STING is Expressed by Hodgkin and Reed Sternberg (HRS) Cells in a Subset of Classical Hodgkin Lymphoma (cHL) and Correlates with Tumor Microenvironment and Immune Response

Abstract

Introduction: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through activation of the adaptor protein STING. The latter, in turn, 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 expression patterns of STING in Hodgkin and Reed-Sternberg (HRS) cells and the potential role of cGAS-STING pathway in anti-tumor immune responses in classical Hodgkin lymphoma (cHL) remain unknown to date. Methods: In this prospective study, STING expression was immunohistochemically analysed in a cohort of 52 previously untreated patients with cHL and available tissue for flow cytometry (FC) analysis and histology as well as available peripheral blood samples for FC. An arbitrary 10% cutoff was used for positivity in HRS. The in vitro system included 6 cHL cell lines (MDAV, L1236, L428, L540, HDLM2, KMH2). Gene (mRNA level) and protein expression/activation of cGAS-STING 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 or were subjected to silencing of STING gene using transient transfection with specific STING siRNA construct. The gene expression of cGAS-STING-associated IFNs and cytokines, including IFN-β, CXCL10, IFN-γ, STING, and a control gene (GAPDH), was analysed with RT-qPCR. Results: STING was positive in HRS cells of cHL in 22 of 52 (42%) patients. STING positivity in HRS significantly correlated with lower numbers of STING+ T-lymphocytes (p < 0.0001) and CD3+ T-lymphocytes (p = 0.04) but higher numbers of CD20+ B-cells assessed by FC in the tissues. In peripheral blood samples assessed by FC at presentation, STING expression by HRS correlated with higher numbers of CD3+ T-cells (p = 0.005) but lower numbers of NK-cells (p = 0.02). STING expression did not correlate with clinical features at presentation, including age, gender, Ann Arbor stage, B-symptoms, tumor burden, bulky disease, anemia, or other hematologic values. 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 agonists stimulated gene expression of IFN-β and/or CXCL10 at a variable level indicating functional cGAS-STING pathway in HRS. Knocking down STING gene resulted in dramatic increase in CXCL10 gene expression in cHL cells. Conclusions: STING is expressed by HRS in a subset of cHL and significantly correlates with the lymphocytic populations in the tumor microenvironment and peripheral blood. The cGAS-STING pathway is functional in HRS suggesting that STING agonists may have therapeutic implications in patients with cHL. Keywords: Diagnostic and Prognostic Biomarkers, Hodgkin lymphoma, Microenvironment No conflicts of interests pertinent to the abstract.