Abstract 2847: Concurrent inactivation of PI3K and PLK1 is synergistic and overcomes acquired resistance to PI3K inhibitors in NOTCH1MUT HNSCC
Authors: Shah Pooja A. , Mazumdar Tuhina , Powell Reid T. , Shen Li , Wang Jing , Stephen Clifford C. , Frederick Mitchell J. , Johnson Faye M. 
Published: April 2023

Targeted therapies are limited for head and neck squamous cell carcinoma (HNSCC), as it is driven by mutations in tumor suppressors, including NOTCH1. We previously identified loss of function NOTCH1 mutations in HNSCC to be sensitive to phosphoinositide-3 kinase (PI3K) inhibitors through sustained Aurora kinase B levels. However, therapy resistance and modest responses are the leading causes of failure for targeted therapies. To address this pressing clinical need, we sought to identify drugs that would enhance the efficacy of PI3K inhibitors. We tested 5768 drugs (0-1µM) with diverse targets in NOTCH1 mutant (NOTCH1MUT - HN31, UMSCC22A, PCI-15B) HNSCC cell lines and copanlisib acquired resistant (CAR) HN31 clones. We determined drug efficacy using two metrics-area over the curve lethal dose (AOC_LD&gt;0, cytotoxic) and area under the growth curve (AUC_GRI&lt;0.9, cytostatic). These metrics are more robust than IC50 values as they use the normalized growth rate inhibition curve and avoid the confounding effect of the rate of cell division. Of 306 drug classes, 100 were effective with at least one drug being cytotoxic or cytostatic in at least one cell line. PLK inhibitors were the most effective class of drugs against NOTCH1MUT HNSCC cell lines (87% effective - 12 cytotoxic, 2 cytostatic, 2 ineffective). We further tested the PLK1-specific inhibitor onvansertib (0-100nM) combined with pan-PI3K inhibitor copanlisib (0-200nM) or dual inhibitor bimiralisib (0-1μM). We observed robust decreases in cell numbers at very low drug concentrations (50nM) with the combinations. We validated these results in vitro in NOTCH1MUT, NOTCH1WT, CAR NOTCH1MUT HNSCC models, and HEK293 by using independent approaches to test for apoptosis. A significant increase in cleaved PARP and cleaved caspase 3, and Annexin V/PI staining (&gt;2x), was evident when PI3K and PLK1 were concurrently inactivated as compared to single agent treatment in HNSCC models. Furthermore, HEK293 cells were unaffected at these doses. We then investigated the role of PLK1 following PI3K inhibition in HNSCC models and found PLK1 protein levels to be downregulated. As PLK1 is downstream of Aurora kinases, we further determined the phospho-PLK1 (p-PLK1) levels in HNSCC models. Interestingly, p-PLK1 levels remained unaltered in NOTCH1WT and CAR NOTCH1MUT cells despite total protein level depletion. However, p-PLK1 levels drastically decreased in NOTCH1MUT HNSCC cells. This finding could explain the importance of PLK1 inactivation in addition to PI3K inhibition as a requirement for increased cell death in HNSCC models. We will further validate this combination in vivo to determine the effect of combined PI3K and PLK1 inhibition on tumor growth and survival. These novel findings may lead to the development of a better therapeutic approach for NOTCH1MUT HNSCC and for patients who develop acquired resistance to targeted therapies.

Citation Format: Pooja A. Shah, Tuhina Mazumdar, Reid T. Powell, Li Shen, Jing Wang, Clifford C. Stephen, Mitchell J. Frederick, Faye M. Johnson. Concurrent inactivation of PI3K and PLK1 is synergistic and overcomes acquired resistance to PI3K inhibitors in NOTCH1MUT HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2847.