The NPM-ALK Oncogenic Kinase Suppresses CGAS-Sting-Associated Anti-Tumor Immune Responses through STAT3 Activation in Anaplastic Large Cell Lymphoma (ALCL)
Authors: Xagoraris Ioanna , Kokaraki Georgia , Plastira Christina , Stathopoulou Konstantina , Leventaki Vasiliki , Drakos Elias , Medeiros Jeffrey , Österborg Anders , Rassidakis George Z. 
Published: November 2021

Abstract
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 level among ALCL cell lines as shown in immunoblots. STING was highly expressed in 36 of 38 (95%) ALK+ ALCL tumors, highlighting its biologic significance in this lymphoma type. Inhibition of ALK activity by Crizotinib resulted in significant increase in IFN-β and CXCL10 gene expression linked to activation/phosphorylation of TBK1 indicating cGAS-STING pathway activation in ALK+ ALCL and Ba/F3-NPM-ALK cells. Silencing of ALK gene with specific ALK siRNA also resulted in a dramatic increase in the CXCL10 gene expression (mRNA level). Similarly, treatment of ALK+ ALCL cells with the XIII STAT3 inhibitor resulted in significantly increased IFN-β and CXCL10 gene expression associated with activation of the cGAS-STING pathway proteins in ALK+ ALCL cells but also in the ALK- ALCL cell line, Mac-1. Incubation of ALK+ ALCL cells with a STING agonist alone led to further activation of the cGAS-STING pathway in ALK+ ALCL cells.
Conclusion: NPM-ALK suppresses STING-associated, anti-tumor immune responses in ALK+ ALCL, through STAT3 activation and regulation of gene expression of type-1 interferons (IFN-β, CXCL10). Thus, combined ALK inhibition (ALK inhibitors) and STING stimulation (STING agonists) may represent a novel investigational therapeutic strategy for these patients.

Disclosures
No relevant conflicts of interest to declare.