Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 May 1;16(9):2550-61.
doi: 10.1158/1078-0432.CCR-10-0279. Epub 2010 Apr 13.

Inhibition of p-STAT3 enhances IFN-alpha efficacy against metastatic melanoma in a murine model

Ling-Yuan Kong  1 , Alexander Gelbard, Jun Wei, Chantal Reina-Ortiz, Yongtao Wang, Eric C Yang, Yared Hailemichael, Izabela Fokt, Arumugam Jayakumar, Wei Qiao, Gregory N Fuller, Willem W Overwijk, Waldemar Priebe, Amy B Heimberger
Affiliations

Inhibition of p-STAT3 enhances IFN-alpha efficacy against metastatic melanoma in a murine model

Ling-Yuan Kong et al. Clin Cancer Res. .

Abstract

Purpose: Melanoma is a common and deadly tumor that upon metastasis to the central nervous system has a median survival duration of <6 months. Activation of the signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy, including immune suppression in melanoma patients. We hypothesized that WP1193, a novel inhibitor of STAT3 signaling, would enhance the antitumor activity of IFN-alpha against metastatic melanoma.

Experimental design: Combinational therapy of STAT3 blockade agents with IFN-alpha was investigated in a metastatic and an established syngeneic intracerebral murine tumor model of melanoma. The immunologic in vivo mechanisms of efficacy were investigated by T-cell and natural killer (NK) cell cytotoxic assays.

Results: IFN-alpha immunotherapy was synergistic with WP1193 showing marked in vivo efficacy against metastatic and established intracerebral melanoma. At autopsy, it was noted that there was a decreased trend in mice with melanoma developing leptomeningeal disease treated with combinational therapy. The combinational approach enhanced both NK-mediated and T-cell-mediated antitumor cytotoxicity.

Conclusions: The immune modulatory effects of STAT3 blockade can enhance the therapeutic efficacy of IFN-alpha immunotherapy by enhancing both innate and adaptive cytotoxic T-cell activities. This combination therapy has the potential in the treatment of metastatic melanoma that is typically refractory to this type of immune therapeutic approach.

PubMed Disclaimer

Figures

Fig 1
Fig 1
A novel small molecule, WP1193, inhibits STAT3 activity. A. Chemical structure of WP1193. B. The combination of IFN-α and WP1193 exerts direct cytotoxic effects on B16 cells. C. and D. WP1193 inhibits the phosphorylation of p-STAT3 in both B16 cells (C) and in splenocytes (D). B16 cells and splenocytes isolated from C57BL/6J mice and were incubated with either the medium, medium supplemented with IFN-α (2000 U/ml), or medium supplemented with both IFN-α and WP1193 (5 or 10 μM). After 2 hours (splenocytes) or 4 hours (B16 cells), cells were lysed, electrophoretically fractionated in 8% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with antibodies to p-STAT3, total STAT3 and β-actin. Semi-quantitative densitometry was used to determine the relative levels of p-STAT3 to STAT3 and β-actin.
Fig 2
Fig 2
In vivo inhibition of Tregs in the bone marrow of mice treated with WP1193, IFN-α, or both. Both WP1193 and IFN-α inhibited Tregs by 31% (P<0.05) and 78% (P<0.01), respectively, compared with the control; inhibition in the peripheral blood was 20% (P<0.05) and 46% (P<0.05), respectively. However, the combination of IFN-α and WP1193 was not synergistic for Treg inhibition in either the bone marrow or the blood. The experiment was been reproduced twice at day 11 and 14 with similar findings.
Fig 3
Fig 3
Cytotoxicity of B16 in vitro produced by splenocytes from mice treated with WP1193, IFN-α, or both. A. The splenocyte effector cells from mice that were treated with either WP1193 or IFN-α induced modest tumor lysis. However, splenocyte effector cells from mice that were treated with the combination of WP1193 and IFN-α potently enhanced B16-specific lysis (P< 0.01). B. Cytotoxicity of B16 in vitro produced by NK and CD8+ T cells treated with WP1193, IFN-α, or both. The NK and CD8+ immune effector cell populations were sorted as described and were treated with either WP1193, IFN-α or both. Similar to the data in Fig 3A, either treatment induced modest tumor lysis. However, both effector immune cell populations treated with the combination of WP1193 and IFN-α potently enhanced B16-specific lysis (P<0.01). This experiment was reproduced twice in its entirety with identical findings.
Fig 3
Fig 3
Cytotoxicity of B16 in vitro produced by splenocytes from mice treated with WP1193, IFN-α, or both. A. The splenocyte effector cells from mice that were treated with either WP1193 or IFN-α induced modest tumor lysis. However, splenocyte effector cells from mice that were treated with the combination of WP1193 and IFN-α potently enhanced B16-specific lysis (P< 0.01). B. Cytotoxicity of B16 in vitro produced by NK and CD8+ T cells treated with WP1193, IFN-α, or both. The NK and CD8+ immune effector cell populations were sorted as described and were treated with either WP1193, IFN-α or both. Similar to the data in Fig 3A, either treatment induced modest tumor lysis. However, both effector immune cell populations treated with the combination of WP1193 and IFN-α potently enhanced B16-specific lysis (P<0.01). This experiment was reproduced twice in its entirety with identical findings.
Fig 4
Fig 4
Regulation of MHC and NK-activating receptors and their respective ligands by WP1193 and IFN-α. Splenocytes or B16 cells were treated with WP1193, IFN-α, or both and MHC, the NK-activating receptors and ligands were subsequently analyzed by flow cytometric analysis. The isotype control is shown by the dashed black line and the respective target antigen by a solid black line. A. B16 cells stained for surface expression of MHC I and II after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α. B. B16 cells stained for surface expression of the NK-activating receptor ligands H60, Rae-1 and CD155 after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α. C. NK cells labeled with anti-NK1.1+ antibody from murine splenocytes stained for surface expression of the NK activating receptors NKG2D, KLRD1, NKp46, and DNAM-1 after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α.
Fig 4
Fig 4
Regulation of MHC and NK-activating receptors and their respective ligands by WP1193 and IFN-α. Splenocytes or B16 cells were treated with WP1193, IFN-α, or both and MHC, the NK-activating receptors and ligands were subsequently analyzed by flow cytometric analysis. The isotype control is shown by the dashed black line and the respective target antigen by a solid black line. A. B16 cells stained for surface expression of MHC I and II after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α. B. B16 cells stained for surface expression of the NK-activating receptor ligands H60, Rae-1 and CD155 after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α. C. NK cells labeled with anti-NK1.1+ antibody from murine splenocytes stained for surface expression of the NK activating receptors NKG2D, KLRD1, NKp46, and DNAM-1 after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α.
Fig 4
Fig 4
Regulation of MHC and NK-activating receptors and their respective ligands by WP1193 and IFN-α. Splenocytes or B16 cells were treated with WP1193, IFN-α, or both and MHC, the NK-activating receptors and ligands were subsequently analyzed by flow cytometric analysis. The isotype control is shown by the dashed black line and the respective target antigen by a solid black line. A. B16 cells stained for surface expression of MHC I and II after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α. B. B16 cells stained for surface expression of the NK-activating receptor ligands H60, Rae-1 and CD155 after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α. C. NK cells labeled with anti-NK1.1+ antibody from murine splenocytes stained for surface expression of the NK activating receptors NKG2D, KLRD1, NKp46, and DNAM-1 after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α.
Fig 4
Fig 4
Regulation of MHC and NK-activating receptors and their respective ligands by WP1193 and IFN-α. Splenocytes or B16 cells were treated with WP1193, IFN-α, or both and MHC, the NK-activating receptors and ligands were subsequently analyzed by flow cytometric analysis. The isotype control is shown by the dashed black line and the respective target antigen by a solid black line. A. B16 cells stained for surface expression of MHC I and II after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α. B. B16 cells stained for surface expression of the NK-activating receptor ligands H60, Rae-1 and CD155 after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α. C. NK cells labeled with anti-NK1.1+ antibody from murine splenocytes stained for surface expression of the NK activating receptors NKG2D, KLRD1, NKp46, and DNAM-1 after exposure to WP1193, IFN-α or the combination of WP1193 and IFN-α.
Fig 5
Fig 5
STAT3 blockade enhances the efficacy IFN-α against metastatic melanoma. A. The number of pulmonary metastasis was quantified 14 days after tumor inoculation. The number of metastasis for tumor-bearing mice without further intervention was 28 ± 12. Neither sub-therapeutic WP1193 (34 ± 17, P=0.31 compared with control) nor IFN-α alone (28 ± 11, P=0.48 compared with control) inhibited B16 pulmonary metastasis (Fig. 5A). However, the number of pulmonary metastasis was significantly reduced for WP1193 + IFN-α combinatorial therapy (9 ± 5) compared to control (P<0.05), WP1193 alone (P<0.05), or IFN-α alone (P<0.05). B. Survival data from C57BL/6J mice treated with WP1193, IFN-α, or both after B16 cells were established in the brain. Median overall survival for mice with intracerebral tumors without further intervention (n=11) was 17 days. C57BL/6J mice with established intracerebral B16 cells treated with a sub-therapeutic dose of WP1193 via oral gavage (n=12) showed a 9% increase in their median survival time to 18.5 days (P<0.04 compared with control). In mice with established tumor treated with IFN-α alone (n=8), there was a 62% increased in median survival to 27.5 days (P< 0.01 compared with control). In those mice with established tumors treated with the combination of IFN-α and WP1193 (n=11), there was a 135% increase in median survival to 40 days that was significantly longer compared with IFN-α alone (P<0.02). In mice that survived long-term subsequent re-challenge by injection of B16 cells into the contralateral hemisphere indicated that minimal immunological memory was induced. This experiment was repeated in its entirety with similar results.
Fig 5
Fig 5
STAT3 blockade enhances the efficacy IFN-α against metastatic melanoma. A. The number of pulmonary metastasis was quantified 14 days after tumor inoculation. The number of metastasis for tumor-bearing mice without further intervention was 28 ± 12. Neither sub-therapeutic WP1193 (34 ± 17, P=0.31 compared with control) nor IFN-α alone (28 ± 11, P=0.48 compared with control) inhibited B16 pulmonary metastasis (Fig. 5A). However, the number of pulmonary metastasis was significantly reduced for WP1193 + IFN-α combinatorial therapy (9 ± 5) compared to control (P<0.05), WP1193 alone (P<0.05), or IFN-α alone (P<0.05). B. Survival data from C57BL/6J mice treated with WP1193, IFN-α, or both after B16 cells were established in the brain. Median overall survival for mice with intracerebral tumors without further intervention (n=11) was 17 days. C57BL/6J mice with established intracerebral B16 cells treated with a sub-therapeutic dose of WP1193 via oral gavage (n=12) showed a 9% increase in their median survival time to 18.5 days (P<0.04 compared with control). In mice with established tumor treated with IFN-α alone (n=8), there was a 62% increased in median survival to 27.5 days (P< 0.01 compared with control). In those mice with established tumors treated with the combination of IFN-α and WP1193 (n=11), there was a 135% increase in median survival to 40 days that was significantly longer compared with IFN-α alone (P<0.02). In mice that survived long-term subsequent re-challenge by injection of B16 cells into the contralateral hemisphere indicated that minimal immunological memory was induced. This experiment was repeated in its entirety with similar results.
Fig 6
Fig 6
The CNS and survival data of C57/BL6 mice with intracerebral melanoma treated with WP1193, IFN-α, or both. A. The neuro-axis of mice at the time of death (treatment failure) was formalin fixed, paraffin embedded, and then stained with hematoxylin and eosin. The top panel demonstrates a whole mount axial section of the CNS from a mouse with death secondary to progression of LMD (shown by the black arrow). The asterisk (*) denotes the brainstem, and # denotes the hippocampus at 2.5× magnification. The bottom panel demonstrates a coronal section of the CNS from a mouse with death secondary to progression of intraparenchymal tumor. The white arrow shows the tumor-brain interface at 2.5× magnification. B. The C57/BL6 mice died of LMD or tumor depending on the treatment. Both the control and the sub-therapeutically WP1193 group died of progressive LMD. In contrast, in those C57/BL6 mice treated with IFN-α or the combination of IFN-α and WP1193, treatment failure-related deaths were secondary to tumor progression rather than LMD.
See this image and copyright information in PMC

References

    1. Sampson JH, Carter JH, Jr, Friedman AH, Seigler HF. Demographics, prognosis, and therapy in 702 patients with brain metastases from malignant melanoma. J Neurosurg. 1998;88:11–20. - PubMed
    1. Kirkwood JM, Manola J, Ibrahim J, et al. A pooled analysis of eastern cooperative oncology group and intergroup trials of adjuvant high-dose interferon for melanoma. Clin Cancer Res. 2004;10:1670–7. - PubMed
    1. Liang S, Wei H, Sun R, Tian Z. IFNα regulates NK cell cytotoxicity through STAT1 pathway. Cytokine. 2003;23:190–9. - PubMed
    1. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K. Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J Exp Med. 2005;202:637–50. - PMC - PubMed
    1. Ravine TJ, Ledinko N. Treatment with human recombinant leukocyte interferons inhibits in vitro invasive ability of human lung carcinoma cells. Clin Exp Metastasis. 1986;4:191–203. - PubMed

Publication types

MeSH terms