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STAT5 and IL-6 dysregulation in mastocytosis
panel) in HMC-1.2 cells and in P815 cells (Figure 6E). Similarly, the JAK2 inhibitor fedratinib, currently in clini- cal trials, nearly ablated IL-6 production in HMC-1.2 cells when in combination with either (or both) PI3K and ERK1/2 inhibitors (Figure 6F), indicating the separate con- tributions of these pathways to persistent IL-6 induction.
Discussion
IL-6 plays important roles in host defense, but if pro- duced in an uncontrolled or persistent manner it may be detrimental and contribute to the development of inflam- matory diseases (e.g. rheumatoid arthritis and inflamma- tory bowel disease) and malignancies.7,8,41 Although little is known about the exact role of IL-6 in mastocytosis and how it is dysregulated, the levels of IL-6 in circulation cor- relate with mast cell burden and tissue involvement, osteoporosis,9,11 and risk of progression.10 Our data suggest a contributory role for mast cells in IL-6 production in mastocytosis and implicate aberrant signaling of D816V- KIT as an initial event promoting persistent IL-6 transcrip- tion and consequent protein secretion. Among these aber- rant signals, we identified increased JAK2 activity and MEK/ERK- and PI3K-derived signals as drivers of IL-6 transcription, the former two by regulating the activa- tion/expression of STAT5. The study provides the first clues into mechanisms leading to persistent IL-6 produc- tion in mastocytosis and potential target molecules for therapeutic intervention.
Increased expression of IL-6 and its signaling through STAT3 in many malignancies can be driven by overexpres- sion of IL-6R, co-receptors and regulators (such as JAK and STAT3), polymorphisms in the IL-6 promoter and/or oncogenic signaling from tyrosine kinase receptors such as EGFR/HER, and may occur when negative regulation is not fully effective.34 Often, in malignant cells, production of IL-6 and constitutive STAT3 activation drive their own expression in feed-forward regulatory loops that are key for tumorigenesis.22,23,34,41 Here, we show that upregulation of IL-6 in mast cells with the D816V-KIT missense variant does not involve the IL-6R and co-receptor gp130 (Online Supplementary Figure S2A) or feedback loops involving STAT3 (Figure 5). Moreover, STAT4, shown to enhance IL- 6 transcription in human fibroblasts,36 had no role in IL-6 upregulation in HMC-1.2 mast cells. Instead, we demon- strate that persistent IL-6 production was dependent on oncogenic D816V-KIT activity and aberrant STAT5 activa- tion, as it was suppressed by tyrosine kinase inhibitors that effectively block D816V-KIT tyrosine kinase activity and by STAT5 silencing or inhibition. In addition, BM mast cells from patients with mastocytosis produced and released IL-6 ex vivo in correlation with D816V-KIT allelic burden (Figure 1). As STAT5 expression and phosphoryla- tion are also up-regulated in BM mast cells of patients with SM,29,38,39 our data suggest that oncogenic STAT5 acti- vation may be a priming event contributing to the elevat- ed serum IL-6 levels in mastocytosis. This does not exclude the involvement of other mechanisms such as IL- 6-mediated feed-forward loops on other cell types in the surrounding tissue, which may drive IL-6 production fur- ther. In addition, it is important to note that, although most of the intracellular IL-6 staining was associated with mast cells in patient’s BM biopsies (Online Supplementary Figure S1), enhanced production of IL-6 in SM may not be
restricted to mast cells as the presence of D816V-KIT may also induce or promote IL-6 production in other clonal cells (Figure 2D and Online Supplementary Table S2). Furthermore, additional signals in the local environment could crosstalk with oncogenic KIT signals in mast cells or other hematopoietic clonal cells in SM with associated multilineage involvement, further contributing to IL-6 dysregulation.
Even though canonical binding sites for STAT3, but not STAT5, are recognized in the IL-6 promoter, stimulation of STAT5 by the IgE receptor in mast cells was reported to mediate IL-6 production37 and constitutively active STAT5 mutants to induce IL-6 expression.42 Whether active STAT5 in D816V-KIT mast cells binds directly to the IL-6 promoter driving transcription, or does so indirectly by binding other transcription factors42 or by causing chro- matin remodeling,43 needs further evaluation. Regardless of this, as STAT5 hyperactivation is critical for neoplastic D816V-KIT mast cell growth and survival39,44 and for nor- mal mast cell development,45 our description of a novel regulatory role for STAT5 on constitutive IL-6 expression supports targeting STAT5 for treatment of patients with mastocytosis and high IL-6 levels. This could also be a potential treatment for patients with other hematologic malignancies such as chronic myelogenous leukemia where serum IL-6 levels represent a predictor of out- come46,47 and pathogenesis is in part driven by constitutive STAT5 activation.48
The activation of STAT5 by D816V-KIT was complex and involved an interplay of JAK2- and MEK/ERK1/2- mediated pathways. JAK2-selective inhibitors such as fedratinib markedly reduced constitutive IL-6 production, while the effectiveness of other pan-jakinib correlated with their relative selectivity for JAK2, suggesting that JAK1 and 3 have no significant role. The specific involve- ment of JAK2 agrees with the notion that STAT5 is a major target for JAK2 in hematopoietic cells29 and that activation of KIT by SCF causes JAK2 phosphorylation.31 Unlike the known effect on JAK2 inhibition, the effect of MEK/ERK1/2 inhibition on STAT5 activity and transcript abundance was unexpected. Phosphorylation in serine/threonine residues of STAT1/3/4 by MAPK was reported to affect STAT1/3/4 activity.33,40 Although serine phosphorylation at the C-terminal tail of STAT5 proteins is essential for leukemogenesis49 and for growth hor- mone-induced gene expression,50 the serine/threonine kinases that mediate this type of phosphorylation or the exact functional implications are not well understood. Surprisingly, in cells with D816V-KIT, inhibition of MEK/ERK1/2 caused a more pronounced effect on STAT5 phosphorylation at tyrosine 694 (a target for JAK2) than at serine 780 (Figure 6), even though JAK2 activity was not affected by the ERK inhibitor (Figure 4). A similar JAK-independent role for ERK in STAT3 tyrosine phos- phorylation was reported in plasma cells for IL-6,27 but the mechanism for such regulation, and what potential kinases or phosphatase may be involved, need further evaluation.
We also found a role for PI3K/AKT-dependent path- ways on IL-6 induction in D816V-KIT mast cells, but in a STAT5-independent manner, indicating that persistent IL-6 expression likely involves multiple transcription fac- tors. Similar to STAT5,39,44 oncogenic growth and survival of mast cells with D816V-KIT is also dependent on con- stitutive activation of AKT.2,30 Thus, combined targeting
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