumdar, Curtis Pickering, Jeffrey Myers, Jing Wang, Mitchell Frederick, Faye Johnson. PDK1 drives susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3913.