Abstract 2992: Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC)
Authors: Sambandam Vaishnavi , Shen Li , Tong Pan , Mazumdar Tuhina , Pickering Curtis , Myers Jeffrey N. , Wang Jing , Frederick Mitchell , Johnson Faye M. 
Published: July 2017

Abstract
Background: Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. However, clinical trials of PI3K/mTOR pathway inhibitors had limited success even in those tumors with pathway alterations, including PIK3CA mutations. To target genomic alterations in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and molecular characteristics in order to identify biomarkers of response.
Methods: We systematically analyzed the association between drug sensitivity and genomic alterations in 59 HNSCC lines.
Results: NOTCH1mut lines are significantly sensitive to PI3K/mTOR pathway inhibitors: GSK2126458 (13/16), BYL719 (6/16), PQR309 (13/16), BKM120 (14/16), BEZ235 (12/16), BAY806942 (14/16) and GDC0980 (13/16 lines). In contrast to PIK3CAmut cell lines, all 7 NOTCH1mut lines tested underwent apoptosis (14.3 fld; P&lt;0.005). After PI3K/mTOR inhibition, NOTCH1mut lines showed significantly reduced clonogenic growth in vitro (0.4/ 0.9 fold in HN31/ PCI15B; P&lt;0.05) and significant tumor growth inhibition in vivo using orthotopic oral xenograft mouse models (1.7 and 2 fold in UMSCC22A and HN31; P&lt;0.01). To determine if NOTCH1 mediates resistance, we conditionally expressed cleaved NOTCH1 by Dox-inducible system in a NOTCH1mut line (UMSCC22A). This rescued PI3K/mTOR inhibitor-induced apoptosis (0.5 fold; P&lt;0.05) and reduced colony formation in vitro. 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 3 NOTCH1mut and 3 NOTCH1WT lines after GSK2126458 treatment. Glutaminase and Glutamate Dehydrogenase were differentially expressed in NOTCH1mut lines. Thus, we hypothesized that PI3K/mTOR inhibition in NOTCH1mut lines induced reactive oxygen species (ROS)-mediated apoptosis via metabolic alterations. Consistent with this hypothesis, NOTCH1mut lines exhibited increased ROS production; Metabolic pathway inhibitors targeting Glycolysis, Pentose Phosphate pathway and Glutaminolysis, in combination with GSK2126458 decreased cell viability in NOTCHWT lines.
Conclusion: In contrast to PIK3CAmut cells, NOTCH1mut HNSCC cells underwent apoptosis after PI3K/mTOR pathway inhibition in vitro and decreased tumor size in vivo. The ectopic activation of NOTCH1 rescued NOTCH1mut HNSCC cells from PI3K/mTOR inhibitor-mediated apoptosis. The underlying mechanism may involve differential effects on tumor metabolism and ROS production. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our future work may uncover previously unknown crosstalk between the PI3K/mTOR and NOTCH pathways in SCCs.
Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Tuhina Mazumdar, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2017-2992