IL-6 stimulation of DNA replication is JAK1/2 mediated in cross-talk with hyperactivated ERK1/2 signaling

Abstract

Myeloproliferative neoplasms (MPNs) are developing resistance to therapy by JAK1/2 inhibitor ruxolitinib. To explore the mechanism of ruxolitinib's limited effect, we examined the JAK1/2 mediated induction of proliferation related ERK1/2 and AKT signaling by proinflammatory interleukin-6 (IL-6) in MPN granulocytes and JAK2V617F mutated human erythroleukemia (HEL) cells. We found that JAK1/2 or JAK2 inhibition prevented the IL-6 activation of STAT3 and AKT pathways in polycythemia vera and HEL cells. Further, we showed that these inhibitors also blocked the IL-6 activation of the AKT pathway in primary myelofibrosis (PMF). Only JAK1/2 inhibitor ruxolitinib largely activated ERK1/2 signaling in essential thrombocythemia and PMF (up to 4.6 fold), with a more prominent activation in JAK2V617F positive granulocytes. Regarding a cell cycle, we found that IL-6 reduction of HEL cells percentage in G2M phase was reversed by ruxolitinib (2.6 fold). Moreover, ruxolitinib potentiated apoptosis of PMF granulocytes (1.6 fold). Regarding DNA replication, we found that ruxolitinib prevented the IL-6 augmentation of MPN granulocytes frequency in the S phase of the cell cycle (up to 2.9 fold). The inflammatory stimulation induces a cross-talk between the proliferation linked pathways, where JAK1/2 inhibition is compensated by the activation of the ERK1/2 pathway during IL-6 stimulation of DNA replication.

Keywords: ERK1/2 signaling; JAK1/2 inhibition; cell cycle; inflammation; myeloproliferative neoplasm.

Figure 1

JAK-STAT signaling stimulation of MAPK and PI3K/AKT pathways in CD34⁺ cells of MPN origin (correspond to Tables 1 and 2). (+p) phosphorylation, (-p) dephosphorylation; → stimulation, ┴ inhibition; white boxes represent down-regulated genes, gray boxes mixed gene expression, black boxes up-regulated genes vs. controls, while white boxes with an intermittent edge represent sporadically expressed genes in MPN vs. control.

Figure 2

Activation of proliferation related signaling pathways by IL-6 in MPN granulocytes. Time scale of IL-6 (20 ng/ml) activation of (A) JAK/STAT3; (B) AKT and (C) ERK1/2 signaling in granulocytes of polycythemia vera (PV, n = 6), essential thrombocythemia (ET, n = 8) and primary myelofibrosis (PMF, n = 8). *p < 0.05, **p < 0.01, ***p < 0.001 vs. non-treated cells (0). IL-6 activation of (D) JAK/STAT3; (E) AKT and (F) ERK1/2 signaling in ET and PMF according to JAK2V617F presence during 1 hour of incubation. (n = 4) Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. non-mutated cells.

Figure 3

IL-6 activation of STAT signaling in PV and PMF as well as inhibition in ET granulocytes. IL-6 (20 ng/ml) activation of JAK/STAT3 signaling in (A) polycythemia vera (PV), (B) essential thrombocythemia (ET) with JAK2V617F (JAK2+), (C) ET without JAK2V617F (JAK2-), (D) primary myelofibrosis (PMF) JAK2+, (E) PMF JAK2-, in the presence of ruxolitinib (Ruxo) and hexabromocyclohexane (Hexa) after 1 hour of incubation. Values are mean ± SEM (n=4). *p < 0.05, **p < 0.01, ***p < 0.001 vs. non-treated cells/Control; ^#the same vs. IL-6 treated cells.

Figure 4

JAK1/2 dependent activation of AKT pathway by IL-6 in PV and PMF besides inhibition in ET granulocytes. IL-6 (20 ng/ml) activation of AKT signaling in (A) HEL cells, (B) polycythemia vera (PV), (C) essential thrombocythemia (ET) with JAK2V617F (JAK2+), (D) ET without JAK2V617F (JAK2-), (E) primary myelofibrosis (PMF) JAK2+, (F) PMF JAK2-, in the presence of ruxolitinib (Ruxo) and hexabromocyclohexane (Hexa) after 1 hour of incubation. Values are mean ± SEM (n=4). *p < 0.05, **p < 0.01, ***p < 0.001 vs. non-treated cells/Control; ^#the same vs. IL-6 treated cells.

Figure 5

IL-6 activation of MAPK pathway in PV and PMF then inhibition in JAK2+ ET granulocytes. IL-6 (20 ng/ml) activation of ERK1/2 signaling in (A) healthy controls, (B) polycythemia vera (PV), (C) essential thrombocythemia (ET) with JAK2V617F (JAK2+), (D) ET without JAK2V617F (JAK2-), (E) primary myelofibrosis (PMF) JAK2+, (F) PMF JAK2-, in the presence of ruxolitinib (Ruxo) and hexabromocyclohexane (Hexa) after 1 hour of incubation. Values are mean ± SEM (n=4). *p < 0.05, **p < 0.01, ***p < 0.001 vs. non-treated cells/Control; ^###p < 0.001 vs. IL-6 treated cells.

Figure 6

IL-6 increased the percentage of MPN granulocytes in S phase of cell cycle mediated by JAK1/2. IL-6 (20 ng/ml) induction of cell cycle in (A) K562 cells (n=3), (B) JAK2V617F mutated HEL cells (n=3), (C) healthy donors (n=3), (D) polycythemia vera (PV, n=4), (E) essential thrombocythemia (ET, n=8) and (F) primary myelofibrosis (PMF, n=8) treated with Ruxolitinib (R) or Hexabromocyclohexane (H) after 16 hours of incubation (gray box). Values are mean±SEM.