Page 219 - Haematologica Vol. 107 - September 2022

LETTER TO THE EDITOR
Deregulated JAK3 mediates growth advantage and hemophagocytosis in extranodal nasal-type natural killer/T-cell lymphoma
Advanced extranodal, nasal-type natural killer/T-cell lym- phoma (NKTCL) is an aggressive malignancy with dismal prognosis, typically associated with hemophagocytic syn- drome that worsens the prognosis.1,2 Hemophagocytic syn- drome results from excessive production of interferon-γ, although the mechanisms leading to interferon-γ overpro- duction in NKTCL cells remain unclear. A deregulation of Janus kinase 3 (JAK3) protein is a general oncogenic event in NKTCL, with up to 80% of patients harboring constitut- ive phosphorylation in the activating Y980 residue, and 7%-35% of patients displaying acquired activating muta- tions, mainly in the pseudokinase domain, which account for its constitutive activation.3-6 Here we demonstrate that constitutively activated JAK3 confers natural killer (NK) cell hypersensitivity to interleukin-2 (IL-2), leading to growth advantage and excessive production of interferon- γ through activation of downstream substrates. We also show that the expression of constitutively activated JAK3A573V in hematopoietic progenitors leads to NK-cell ex- pansion in mice. Finally, using an original in vivo murine model based on the expression of JAK3A573V in primary NK cells from Rag2-/- mice, we observed that transplanted wild-type recipients reproduce the typical features of NKTCL including NK-cell expansion and hemophagocytic syndrome.
To get insights into the role of JAK3 in NKTCL pathophysi- ology, we hypothesized that JAK3 deregulation could lead to an excessive sensitivity to cytokines. In preliminary ex- periments, we treated control NK cells and the NK-cell line MEC04, derived from a NKTCL (harboring the JAK3A573V mutation), and NKL and KHYG-1 cell lines, derived from NK-cell leukemias, all harboring a constitutively phos- phorylated JAK3 on Y980 residue,3 with recombinant IL-2 (rIL-2) as a representative cytokine that stimulates a re- ceptor involving the common γc/JAK3 axis. Cells were ex- posed to 100 U/mL of rIL-2 for 2 days and counted daily using the trypan blue exclusion assay. A rapid and signifi- cant increase in the number of viable cells was observed throughout time when compared to control NK cells iso- lated from healthy donors or to MEC04 cells cultured without rIL-2 (Online Supplementary Figure S1A). After 2 days of culture, there was a more than 4-fold increase in the number of MEC04 cells in comparison with normal NK cells (P<0.0001). Similar results were observed for NKL and KHYG1 cells (Online Supplementary Figure S1). Strik- ingly, this proliferative effect observed after exposure to
rIL-2 was totally abrogated when cells were exposed to the JAK3 inhibitor CP-690550 (Online Supplementary Fig- ure S1A), providing further evidence that JAK3 signaling is important in NKTCL oncogenesis.
To confirm the importance of JAK3 signaling in NKTCL, MEC04 cells were stimulated with rIL-2 for 30 minutes and subjected to western-blot analysis for the phos- phorylation of the tyrosine 705 (Y705) residue of Signal transducer and activator of transcription 3 (STAT3) onco- gene.3,7 We found that before stimulation with rIL-2, Y705- STAT3 showed constitutive baseline phosphorylation in MEC04 cells when compared to control NK cells. After stimulation with rIL-2, Y705-STAT3 phosphorylation was increased in MEC04 cells. This phenomenon was ab- rogated when cells were exposed to CP-690550 before rIL-2 stimulation (Online Supplementary Figure S1B). Col- lectively, these results provide evidence for a synergistic role between JAK3 deregulation and IL-2 signaling in the growth of NK-cell lines.
To explore the involvement of deregulated JAK3 in the hy- persecretion of interferon-γ, we used NKL and KHYG-1 cells as they produce the highest amounts of interferon- γ. Cells were cultured in the presence or absence of rIL-2, and interferon-γ in the supernatant was measured after 48 h of culture. The two cell lines spontaneously secrete low levels of interferon-γ in the absence of rIL-2. In the presence of rIL-2, the amount of secreted interferon-γ/106 viable cultured cells increased dramatically. This effect was almost completely abrogated when cells were con- comitantly cultured with CP-690550 (Online Supplemen- tary Table S1). Accordingly, the phosphorylation of target proteins STAT3, AKT and ERK1/2 was decreased in the presence of JAK3 inhibitors (Online Supplementary Figure S1C). To confirm further the role of JAK3 signaling in in- terferon-γ secretion, we selectively knocked-down JAK3 in NKL cells using specific siRNA. The decreased ex- pression of JAK3 induced a rapid decrease in cell viability (Online Supplementary Figure S1D), with dephosphoryla- tion of target proteins (Online Supplementary Figure S1E), as well as a major decrease in interferon-γ secretion after 2 days of cell culture (27±2.1 pg/mL/106 viable cells in the presence of JAK3-targeting siRNA vs. 127±3.4 pg/mL/106 viable cells in the presence of scrambled siRNA, P<0.001). Furthermore, the phosphorylation of AKT and ERK, two substrates downstream of JAK3, was inhibited by GDC- 0941 and UO126, respectively, as assessed after culture
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