Abstract 302: Loss of head and neck squamous cell carcinoma MK2 increases STING activation through increased micronuclei formation
Authors: Spiess Colby , Millington Grace , Smith Hannah , Okwuone Dakota D. , Berggren Kiersten , Morgan Deri , Lominska Chris , Thomas Sufi M. , Wang Jingxin , Markiewicz Mary , Gan Gregory N. 
Published: April 2023

Our previous work has shown that the phosphorylation of MK2, a downstream kinase of the p38 pathway, is a prognostic feature of head and neck cancer (HNC) where patients with higher MK2 phosphorylation had lower overall survival. Studies with a MK2 inhibitor demonstrate control of HNC growth in a mouse model that was synergistic with radiotherapy (RT) in inhibiting tumor growth. The mechanism of action that MK2 inhibition combined with RT has on modulating HNC growth is unknown. A better understanding of this mechanism might reveal how to effectively deploy MK2 inhibitors in HNC treatment. Here we describe our studies in which we suppressed MK2 expression in HNC cell lines and found increased genomic instability in response to radiotherapy.

Tu167 and Cal27 HNC cells were transfected with a stable shRNA construct that suppressed ~90% of the MK2 expression compared to a control shRNA construct (SCR) as determined by immunoblot. Loss of MK2 did not significantly affect cell growth nor did it affect cells from forming colonies. However, exposing the cells to 10 Gy radiation caused the formation of micronuclei that was increased in the shRNA cells compared to SCR at 48 hours (Cal 27 SCR + 10 Gy = 0.5 ± 0.3 Micronuclei/cell v Cal27 shRNA + 10 Gy = 1.6 ± 0.1. Micronuclei/cell P&lt;0.01; Tu167 SCR + 10 Gy = 1.1 ± 0.2 Micronuclei/cell; Tu167 shRNA + 10 Gy = 2.3 ± .3; Micronuclei/cell p&lt;0.001). Immunocytochemical techniques were used to image cells at 60x magnification. In conjunction with increased micronuclei formation, we saw an increase expression in both the DNA sensor cGAS, and phosphorylation of STING in the shRNA cells compared to SCR. The increase in micronuclei caused by radiation was largely inhibited by cytochalasin B which also inhibited the increase in STING phosphorylation. There was no difference in the staining of γH2AX after 2 hours following RT but after 48 hours the γH2AX staining was concentrated in the micronuclei which were more numerous in the shRNA cells.

These data provide evidence that MK2 plays a role in controlling the DNA damage response, and we show for the first time that loss of MK2 increases the number of micronuclei formed causing an enhanced activation of the cGAS-STING pathway, an innate immune DNA sensing pathway. This suggests that inhibition of MK2 may enhance RT effectiveness by activating STING and facilitating an anti-tumor immune response.

Citation Format: Colby Spiess, Grace Millington, Hannah Smith, Dakota D. Okwuone, Kiersten Berggren, Deri Morgan, Chris Lominska, Sufi M. Thomas, Jingxin Wang, Mary Markiewicz, Gregory N. Gan. Loss of head and neck squamous cell carcinoma MK2 increases STING activation through increased micronuclei formation [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 302.