The NPM-ALK Oncogenic Kinase Suppresses CGAS-Sting-Associated Anti-Tumor Immune Responses through STAT3 Activation in Anaplastic Large Cell Lymphoma (ALCL)

Background: ALK+ anaplastic large cell lymphoma (ALCL) is a distinct T-cell non-Hodgkin lymphoma type that frequently carries the t(2;5) resulting in overexpression and activation of NPM-ALK chimeric kinase, which activates multiple oncogenic pathways including JAK-STAT3 pathway. The presence of cytosolic DNA of either exogenous or endogenous origin activates the cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, which activates the adaptor protein STING. The latter then activates the TBK1 and IKK kinases, which activate through phosphorylation the transcription factors IRF3 and NF-κB, respectively. IRF3 and NF-κB induce expression of interferons (e.g. IFN-β) and cytokines leading to activation of innate immune responses. The potential role of NPM-ALK oncogenic kinase in cGAS-STING-related anti-tumor immune responses in ALK+ ALCL is unknown to date. Therefore, the present study aimed to investigate the biologic impact of NPM-ALK on cGAS-STING activation status and expression of relevant interferon genes in ALK+ ALCL. Methods: The in vitro system included 5 ALK+ (Karpas 299, SUPM2, DEL, SUDHL1, L82) and 2 ALK- (Mac-1, Mac-2a) ALCL cell lines, as well as Ba/F3 cells stably transfected with NPM-ALK (Ba/F3-NPM-ALK) or a control (Ba/F3-MIG) plasmid. Expression and activation (phosphorylation) of cGAS-STING pathway proteins at baseline and experimental conditions were analysed by RT-PCR and Western blot at the RNA and protein level, respectively. Inhibition of ALK and STAT3 activity was performed using Crizotinib and the selective XIII STAT3 inhibitor, respectively. Silencing of ALK gene was performed using transient transfection with ALK siRNA and the Amaxa Nucleofector Technology. A STING agonist and TBK inhibitor (Amlexanox) were also used alone or in combination with other agents. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of interferon beta (IFN-β), CXCL10, and interferon gamma (IFN-γ), as well as a control gene (GAPDH), with quantitative RT-PCR. The patient study group included 38 previously untreated patients with ALK+ ALCL. Immunohistochemical analysis for STING protein expression was performed using a monoclonal antibody (Cell Signaling) and standard protocols. An arbitrary 10% cutoff was used to define positivity. Results: STING gene was highly expressed at both the mRNA and protein level in ALK+ and ALK- ALCL cell lines, however, cGAS-STING pathway proteins were activated at a variable