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STAT5 and IL-6 dysregulation in mastocytosis
from D816V-KIT can promote ligand-independent IL-6 production without involving some of the most common autocrine feed-forward loops described in malignant cells.
The constitutive release of IL-6 by unstimulated HMC- 1.2 was enhanced by stimuli such as complement compo- nent 5a (C5a), IL-1b, 10% FBS and PMA/ionomycin (Online Supplementary Figure S3A, left panel) suggesting that the production and secretion of IL-6 due to D816V- KIT can synergize with other complementary signals or environmental cues. Of interest, the intracellular content of IL-6 protein was only approximately 10% of the total IL-6 released (Online Supplementary Figure S3A), and although dasatinib did not affect this percentage, it also reduced the total intracellular content. Thus, dasatinib inhibited the transcription, intracellular content and release of IL-6 (Figure 3B and C, and Online Supplementary Figure S3A, right panel), consistent with the conclusion that D816V-KIT signals cause constitutive de novo produc- tion and release of IL-6 without regulating storage. In addition, we demonstrate that IL-6 protein released by HMC-1.2 cells is biologically active since conditioned media of these cells caused IL-6R-mediated STAT3 phos- phorylation in LAD2 cells which express and respond to IL-6R activation12 (Online Supplementary Figure S3B).
D816V-KIT-induced IL-6 production is dependent on JAK2, ERK and PI3K pathways
Mitogen-activated protein kinase (ERK1/2 and p38) and PI3K pathways are part of the oncogenic signals derived from D816V-KIT activity.2,25 Since these pathways may affect IL-6 expression,26-28 we investigated their potential roles in D816V-KIT-induced IL-6 production. While inhibi- tion of p38 with SB203580 had minor effects on IL-6 syn- thesis, inhibition of the ERK1/2 pathway using a MEK1/2 inhibitor (U0126) (Figure 4A) or inhibition of the PI3K pathway by the PI3Kα/δ/β inhibitor LY294002 (Figure 4B), caused a 50-60% reduction, respectively, in IL-6 pro- duction at the message (Figure 4B, left panel) and protein levels (Figure 4B, right panel).
The JAK/STAT axis is also known to be prominently up- regulated by D816V-KIT activity.29,30 JAK2 is activated by SCF31 leading to STAT phosphorylation and translocation into the nucleus, where it exerts its transcriptional activity.32 Inhibition of JAK2 by the JAK2 selective inhibitor fedratinib (TG101348) markedly blocked IL-6 constitutive transcription and cytokine release (Figure 4C, left and right panels, respectively). Ruxolitinib which inhibits JAK1 in addition to JAK2, similarly inhibited IL-6 expression, although at higher concentrations than fedra- tinib (Figure 4D). However, tofacitinib, a pan-JAK inhibitor preferential for JAK3 and to a lesser extent JAK1, was less effective (Figure 4D). Similar to HMC-1.2 cells, in mouse P815 cells inhibition of MEK/ERK1/2, PI3K or JAK2 pathways markedly reduced IL-6 release in mouse P815 cells (Figure 4E). The data thus implicate JAK2 in D816V- KIT induced IL-6 production.
Since JAK2 has also been reported in certain cells to acti- vate PI3K or ERK,32,33 we further investigated the potential inter-relationships between JAK2 and the ERK and PI3K pathways. While, as expected, inhibition of KIT by dasa- tinib blocked the phosphorylation of JAK2, AKT and ERK1/2 signaling pathways downstream of the receptor by 40-80% (Figure 4F), inhibitors of JAK2, PI3K/AKT or MEK1/2/ERK1/2 reduced phosphorylation of their respec- tive targets but did not show significant effects on any of
the others, suggesting that these signals are activated inde- pendently from each other.
D816V-KIT-induced IL-6 production is dependent on STAT5
JAK phosphorylates STAT, and STAT family members such as STAT3,34,35 STAT436 and STAT537 have been impli- cated in the regulation of IL-6 transcription in various cells and conditions. As the levels of expression or phosphory- lation of STAT3, STAT4 and STAT5 (Online Supplementary Figure S4A-C) are increased in HMC-1.2 and other cells with D816V-KIT30 and STAT4 and STAT5 are up-regulated in BM mast cells from patients with SM,29,38,39 we investi- gated their possible involvement in the induction of IL-6 transcription by silencing STAT3-5 expression using sh- RNA or si-RNA.
Sh-RNA-mediated STAT3 silencing resulted in >75% reduction in STAT3 at the messenger and protein levels (Figure 5A) but did not affect IL-6 production by HMC-1.2 (Figure 5A, red bar). Similarly, a selective small inhibitor molecule for STAT3, C188-9, at concentrations that caused >80% reduction in STAT3 phosphorylation (Online Supplementary Figure S5A, top panel) did not alter constitu- tive IL-6 production by these cells (Online Supplementary Figure S5A, bottom panel). Neutralizing antibodies for the IL-6R, which signals through STAT3, were also ineffective on IL-6 production (Online Supplementary Figure S2A). Reduction of STAT4 message and protein by >50% using sh-RNA knockdown was also inconsequential for IL-6 persistent production by these cells (Figure 5B).
However, specific reduction in the mRNA for STAT5A or STAT5B messages by >50% and in protein expression (Figure 5C) using STAT5-specific si-RNA pools, resulted in concomitant reductions in IL-6 transcription (Figure 5D). Simultaneous knockdown of STAT5A and B did not accomplish significantly greater effects on IL-6 expression than silencing each individually (Figure 5C and D), sug- gesting a redundant function for the two isoforms. The selective STAT5 inhibitor (CAS 285986-31-4), at a concen- tration that inhibited STAT5 phosphorylation by 50% (50 mM) (Online Supplementary Figure S5B, top panel) also significantly reduced IL-6 transcription and IL-6 protein synthesis by 50% (Online Supplementary Figure S5B, bottom panel, and S5C), a result that was also confirmed in P815 cells (Online Supplementary Figure S5D). As STAT5 is over- expressed and hyperactivated in cells carrying D816V (Online Supplementary Figure S4C)29,38,39 and STAT5 mRNA was not depleted even when both STAT5A and B were silenced (Figure 5C), we treated STAT5-knockdown cells with the STAT5 inhibitor, which showed a reduction in IL-6 mRNA expression by 80% (Online Supplementary Figure S5E) consistent with the reduction observed by inhibition of JAK2. The results point towards JAK2/STAT5 as a major pathway leading to the constitutive expression of IL-6 in D816V-KIT expressing cells.
ERK contributes to STAT5 phosphorylation and expression while the effect of the PI3K pathway on IL-6 is STAT5-independent
As MAP kinases have been implicated in the phospho- rylation and the activation of STAT family members,40 we sought to determine whether ERK1/2 may contribute to the induction of IL-6 by enhancing STAT5 phosphoryla- tion. While inhibition of ERK1/2 did not affect JAK2 phos- phorylation (Figure 4F), it substantially inhibited STAT5
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