Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. To identify novel biomarkers of response to PI3K/mTOR pathway inhibitors 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 genomic alterations. We identified that NOTCH1mut lines were significantly more sensitive to PI3K/mTOR pathway inhibitors than NOTCHWT lines: GSK2126458 (12/14 NOTCH1Mut lines), BYL719 (6/14), PQR309 (12/14), BKM120 (14/16), BEZ235 (12/16), BAY806942 (13/14) and GDC0980 (5/14 lines). In contrast to PIK3CAmut cell lines that experienced cell cycle arrest, after PI3K/mTOR pathway inhibition, NOTCH1mut lines underwent significant apoptosis in addition to G1/S cell cycle arrest. NOTCH1mut lines also showed reduced clonogenic growth in vitro and tumor growth inhibition in vivo in both oral orthotopic and subcutaneous xenograft mouse models. NOTCH1 knock out (KO) by CRISPR-Cas9 system in a NOTCH1WT line (PJ34) rendered it more sensitive to PI3K/mTOR inhibition.
After PI3K/mTOR inhibition, PJ34-NOTCH1 KO showed significant reduction in clonogenic growth (1.57-fold; P<0.05) and increased apoptosis (4.3-fold; P<0.05) compared to the parental line.
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. Several proteins related to cell cycle were differentially regulated in NOTCH1mutcells compared to wild type lines. Notably, both mRNA and protein levels of Aurora B were significantly decreased in NOTCH1mutcells but not in NOTCHwt cells following PI3K/mTOR inhibition. Aurora B is an important cell cycle regulator and deregulation of Aurora kinases leads to defective chromosomal segregation and mitotic catastrophe in numerous cancers. Aurora kinase inhibitors as single agent are highly effective in a panel of NOTCHwt cell lines as demonstrated by decreased colony formation ability and proliferation as well as G2/M arrest and apoptosis.
Inhibition of Aurora kinases in combination with PI3K inhibitors displayed synergy (Combination Index<1) in 64% of NOTCH1 wild type lines (26/44) and 66% of NOTCH1mutcell lines (8/12) also exhibited increased sensitivity as assessed by Cell-titer Glo assay. Aurora B knock down and over expression studies are underway to validate the finding.
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 present work may uncover potential combination therapies for HNSCC.
Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Shaohua Peng, Tuhina Mazumdar, Ratnakar Singh, Curtis R. Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. PI3K/mTOR pathway inhibition induces Aurora B mediated cell death in NOTCH1 mutant head and neck squamous (HNSCC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2977.