Abstract
Introduction: Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) is a distinct aggressive T-cell non-Hodgkin lymphoma (NHL) type, commonly seen in pediatric patients and young adults. ALK+ ALCL is characterized by overexpression and activation of ALK kinase due to chromosomal translocations of ALK gene locus. The t(2;5)(p23;q35), the most frequent ALK gene translocation, results in overexpression and activation of the NPM/ALK oncogenic kinase, which in turn activates many oncogenic pathways involved in lymphomagenesis. Recently, we showed that NPM/ALK also suppresses anti-tumor immune responses via the cGAS-STING pathway (Xagoraris et al, Blood 2024;144: 2999); however, the role of the transcription factors IRF3 and IRF7 that operate downstream of cGAS-STING and induce the expression of interferons (IFNs) and cytokines is unknown to date. Furthermore, interleukin 10 (IL10) and its receptors (IL10R) are known to be upregulated in ALK+ ALCL due to STAT3 activation, which confer resistance to ALK inhibitors (Prokoph et al, Blood 2020;136:1657) and, based on its function in other cell systems, might contribute to suppression of innate immune responses.
Methods: The in vitro study model 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 (BaF3-NPM/ALK) or control (BaF3-MIG) plasmid. The mRNA and protein levels of various gene products were assessed by quantitative RT-PCR (RT-qPCR) and Western blot analysis in all sets of experiments. Silencing of ALK, STAT3, IRF3, IRF7 and IL10 genes was performed using transient transfections with Nucleofector system (Lonza). Ceritinib and Lorlatinib were used as a next generation ALK inhibitors in vitro. Selective STAT3 inhibitors and a STING agonist were utilized as well. The levels of secreted cytokines, chemokines and growth factors, were assessed in cell culture supernatants using the Proteome Profiler Human XL Cytokine Array (105 proteins, R&D systems) and targeted proteomics with the Olink Target 48 Human Cytokine panel. For the in vivo study, a mouse T-cell NHL cell line (EL4) 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: Treatment of human ALK+ ALCL cells with the next generation ALK inhibitors, Lorlatinib and Ceritinib, as well as STAT3 inhibitors resulted in a dramatic decrease of IL10 mRNA level, which was associated with upregulation of IFN-β gene expression. Conversely, silencing of IL10 gene led to increased IRF3 gene expression suggesting that the NPM/ALK-STAT3-IL10 axis downregulates IRF3 expression and function in ALK+ ALCL. Similarly, BaF3-NPM/ALK stable clones showed significantly lower mRNA and protein levels of murine IRF3 as compared to control BaF3-MIG stable transfectants. In addition to NPM/ALK-STAT3-IL10 axis, IRF3 and IRF7 expression and activation are strongly dependent on the presence of STING, since stimulation of the murine EL4 cells (T-cell NHL) with a STING agonist dramatically increased phosphorylated (activated) and total IRF3 and IRF7 protein levels in STING-proficient but not STING-deficient clonesin vitro. Using the Human XL Cytokine Array, transient silencing of IRF3 gene resulted in decreased secretion of IL10, IL17, IGFBP-3, CD147 in ALK+ ALCL cell culture supernatants. Moreover, using the Olink Target 48 Human Cytokine panel, IRF3 gene silencing was linked to dramatic increase of MMP12 and CCL2 secretion in cell culture supernatants in ALK+ but not in ALK- ALCL cells. Similar results were obtained following transient IRF7 gene silencing. By contrast, decreased secretion of CXCL9 and IL6 was observed in ALK- but not in ALK+ ALCL cell culture supernatants after knocking down IRF3 gene.Conclusions: Our findings provide evidence that the IRF3 and IRF7 transcription factors are dependent on functional cGAS-STING pathway and substantially contribute to anti-tumor immune responses in ALK+ ALCL. As NPM/ALK-STAT3-IL10 axis suppresses IRF3/7 - mediated immunomodulatory effects, targeting this oncogenic axis combined with stimulation of the cGAS-STING pathway may represent an efficient therapeutic strategy for patients with refractory or relapsing ALK+ ALCL.
