Targeting myeloid-derived suppressor cells using a novel adenosine monophosphate-activated protein kinase (AMPK) activator

Abstract

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of early myeloid cells that accumulate in the blood and tumors of patients with cancer. MDSC play a critical role during tumor evasion and promote immune suppression through variety of mechanisms, such as the generation of reactive oxygen and nitrogen species (ROS and RNS) and cytokines. AMPactivated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that regulates energy homeostasis and metabolic stress. However, the role of AMPK in the regulation of MDSC function remains largely unexplored. This study was designed to investigate whether treatment of MDSC with OSU-53, a PPAR-inactive derivative that stimulates AMPK kinase, can modulate MDSC function. Our results demonstrate that OSU-53 treatment increases the phosphorylation of AMPK, significantly reduces nitric oxide production, inhibits MDSC migration, and reduces the levels of IL-6 in murine MDSC cell line (MSC2 cells). OSU53 treatment mitigated the immune suppressive functions of murine MDSC, promoting T-cell proliferation. Although OSU-53 had a modest effect on tumor growth in mice inoculated with EMT-6 cells, importantly, administration of OSU53 significantly (p < 0.05) reduced the levels of MDSC in the spleens and tumors. Furthermore, mouse MDSC from EMT-6 tumor-bearing mice and human MDSC isolated from melanoma patients treated with OSU-53 showed a significant reduction in the expression of immune suppressive genes iNOS and arginase. In summary, these results demonstrate a novel role of AMPK in the regulation of MDSC functions and provide a rationale of combining OSU-53 with immune checkpoint inhibitors to augment their response in cancer patients.

Keywords: AMPK; Immunotherapy; MDSC; OSU-53; iNOS.

Figure 1.

Effect of OSU-53 on the apoptosis of MSC2 cells. (A) MSC2 cells were treated with the indicated doses of OSU-53 or DMSO overnight and analyzed by FACS. (B) Monocytes isolated from healthy donors (n = 2) were treated overnight with the indicated doses of OSU-53 and DMSO (control). The cells were stained with AnnexinV/PI and analyzed via FACS to determine the percentage of apoptotic cells. Values represent mean ± SD.

Figure 2.

Increased expression of phosphorylated AMPK in MDSC treated with OSU53. (A) Immunoblot showing the expression of p-AMPK and total AMPK in MSC2 cells treated with the indicated doses of OSU-53 or DMSO for 12 h. Protein lysates from MSC2 cells were probed with p-AMPK, total AMPK, and ß-Actin antibodies.

Figure 3.

Reduction iNOS and arginase expression in murine and human MDSC treated with OSU-53. (A) MSC2 cells were treated with indicated doses of OSU-53 or DMSO (control) overnight and then stimulated with LPS. The levels of nitrite were measured in the supernatant using Griess reagent. (B) Fold change in NOS2 (iNOS) gene expression in MSC2 cells was treated with indicated doses of OSU-53 or DMSO. (C-D) iNOS and arginase gene expression in MDSC from EMT-6 tumor-bearing mice treated OSU-53 (5 µM) or DMSO. MDSC isolated from melanoma patients were treated overnight with DMSO or OSU-53 (5 µM). The expression of (E) inducible nitric oxide synthase and (F) arginase was determined using real time PCR. The bars show fold change in gene expression compared to DMSO (controls). Values represent mean ± SD. (p < 0.05).

Figure 4.

Reduced MSC2 migration in response to cytokines produced by tumor cells. (A) Schematic showing the experimental setup. MSC2 cells cultured in serum free media were treated with the indicated doses of OSU-53 for 12 h. The MSC2 cells were added into the transwell insert and migration was allowed to precede overnight in response to conditioned media from mouse EMT6-HER-2⁺ tumor cells. (B) Representative image of inserts showing MSC2 cells after migration. (C) Quantification of the number of migrated MSC2 cells. Values represent mean ± SD.

Figure 5.

Levels of IL-6 and TNF-α in MSC2 cells treated with OSU-53. MSC2 cells were treated with the indicated doses of OSU-53 for 12 h, following which the cell free supernatant were collected and analyzed for levels of TNF-α and IL-6 by ELISA. (A) TNFα (pg/mL). (B) IL-6 (pg/mL).

Figure 6.

OSU-53 reduces MDSC mediated suppression of T-cell proliferation. MDSC isolated from tumor-bearing mice were treated with indicated doses DMSO or OSU-53 and co-cultured (2:1) with CFSE-labeled T cells activated with anti-CD3/CD28 beads for 3-days and proliferation was assessed by flow cytometry for CFSE staining. Cells were stained with anti-CD4⁺ or anti-CD8⁺ antibodies. Shown are representative histogram (left panels) and bar graphs (right panels). Results are from one representative experiment.

Figure 7.

OSU-53 reduces MDSC frequency in tumor-bearing mice. Female Balb/c mice were inoculated with EMT-6-HER2 breast cancer cells in the mammary fat pad. The mice received OSU-53 (100 mg/kg) or vehicle via oral gavage for a period of 2 weeks. (A) Tumor growth in mice treated with OSU-53 or vehicle. (B) Weight of spleens from mice (C–D) FACS analysis of splenocytes and single cell suspensions prepared from tumors stained with CD11b and Gr1 and antibodies. Each group consisted of five mice. Values are the mean ± SE of tumor volumes at each time point, (p < 0.05).