Abstract 5929: HuR-targeted therapy promotes the adaptive immune response in medulloblastoma through the cGAS-Sting pathway

Abstract

Abstract HuR is an RNA binding protein overexpressed in multiple human cancers and is involved in tumorigenesis, invasion, and metastasis. Genetic and pharmacologic inhibition of HuR demonstrated antitumor activity establishing HuR as a molecular target for cancer therapy. However, little is known about HuR’s role in medulloblastoma (MB). In the present study, we investigated the effect of CMLD2, a small molecule inhibitor of HuR, on MB cell growth and on the adaptive immune response by assessing the cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway. The cytoplasmic double-stranded DNA (dsDNA) sensing cGAS-STING signaling pathway plays pivotal role in host defense against cancers. Activation of the cGAS-STING signaling cascade induces the expression of pro-inflammatory cytokines and type I interferons promoting anti-tumor immunity. Human MB Daoy cells were treated with DMSO (control) or CMLD2 (20uM and 30uM). At 48 and 72 h after treatment, cells were collected and cell viability was determined using Trypan blue assay. A time and dose-dependent cytotoxicity was observed with 22.5% and 32.5% reduction in cell number with 20uM treatment (p<0.01) and 38.3% and 50.2% reduction with 30uM treatment (p<0.001) at 48 h and 72 h respectively compared to DMSO-treated control. Western blot analysis showed CMLD2 treatment inhibited HuR and markedly increased the expression of cGAS-STING pathway proteins (p-STING Ser366, p-TBK1 Ser172, p-IRF3Ser396 and pNF-KBSer536) at the two drug concentrations tested albeit greater increase in cGAS-STING protein expression was observed with 20uM treatment. Analysis for chemokines (CXCL10 and CCL5) and cytokine (IL-6) associated with the cGAS-STING pathway in cell culture supernatant collected from 20uM CMLD2 treatment showed significant increase in CXCL10 (1.96 ± 0.12 and 2.12 ± 0.06 fold increase at 48 h and 72 h; p<0.01) and CCL5 (1.21 ± 0.09 and 1.86 ± 0.05 fold increase at 48 h and 72 h; p=ns at 48 h; p<0.01 at 72 h) over DMSO-treated control. IL-6 expression significantly increased at 72h (1.98± 0.24 fold, p<0.001) but not at 48h after 20uM CMLD2 treatment compared to DMSO treated control. Our study results demonstrate CMLD2 mediated HuR targeting induces MB cell cytotoxicity and concurrently activates the cGAS-STING pathway. Testing of CMLD2 treatment in additional MB cell models and studying the impact of the cGAS-STING activation on immune cells (T cells, NK cells and macrophages) will aid in developing novel HuR-targeted treatment strategies in MB. Funding: The study was supported in part by a grant received from the National Cancer Institute of the National Institutes of Health (R01 CA 282735-01) and funds from the Jim and Christy Everest Endowed Chair in Cancer Developmental Therapeutics. Citation Format: Meghna Mehta, Rajeswari Raguraman, Anupama Munshi, Rajagopal Ramesh. HuR-targeted therapy promotes the adaptive immune response in medulloblastoma through the cGAS-Sting pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5929.