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. 2016 Oct 18;7(42):68489-68502.
doi: 10.18632/oncotarget.12095.

RPN13/ADRM1 inhibitor reverses immunosuppression by myeloid-derived suppressor cells

Ruey-Shyang Soong  1   2   3 , Ravi K Anchoori  4 , Benjamin Yang  1 , Andrew Yang  1 , Ssu-Hsueh Tseng  1 , Liangmei He  1 , Ya-Chea Tsai  1 , Richard B S Roden  1   5   4 , Chien-Fu Hung  1   4
Affiliations

RPN13/ADRM1 inhibitor reverses immunosuppression by myeloid-derived suppressor cells

Ruey-Shyang Soong et al. Oncotarget. .

Abstract

Myeloid-derived-suppressor cells (MDSCs) are key mediators of immune suppression in the ovarian tumor microenvironment. Modulation of MDSC function to relieve immunosuppression may enhance the immunologic clearance of tumors. The bis-benzylidine piperidone RA190 binds to the ubiquitin receptor RPN13/ADRM1 on the 19S regulatory particle of the proteasome and directly kills ovarian cancer cells by triggering proteotoxic stress. Here we examine the effect of RA190 treatment on the immunosuppression induced by MDSCs in the tumor microenvironment, specifically on the immunosuppression induced by MDSCs. We show that RA190 reduces the expression of Stat3 and the levels of key immunosuppressive enzymes and cytokines arginase, iNOS, and IL-10 in MDSCs, while boosting expression of the immunostimulatory cytokine IL-12. Furthermore, we show that the RA190-treated MDSCs lost their capacity to suppress CD8+ T cell function. Finally, we show that RA190 treatment of mice bearing syngeneic ovarian tumor elicits potent CD8+ T cell antitumor immune responses and improves tumor control and survival. These data suggest the potential of RA190 for ovarian cancer treatment by both direct killing of tumor cells via proteasome inhibition and relief of MDSC-mediated suppression of CD8 T cell-dependent antitumor immunity elicited by the apoptotic tumor cells.

Keywords: MDSCs; RPN13; Stat3; immunosuppression; proteasome.

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Conflict of interest statement

CONFLICTS OF INTEREST

Under a licensing agreement between Pontifax and the Johns Hopkins University, Drs. Anchoori and Roden are entitled to royalties on an invention described in this article. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies.

Figures

Figure 1
Figure 1. Impact of RA190 treatment or RPN13 knock down on P-Stat3 and Stat3 levels in MDSCs in vitro.
(A) Immunoblot of P-Stat3 and Stat3 expression level of MDSCs treated with PBS, RA190 (2 μM), and the inactive analog RA190R (2 μM). (B) Expression of P-Stat3 and Stat3 in MDSCs treated with various doses of RA190 for 8 hours. (C) Time course of P-Stat3 and Stat3 expression in MDSCs following RA190 treatment. (D) Lentivirus expressing Rpn13 shRNA was used to infect MDSCs and knock down Rpn13 expression. Immunoblot showing Rpn13 protein expression in MDSCs treated with control shRNA and Rpn13 shRNA as well as expression level of P-Stat3 and Stat3.
Figure 2
Figure 2. Impact of RA190 treatment upon IL-10, IL-12, and Gr-1 expression in MDSCs isolated from spleen and tumor microenvironment
(A–D) Splenocytes of ID8-Luc tumor bearing mice were treated with or without RA190 (2 μM) for 48 hours and IL-10 and IL-12 expression were assessed in CD11b+ Gr-1+ cells. (A) Representative flow cytometry of IL-10 in MDSCs in spleen. (B) Bar graph showing IL-10 expression in MDSCs in spleen. (C) Representative flow cytometry of IL-12 in MDSCs. (D) Bar graph showing IL-12 expression in MDSCs in spleen. (E-K) Ascites were collected from the peritoneum of ID8-Luc tumor-bearing mice and treated with or without RA190 (2 μM) for 48 hours, and IL-10 and IL-12 expression levels were examined in CD11b+ Gr-1+ cells in the tumor microenvironment. (E) Representative flow cytometry of IL-10 in MDSCs in ascites. (F) Bar graph showing IL-10 expression level of MDSCs in the ascites. (G) MDSC population distribution after PBS or RA190 treatment. (J) Bar graph showing the percentage of CD11b+Gr-1Hi cells and CD11b+Gr1Low cells among ascites. (H–I) Representative flow cytometry analysis of IL-12 in Gr-1Hi and Gr-1Low MDSC populations. (K) Bar graph showing IL-12 expression in CD11b+Gr1Hi and CD11b+Gr-1Low populations in various treatment groups. Values are shown as mean ± SD (*P = 0.05, **P = 0.01, ns, not significant).
Figure 3
Figure 3. Arginase and iNOS levels in MDSCs isolated from spleen and tumor microenvironment following RA190 treatment or RPN13 knock down in vitro
(A and B) MDSCs from spleens and ascites of ID8-Luc tumor-bearing mice were treated with or without RA190 (2 μM) in vitro for 24 hours. The levels of Arginase and iNOS were assessed by flow cytometry. (A) Bar graph showing arginase expression in CD11b+Gr1+ cells isolated from spleen and ascites. (B) Bar graph showing iNOS expression in CD11b+Gr1+ cells isolation from spleen and ascites. (C and D) Lentivirus expressing Rpn13 shRNA was used to infect MDSCs and knock down Rpn13 expression. Arginase and iNOS expression in MDSCs receiving no treatment, infected with lentivirus expressing control shRNA, infected with lentivirus expressing Rpn13 shRNA, or treated with RA190 (2 μM) were assessed by flow cytometry. (C) Bar graph showing the percentage of arginase expressing CD11b+Gr-1+ cells in different groups. (D) Bar graph showing the percentage of iNOS expressing CD11b+Gr-1+ cells in different groups. Values are shown as mean ± SD (*P = 0.05, **P = 0.01, ns, not significant).
Figure 4
Figure 4. T cell proliferation after co-culturing with MDSCs treated with RA190 or RPN13 knock down in vitro.
(A and B) OT-1 T cells stimulated by SIINFEKL peptide loaded on irradiated TC-1 cells were labeled with CFSE, and then co-cultured with RA190-treated MDSCs. (A) Representative flow cytometry of T cell proliferation as measured by CFSE dilution in unstimulated T cells, stimulated T cells, stimulated T cells co-cultured with MDSCs, and stimulated T cells co-cultured with RA190-treated MDSCs. (B) Bar graph depicting the percentage of proliferated OT-1 T cells. (C and D) OT-1 T cells stimulated by SIINFEKL peptide loaded on irradiated TC-1 cells were labeled with CFSE, and then co-cultured with Rpn13 knocked down MDSCs. (C) Representative flow cytometry of T cell proliferation measured by CFSE dilution in unstimulated T cells, stimulated T cells, stimulated T cells co-cultured with MDSCs infected with control shRNA, and stimulated T cells co-cultured with MDSCs infected with Rpn13 shRNA. (D) Bar graph showing the percentage of proliferated T cells in various treatment groups. Values are shown as mean ± SD (*P = 0.05, **P = 0.01, ns, not significant).
Figure 5
Figure 5. Impact of RA190 treatment upon P-Stat3, arginase and iNOS expression levels in MDSCs, and T cell populations in the tumor microenvironment
C57BL/6 mice (n = 5/group) were injected with ID8-OVA-Luc tumor cells (2 × 106) intraperitoneally. Mice were treated with RA190 (10 mg/kg/day) beginning on day 11 for five consecutive days and again on day 18 for five consecutive days. (A–B) MDSCs were isolated from ascites at day23 and analyzed for P-Stat3, arginase, and iNOs expression by intracellular staining and flow cytometry. (A) Bar graph showing mean fluorescent intensity (MFI) of P-Stat3 in the tumor microenvironment of vehicle and RA190-treated groups. (B) Bar graph showing percentage of Arginase+ and iNOS+ MDSCs in the tumor microenvironment in various groups. (C–D) Ascites were collected from mice treated with vehicle or RA190 and flow cytometry was used to assess different T cell populations in the tumor microenvironment. (C) Bar graph showing the absolute number of CD8+IFNγ+ T cells in the tumor microenvironment. (D) Bar graph showing the absolute number of CD4+CD25+Foxp3+ T cells in the tumor microenvironment. Values are shown as mean ± SD (*P = 0.05, **P = 0.01, ns, not significant).
Figure 6
Figure 6. Role of CD8+ T cells in the antitumor effect of RA190 treatment
Groups of ID8-OVA-Luc tumor-bearing mice (n = 5) were treated with vehicle or RA190 according to the similar schedule described in Figure 5. To deplete CD8+ T cells, a group of mice (n = 5) treated with RA190 were also injected intraperitoneally with anti-CD8 monoclonal antibody (100 μg/mouse) on days 11, 12 and 13 after tumor challenge and every other week subsequently. (A) Line graph showing tumor growth as measured by bioluminescence intensity in various treatment groups. (B) Kaplan–Meier survival analysis of mice in various treatment groups. Values are shown as mean ± SD (*P = 0.05, **P = 0.01, ns, not significant).
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