Abstract 393: NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma
Authors: Johnson Faye M. , Sambandam Vaishnavi , Shen Li , Zhang Ming , Saigal Rishi , Tong Pan , Mazumdar Tuhina , Byers Lauren A. , Pickering Curtis , Myers Jeffrey N. , Wang Jing , Frederick Mitchell 
Published: July 2016

Recently published whole exome sequencing studies in head and neck squamous cell carcinoma (HNSCC) tumors revealed that few had therapeutically targetable alterations using current strategies. This finding defines translational gap between genomics and HNSCC treatment. One potential targetable alteration is PIK3CA mutations. However, clinical trials testing PI3K/mTOR pathway inhibitors have had limited success and these inhibitors only lead to cell cycle arrest in PIK3CA mutant HNSCC cell lines. Thus, there is a critical need to identify therapeutic vulnerabilities for common mutation groups, including tumor suppressors, in HNSCC. One of these molecular subgroups is NOTCH1 which is the second most frequently mutated gene in HNSCC, with a 10-15% prevalence of inactivating mutations. Although there are several studies underscoring the importance of NOTCH1 as a tumor suppressor in HNSCC, none has identified a therapy that targets NOTCH1 mutant (mut) HNSCC. Our objective was to identify predictive biomarkers of sensitivity to PI3K/mTOR inhibitors by integrating drug and multiple-omics data.

Cell viability with six PI3K/mTOR inhibitors in 68 HNSCC lines was measured by the CellTiter Glo assay. The peak plasma concentration of each drug was used as the cut-off to determine sensitivity. We observed a striking correlation between NOTCH1mut and sensitivity to PI3K/mTOR pathway inhibitors. When fisher's exact test was performed, NOTCH1mut lines were more sensitive to GSK2126458 (P&lt;0.027), BYL719 (P&lt;0.004) and PQR309 (P&lt;0.014) than NOTCH1 wild type cell lines. NOTCH1 was also identified as an upstream regulator in sensitive cell lines by Ingenuity® Pathway Analysis. Basal NOTCH1 protein expression was higher in HNSCC lines resistant to PI3K/mTOR inhibition using unsupervised hierarchical clustering of Reverse Phase Protein Array data.

NOTCH1mut lines underwent more apoptosis after GSK2126458 treatment compared to NOTCH1wt lines (PCI15B- 48.1 fold; P&lt;0.05, HN31- 46.9 fold; P&lt;0.05). There was also increased accumulation of cells in G1 after GSK2126458 treatment in NOTCH1mut lines (PCI15B-1.3 fold, P&lt;0.05; HN31- 1.4 fold, P&lt;0.05). To check if inhibition of NOTCH1 pathway inhibition sensitizes NOTCH1wt lines to PI3K/mTOR inhibition, resistant NOTCH1wt lines were treated with Gamma secretase inhibitors and GSK2126458. The combination led to significantly decreased cell viability (DAPT- 1.5 fold and YO010227- 1.7 fold). The combination studies will be further expanded to 38 NOTCH1wt lines. On-going studies include assessment of drug sensitivity in vivo, mechanistic studies and the effect of genetic manipulation of NOTCH1 signaling on sensitivity to PI3K/mTOR inhibitors.

Our data suggests that loss of active NOTCH1 signaling confers sensitivity to PI3K/mTOR inhibition. If the combination of NOTCH1 and PI3K/mTOR inhibition leads to apoptosis, this combination could be translated into the clinic.

Citation Format: Faye M. Johnson, Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Pan Tong, Tuhina Mazumdar, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick. NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393.