Abstract 369: Identification of pathways that enhance cell death in NOTCH1-mutant HNSCC
Authors: Shah Pooja A. , Mazumdar Tuhina , Powell Reid T. , Shen Li , Wang Jing , Stephen Clifford C. , Frederick Mitchell J. , Johnson Faye M. 
Published: July 2021

Head and neck squamous carcinoma (HNSCC) is dominantly driven by mutations in tumor suppressor genes making it challenging to devise biomarker-based targeted therapy. In order to meet this pressing clinical need, our group recently demonstrated that HNSCCs with loss of function NOTCH1 mutations were more sensitive to phosphoinositide-3 kinase (PI3K) pathway inhibition than their wild-type counterparts.Modest clinical responses and acquired resistance are leading causes of failure for molecular targeted therapies that are otherwise well tolerated. In order to address this challenge and identify drugs that could work in combination with PI3K inhibitors against NOTCH1-mutant HNSCC, we performed a high throughput screen of 5669 drugs (0-1µM) with diverse targets. We used a laser-based confocal imaging platform to determine actual cell numbers using DAPI staining before and after drug treatment of NOTCH1-mutant HNSCC cells (HN31, UMSCC22A). We used 2 metrics of efficacy to identify potential candidates to combine with PI3K inhibition: an arbitrary cut off value of ≤ 0.9 for the area under the curve, growth rate index and area over the curve lethal dose (AOC_LD). We calculate both metrics using the normalized growth rate inhibition curve in order to avoid the confounding effect of the rate of cell division. The AOC_LD is &gt; 0 only when there are fewer cells after treatment than before (i.e., there was cell death). Of the resulting 340 candidates, we excluded chemotherapy, PI3K inhibitors, and non-specific drugs. When several candidates targeted the same pathway, we chose 2-3 of the most specific drugs to use in the combination screen. We combined the resulting 74 drugs with PI3K inhibitors, bimiralisib (0-1µM) or copanlisib (FDA approved, 0-100nM), for 72 h. Synergistic effects from these combinations were assessed using Bliss, HAS, Zip, and Loewe models. Trametinib (MEK inhibitor) and copanlisib were synergistic with the combination leading to 0.90 fraction of cells affected at concentrations of 30nM and 100nM respectively. These concentrations are target-specific and clinically achievable. Likewise, low concentrations of inhibitors of EGFR (10nM afatinib, 50nM AZ5104), HER2 (25nM sapitinib, 10nM poziotinib), and PLK1 (50nM BI2536, 50nM volasertib) were both effective and additive to synergistic with PI3K inhibitors. These combinations will further be validated in vitro using an independent approach to test for apoptosis (cleaved PARP and caspase-3 induction and TUNEL assay) and cell counts in multiple NOTCH1 mutant HNSCC cell lines. We will test the most promising combination in vivo. We have identified four drug classes that not only maximize the killing of NOTCH1-mutant HNSCC, but may also prevent resistance at clinically relevant concentrations. The identified pathways may give us insight into mechanisms of resistance. If validated, these combinations may lead to the first biomarker-specific, targeted therapy for HNSCC.

Citation Format: Pooja A. Shah, Tuhina Mazumdar, Reid T. Powell, Li Shen, Jing Wang, Clifford C. Stephen, Mitchell J. Frederick, Faye M. Johnson. Identification of pathways that enhance cell death in NOTCH1-mutant HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 369.