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, 1 (12), e46

The JAK Inhibitor AZD1480 Regulates Proliferation and Immunity in Hodgkin Lymphoma

The JAK Inhibitor AZD1480 Regulates Proliferation and Immunity in Hodgkin Lymphoma

E Derenzini et al. Blood Cancer J.

Erratum in

  • Blood Cancer J. 2014;4:e231

Abstract

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been reported to promote proliferation and survival of Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma (HL). We investigated the activity of the JAK inhibitor AZD1480 in HL-derived cell lines and determined its mechanisms of action. AZD1480 at low doses (0.1-1 μ) potently inhibited STATs phosphorylation, but did not predictably result in antiproliferative effects, as it activated a negative-feedback loop causing phosphorylation of JAK2 and extracellular signal-regulated kinases 1 and 2 (ERK1/2), and increased IP-10, RANTES and interleukin (IL)-8 concentrations in the supernatants. Inhibition of the ERK activity by mitogen-activated extracellular signal regulated kinase (MEK) inhibitors (UO126 and PD98059) enhanced the cytotoxic activity of AZD1480. Interestingly, submicromolar concentrations of AZD1480 demonstrated significant immunoregulatory effects by downregulating T-helper 2 cytokines and chemokines, including IL-13 and thymus- and activation-regulated chemokine, and the surface expression of the immunosuppressive programmed death ligands 1 and 2. Higher concentrations of AZD1480 (5 μ) induced G2/M arrest and cell death by inhibiting Aurora kinases. Our study demonstrates that AZD1480 regulates proliferation and immunity in HL cell lines and provides mechanistic rationale for evaluating AZD1480 alone or in combination with MEK inhibitors in HL.

Figures

Figure 1
Figure 1
Baseline JAK/STAT pathway activation status and effects of AZD1480 in HL cell lines. (a) Baseline activation of the JAK/STAT pathway in the HL cell lines HD-LM2, L-428, KM-H2 and L-540. PBMCs from three healthy donors were used for comparison. (b) Western blot assay of the four HL cell lines treated with increasing doses of AZD1480 (0.1–5 μm) for 72 h. (c) MTS assay of the four HL cell lines treated with AZD1480. Cells were incubated with increasing concentrations of AZD1480 (0.1–10 μm) for 24, 48 and 72 h. The value for each cell line is the mean of three independent experiments performed in triplicate. Error bars represent s.e.m. (d) IC50 values after HL cell lines were incubated with increasing doses of AZD1480 for 72 h.
Figure 2
Figure 2
AZD1480 induces apoptosis in HL cell lines. (a) Representative experiment demonstrating the effect of two different doses of AZD1480 (1 or 5 μm for 72 h) on apoptosis as determined by annexin V-binding assay. The percentage of dead cells is shown in the upper right quadrant. (b) Summary of the results of dual annexin V and propidium iodide (PI) staining. Each value is the mean of three independent experiments performed in triplicate. *P<0.05; **P<0.005; NS, not significant. Error bars represent s.e.m. (c) Immunoblotting showing activation of the intrinsic apoptotic pathway in HL cell lines incubated with AZD1480 (1–5 μm) for 72 h. Consistent with the data in (a) and (b), cleavage of poly (adenosine diphosphate ribose) polymerase (PARP) and activation of caspases 9 and 3 were observed in all the cell lines exposed to 5 μm AZD1480. In L-540 and L-428, caspase cleavage was observed also with 1 μm AZD1480.
Figure 3
Figure 3
AZD1480 induces paradoxical hyperphosphorylation of JAK2, TYK2 and MAP Kinases (ERK, p38) in HL cells. (a) Representative western blot assay of the three HL cell lines treated with increasing concentrations of AZD 1480 (0.1–5 μm for 72 h). Whole-cell lysates examined for p-JAK2 (two antibodies against Y1007/1008 at the activation loop obtained from different clones (see Materials and Methods)), JAK2, p-TYK2 Y1054/1055, TYK2, p-JAK3 Y980 and JAK3. (b) Representative western blot assay showing increased levels of ERK, p38 and SHP-2 phosphorylation in HD-LM2 and L-428, after treatment for 72 h with increasing doses of AZD1480 (0.1–5 μm). In contrast, L-540 showed a dose-dependent inhibition of ERK and p38 activation without increased SHP-2 phosphorylation. SOCS-3 levels decreased in all the cell lines. (c) After incubation with 1 μm AZD1480 for 72 h, cell culture supernatants were analyzed for IL-8, IP-10 and RANTES levels by enzyme-linked immunosorbent assay. In the bar graphs, each value is the mean of three independent experiments performed in triplicate. *P<0.05; **P<0.005; NS, not significant. (d) Effect of MEK inhibitors used with or without AZD1480 in HL cells. Combination index analysis performed using CalcuSyn software, showing that AZD1480 (1 μm) acted synergistically (CI <1) with UO126 or PD98059 (10–100 μm) in HD-LM2 and L-428 cells at 72 h. No synergistic effect was observed in L-540 cells. (e) Representative western blot assay of HL cells after treatment with 25-μm UO126 or PD98059 with or without 1-μm AZD1480 for 72 h.
Figure 4
Figure 4
Immunoregulatory effects of AZD1480 in HL cells. (a) Representative experiment demonstrating the effect of AZD1480 on PD-L1 and PD-L2 expression in HD cell lines. Cells were incubated with medium or AZD1480 1 μm for 72 h, and PD-L1 and PD-L2 expression levels were examined by flow cytometry. The bar graphs summarize the results from three independent experiments. Each value represents the mean of three independent experiments. *Denotes P-value less than 0.05, and **denotes P-value less than 0.005. (b) Cells were incubated with medium or AZD1480 1 μm for 72 h, and supernatants were examined for cytokine levels by enzyme-linked immunosorbent assay. The bar graphs summarize the results from three independent experiments. Each value represents the mean of three independent experiments. *Denotes P-value less than 0.05 and **denotes P-value less than 0.005.
Figure 5
Figure 5
AZD1480 induces G2/M cell cycle arrest by inhibition of Aurora A in HL cell lines. (a) Cells were incubated with AZD1480 (1 or 5 μm) for 24 h, and the cell cycle was analyzed by flow cytometry. AZD1480 induced an increase in the G2/M fraction only when a high concentration (5 μm) was used. (b) Bar graphs summarizing cell cycle analysis results; each value is the mean of three independent experiments. (c) Baseline expression status of Aurora kinases and histone H3 in HL cell lines. Whole-cell lysates of untreated HL cells were examined by western blotting for Aurora A, Aurora B, histone H3 and p-histone H3 (Ser10). (d) Representative western blot assay showing the effect of treatment with 1 and 5 μm AZD1480 (with or without MG132 20 μm) for 3 h on Aurora A, Aurora B and histone H3 phosphorylation (Ser 10) in HL cells. Cells were pretreated with nocodazole 400 ng/ml for 18 h to achieve a mitotic block.
Figure 6
Figure 6
Model for AZD1480 activity in HL cells. A model showing the dual, dose-dependent mechanism of action of AZD1480 in HL cells. At low doses (0.1–1 μm), AZD1480 inhibits the JAK/STAT pathway, showing predominantly immunoregulatory effects (downregulation of IL-6, IL-13, TARC, PD-L1 and PD-L2). At high doses (5 μm), AZD1480 also inhibits the Aurora kinases, promoting G2/M arrest and apoptosis in HL cells.

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