Background: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through production of the second messenger cGAMP and subsequently activation of the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The potential role of cGAS-STING pathway in anti-tumor immune responses in cHL remains unknown to date.
Methods: STING expression was immunohistochemically analysed in a pilot study group of 32 untreated patients with cHL and available tissue. The in vitro system included 6 cHL cell lines (MDA-V, L1236, L428, L540, HDLM2, KMH2). Gene expression (RNA level) and protein expression and activation (phosphorylation) of cGAS-STING pathway components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot, respectively. The cHL cell lines were treated with a STING agonist and TBK1/IKK inhibitor (Amlexanox) alone or in combination with other agents. Silencing of STING gene was performed using transient transfection with specific STING siRNA. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of IFN-β, CXCL10, IFN-γ, and a control gene (GAPDH) with RT-qPCR.
Results: Using an arbitrary 10% cutoff, STING was positive in the neoplastic Hodgkin & Reed-Sternberg (HRS) cells of cHL in 20 of 32 (63%) patients with a membranous and cytoplasmic pattern. STING expression at the mRNA and protein level was substantially higher in L1236, L428 and HDLM2 compared to other cHL cell lines. Treatment with STING agonist alone stimulated gene expression of IFN-β and/or CXCL10 at a variable level depending on the cell line indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Knocking down STING gene resulted in decreased CXCL10 and type 1 IFN gene expression by cHL cells. Amlexanox treatment also resulted in downregulation of IFN-β or CXCL10 gene expression in vitro.
Conclusion: STING agonists and Amlexanox modulate gene expression of type 1 IFNs in cHL with direct therapeutic implications. A large cohort of cHL patients is currently being analysed for the expression and prognostic significance of STING, and the final results will be available at the time of ISHL 2022.
Ioanna Xagoraris, Konstantina Stathopoulou, Persa Xyderou, Marzia Palma, Elias Drakos, Theodoros P. Vassilakopoulos, Georgios Z. Rassidakis