Abstract B19: Susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition via PDK1

Abstract

Abstract Genomic alterations in the PI3K/mTOR pathway occur in 54% of head and neck squamous cell carcinoma (HNSCC) patients. To identify novel biomarkers of response to PI3K/mTOR pathway inhibitors in HNSCC, we tested the efficacy of seven PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and genomic alterations. We identified that NOTCH1MUT lines were significantly sensitive to the following PI3K/mTOR pathway inhibitors: GSK2126458 (12/14 lines), BYL719 (6/14), PQR309 (12/14), BKM120 (14/16), BEZ235 (12/16), BAY806942 (13/14), and GDC0980 (5/14). In contrast to PIK3CAMUT cell lines, NOTCH1MUT lines underwent significant apoptosis in addition to G1/S cell cycle arrest after PI3K/mTOR pathway inhibition. NOTCH1MUT lines also showed significantly reduced clonogenic growth in vitro and significant tumor growth inhibition in vivo in both oral orthotopic and subcutaneous xenograft mouse models. We employed CRISPR-Cas9 gene editing technology to knock out NOTCH1 gene in two NOTCH1WT lines, PJ34 and UMSCC49. After GSK2126458 treatment, NOTCH1-KO lines showed decreased cell viability compared to parental lines. Both NOTCH1 KO lines also showed increased cleaved PARP and cleaved caspase 3 by Western blot analysis after PI3K/mTOR inhibition. With GSK2126458 and BEZ235 treatment, both PJ34 and UMSCC49 NOTCH1 knock out lines showed significantly decreased number of colonies compared to the parental lines. As no canonical pathways account for the underlying mechanism of sensitivity, we measured the level of 301 proteins by reverse phase protein array (RPPA) in three NOTCH1MUT and three NOTCH1WT lines after GSK2126458 treatment. Several proteins were differentially regulated in NOTCH1MUT cells compared with wild-type lines including PDK1, FoxM1 and phospho-ERK. We then investigated PDK1 because it is activated by PI3K and can regulate FOXM1, ERK, and p70 ribosomal protein S6 kinase (p70S6K), which were also inhibited more robustly in NOTCH1MUT HNSCC. To test the role of PDK1 in PI3K/mTOR inhibition, we overexpressed PDK1 in NOTCH1MUT cells and the presence of PDK1-GFP was confirmed by Western blot analysis. PDK1 overexpression in NOTCH1MUT cells successfully led to decreased apoptosis after GSK2126458 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 cleaved Caspase 3 levels by Western blot analysis. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and in squamous cell carcinomas of the lung, esophagus, and other sites, may serve as a biomarker for response. Our present work may uncover important combination therapies for HNSCC. Citation Format: Vaishnavi Sambandam, Mitchell J. Frederick, Li Shen, Pan Tong, Xiayu Rao, Shaohua Peng, Tuhina Mazumdar, Quili Li, Curtis R. Pickering, Jeffery N. Myers, Jing Wang, Faye M. Johnson. Susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition via PDK1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B19.