NPM-ALK Oncoprotein Suppresses Anti-Tumor Immune Responses Partially through Cgas-Sting Pathway in ALK+ Anaplastic Large Cell Lymphoma
Authors: Xagoraris Ioanna , Yang Ying , Trogrlic Dora , Mansson Alma , Plastira Christina , Leventaki Vasiliki , Qian Hong , Lundqvist Andreas , Rassidakis George Z. 
Published: December 2024

Introduction
Anaplastic lymphoma kinase positive (ALK+) anaplastic large cell lymphoma (ALCL) is a distinct type of aggressive T-cell non-Hodgkin lymphoma (T-NHL) with high frequency in childhood and young adults. ALK+ ALCL is characterized by overexpression and activation of ALK due to chromosomal translocations with the most frequent being the t(2;5)(p23;q35) resulting in expression of the NPM-ALK oncogenic kinase. The latter activates multiple oncogenic pathways involved in lymphomagenesis. The cGAS-STING pathway triggers innate immune responses through activation of TBK1 and IKK kinases and their targets, the transcription factors IRF-3 and NF-κB, which induce the expression of interferons (IFNs) and cytokines. The potential role of NPM-ALK in regulation of anti-tumor immune responses is largely unknown to date.
Methods
The in vitro system included four ALK+ (Karpas 299, DEL, SUPM2, L82) and two ALK- ALCL (Mac1, Mac2a) cell lines as well as murine BaF3 cells stably transfected with NPM-ALK or control (MIG) plasmid. Quantitative reverse transcription (qRT)-PCR and Western blot analysis were used for the assessment of mRNA and protein levels, respectively. Silencing of various genes was performed using transient transfections with Nucleofector system (Lonza). Ceritinib was used as a next generation ALK inhibitor in cultured lymphoma cells. Proteome Profiler Human XL Cytokine Array (109 proteins, R&D Systems) and functional 51Cr-based NK-cell killing assays were utilized to assess the cytokine profile and NK-mediated anti-tumor responses in human ALCL cell lines, respectively. A mouse T-cell NHL cell line (EL-4) either STING-proficient or -deficient (knocked down using CRISPR-Cas9) was injected in syngeneic, immunocompetent mice and plasma was collected at day 6 and at the time of sacrifice (day 14-19) for proteomic analysis using the Olink Target 48 Mouse Cytokine Panel.
Results
Stable transfection of NPM-ALK in BaF3 resulted in upregulation of type I IFN gene expression associated with activation of the cGAS-STING pathway. Treatment with Ceritinib resulted in a dramatic increase of IFN-β (&gt;100-fold) and CXCL10 (&gt;30-fold) mRNA levels in ALK+ ALCL cells as well as in murine BaF3/NPM-ALK cells that was linked to significantly altered cytokine profile in vitro, including upregulation of IL-17, CCL5, MCP-1 and TARC, and downregulation of IL-10, IL-1R4, Blys and CD30. Silencing of ALK gene also resulted in upregulation of IFN-β and CXCL10 gene expression in ALK+ ALCL and BaF3/NPM-ALK cells. Similarly, silencing of STAT3, an important downstream target of NPM-ALK, also led to a significant increase in the IFN-β (&gt;120-fold) and CXCL10 mRNA levels in ALK+ ALCL cells associated with increased activation (phosphorylation) of the cGAS-STING-associated kinase TBK1 and its downstream target IRF3. Similar results were obtained after STAT3 gene silencing. Stimulation of the cGAS-STING pathway by a STING agonist led to upregulation of type I IFN gene expression and increased NK-cell killing suggesting that cGAS-STING pathway is functional in ALK+ and ALK- ALCL cells in vitro. Plasma proteomics performed on the in vivo mouse model (EL-4) showed significantly decreased levels of IL-17, TNF, IL-2, IL-22 and CXCL11, in mice that developed STING-deficient as compared to STING-proficient T-NHL. Taken together, NPM-ALK seems to modulate anti-tumor immune responses partially mediated by STAT3 and cGAS-STING pathways.
Conclusion
NPM-ALK oncogenic kinase suppresses cGAS-STING-associated anti-tumor immune responses through STAT3 activation and regulation of IFN-β and CXCL10 gene expression in ALK+ ALCL. STING-deficiency in the T-NHL cells seems to affect the chemokine and cytokine profile in vivo. Modulation of cGAS-STING activity, in addition to ALK inhibition, might represent a novel target for investigational therapy in ALK+ ALCL, and likely other T-cell NHLs.