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Cited by 195Protein Tyrosine PhosphatasesCancer Immunotherapy and BiomarkersImmunotherapy and Immune Responses

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SHP2 inhibition triggers anti-tumor immunity and synergizes with PD-1 blockade

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Zhao, Mingxia, Guo, Wenjie, Wu, Yuanyuan, Yang, Chenxi, Zhong, Liang, Deng, Guoliang, Zhu, Yuyu, Liu, Wen, Gu, Yanhong, Lu, Yin, Kong, Lingdong, Meng, Xiangbao, Xu, Qiang, Sun, Yang·2019·Acta Pharmaceutica Sinica B
DOI 10.1016/j.apsb.2018.08.009PMC PMC6437555
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Abstract

Tyrosine phosphatase SHP2 is a promising drug target in cancer immunotherapy due to its bidirectional role in both tumor growth promotion and T-cell inactivation. Its allosteric inhibitor SHP099 is known to inhibit cancer cell growth both in vitro and in vivo. However, whether SHP099-mediated SHP2 inhibition retards tumor growth in vivo via anti-tumor immunity remains elusive. To address this, a CT-26 colon cancer xenograft model was established in mice since this cell line is insensitive to SHP099. Consequently, SHP099 minimally affected CT-26 tumor growth in immuno-deficient nude mice, but significantly decreased the tumor burden in CT-26 tumor-bearing mice with intact immune system. SHP099 augmented anti-tumor immunity, as shown by the elevated proportion of CD8+IFN-γ+ T cells and the upregulation of cytotoxic T-cell related genes including Granzyme B andPerforin, which decreased the tumor load. In addition, tumor growth in mice with SHP2-deficient T-cells was markedly slowed down because of enhanced anti-tumor responses. Finally, the combination of SHP099 and anti-PD-1 antibody showed a higher therapeutic efficacy than either monotherapy in controlling tumor growth in two colon cancer xenograft models, indicating that these agents complement each other. Our study suggests that SHP2 inhibitor SHP099 is a promising candidate drug for cancer immunotherapy.

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Graphical abstract Graphical abstract The SHP2 allosteric inhibitor, SHP099, is a promising drug candidate for cancer immunotherapy. SHP2 inhibition both enhances and normalizes anti-tumor immunity, and the combination of SHP099 and anti-PD-1 is a potentially robust therapeutic strategy for cancer control.
Graphical abstract Graphical abstract The SHP2 allosteric inhibitor, SHP099, is a promising drug candidate for cancer immunotherapy. SHP2 inhibition both enhances and normalizes anti-tumor immunity, and the combination of SHP099 and anti-PD-1 is a potentially robust therapeutic strategy for cancer control.CC BY-NC-ND · PubMed Central
Figure 1 SHP2 inhibition decreases tumor load in CT-26 xenograft BALB/c mice. The mice were subcutaneously injected with 1 × 106 CT-26 colon cancer cells, and treated with 5-Fu (25 mg/kg i.p. every other day) and SHP099 or PHPS1 (5 mg/kg i.p. every day) from day 3 onwards. (A) Representative images. (B) Body weight of the mice. (C) Mean tumor weights of different groups. (D) Mean tumor volumes of different groups. (E) Tumor volumes of individual mice. Data are presented as mean ± SEM of 6 mice per group. *P < 0.05, **P < 0.01 vs. PBS.
Figure 1 SHP2 inhibition decreases tumor load in CT-26 xenograft BALB/c mice. The mice were subcutaneously injected with 1 × 106 CT-26 colon cancer cells, and treated with 5-Fu (25 mg/kg i.p. every other day) and SHP099 or PHPS1 (5 mg/kg i.p. every day) from day 3 onwards. (A) Representative images. (B) Body weight of the mice. (C) Mean tumor weights of different groups. (D) Mean tumor volumes of different groups. (E) Tumor volumes of individual mice. Data are presented as mean ± SEM of 6 mice per group. *P < 0.05, **P < 0.01 vs. PBS.CC BY-NC-ND · PubMed Central
Figure 2 SHP2 inhibition promotes cytotoxic T-cell cytokine/granule production in CT-26 xenograft BALB/c mice. (A) H & E staining of tumor sections. (B) TUNEL assay for apoptotic cells in tumor tissue. (C) IHC for detecting IFN-γ and GZMB in the tumor sections. (D) qPCR for Ifn-γ, Prf and GzmB expression in tumors. Data are presented as mean ± SEM of 6 mice per group. (E) and (F) Intracellular staining for IFN-γ and GZMB production in tumor infiltrating CD8+ T cells. Data are presented as mean ± SEM of 3 mice per group. *P < 0.05, **P < 0.01 vs. PBS. Prf, perforin; GzmB, granzyme B. Scale bar, 10 μm.
Figure 2 SHP2 inhibition promotes cytotoxic T-cell cytokine/granule production in CT-26 xenograft BALB/c mice. (A) H & E staining of tumor sections. (B) TUNEL assay for apoptotic cells in tumor tissue. (C) IHC for detecting IFN-γ and GZMB in the tumor sections. (D) qPCR for Ifn-γ, Prf and GzmB expression in tumors. Data are presented as mean ± SEM of 6 mice per group. (E) and (F) Intracellular staining for IFN-γ and GZMB production in tumor infiltrating CD8+ T cells. Data are presented as mean ± SEM of 3 mice per group. *P < 0.05, **P < 0.01 vs. PBS. Prf, perforin; GzmB, granzyme B. Scale bar, 10 μm.CC BY-NC-ND · PubMed Central
Figure 3 SHP2 inhibition has no effect on tumor size in CT-26 xenograft BALB/c nude mice. The mice were subcutaneously inoculated with 1×106 CT-26 colon cancer cells, and treated with 5-Fu (25 mg/kg i.p. every other day) and SHP099 (5 mg/kg i.p. every day) from day 3 onwards. (A) Representative images. (B) Mean tumor volumes of different groups. (C) Mean tumor weights of different groups. (D) Tumor volumes of individual mice. (E) Mean body weight of mice. Data in (B), (C), and (E) are presented as mean ± SEM of 8 mice per group. (F) H & E staining of tumor sections. (G) TUNEL assay for apoptotic cells in tumor tissue. (H) IHC for PCNA in tumor sections. (I) MTT viability assay for CT-26 colon cancer cells. Data are presented as mean ± SEM of three different experiments. *P < 0.05, **P < 0.01 vs. PBS. NS represents no significance vs. SHP099 (0 μmol/L). Scale bar, 10 μm.
Figure 3 SHP2 inhibition has no effect on tumor size in CT-26 xenograft BALB/c nude mice. The mice were subcutaneously inoculated with 1×106 CT-26 colon cancer cells, and treated with 5-Fu (25 mg/kg i.p. every other day) and SHP099 (5 mg/kg i.p. every day) from day 3 onwards. (A) Representative images. (B) Mean tumor volumes of different groups. (C) Mean tumor weights of different groups. (D) Tumor volumes of individual mice. (E) Mean body weight of mice. Data in (B), (C), and (E) are presented as mean ± SEM of 8 mice per group. (F) H & E staining of tumor sections. (G) TUNEL assay for apoptotic cells in tumor tissue. (H) IHC for PCNA in tumor sections. (I) MTT viability assay for CT-26 colon cancer cells. Data are presented as mean ± SEM of three different experiments. *P < 0.05, **P < 0.01 vs. PBS. NS represents no significance vs. SHP099 (0 μmol/L). Scale bar, 10 μm.CC BY-NC-ND · PubMed Central
Figure 4 SHP2 deficiency in T-cells augments anti-tumor responses. T-cell specific SHP2 knockout mice (Ptpn11Cd4–/–) were subcutaneously injected with 1 × 106 MC-38 colon cancer cells. (A) Representative images. (B) Mean tumor volumes of different groups. (C) Tumor volumes of individual mice. (D) Mean tumor weights of different groups. (E) qPCR assay for Ifn-γ, GzmB and Prf mRNA expression in the tumors. Data in (B), (D), and (E) are presented as mean ± SEM of 8 mice per group. (F) H & E staining of tumor sections. (G) TUNEL assay for apoptotic cells in tumor tissue. (H) IHC for IFN-γ and GZMB in tumor sections. (I) Intracellular staining for cytotoxic T-cell related cytokine/granule production in the tumor infiltrating CD8+ T cells. Data are presented as mean ± SEM of 3 mice per group. *P < 0.05, **P < 0.01 vs. WT. Prf, perforin; GzmB, granzyme B. Scale bar, 10 μm.
Figure 4 SHP2 deficiency in T-cells augments anti-tumor responses. T-cell specific SHP2 knockout mice (Ptpn11Cd4–/–) were subcutaneously injected with 1 × 106 MC-38 colon cancer cells. (A) Representative images. (B) Mean tumor volumes of different groups. (C) Tumor volumes of individual mice. (D) Mean tumor weights of different groups. (E) qPCR assay for Ifn-γ, GzmB and Prf mRNA expression in the tumors. Data in (B), (D), and (E) are presented as mean ± SEM of 8 mice per group. (F) H & E staining of tumor sections. (G) TUNEL assay for apoptotic cells in tumor tissue. (H) IHC for IFN-γ and GZMB in tumor sections. (I) Intracellular staining for cytotoxic T-cell related cytokine/granule production in the tumor infiltrating CD8+ T cells. Data are presented as mean ± SEM of 3 mice per group. *P < 0.05, **P < 0.01 vs. WT. Prf, perforin; GzmB, granzyme B. Scale bar, 10 μm.CC BY-NC-ND · PubMed Central
Figure 5 SHP2 inhibition synergizes with PD-1 blockade in MC-38 xenograft mice. The mice were subcutaneously inoculated with 1×106 MC-38 colon cancer cells, and treated with anti-PD-1 (5 mg/kg i.p. once every three days) and/or SHP099 (5 mg/kg i.p. every day) from day 3 onwards. All results presented are of day 13 post-inoculation. (A) Representative images of tumors. (B) Mean tumor volumes of different groups. (C) Mean tumor weights of different groups. (D) Tumor volume of individual mice. (E) Mean body weight of mice. (F) H & E staining of tumor sections. (G) TUNEL assay for apoptotic cells in tumor sections. (H) qPCR assay for the Ifn-γ, GzmB, Prf and FasL mRNA levels in tumor tissues. Data in (B), (C), (E), and (H) are presented as mean ± SEM of 6 mice per group. (I) IHC for IFN-γ and GZMB in tumor sections. (J) Intracellular staining for IFN-γ production in tumor infiltrating CD8+ T cells. Data in (J) are presented as mean ± SEM of 3 mice per group. (K) MTT viability assay for MC-38 colon cancer cells. Data are presented as mean ± SEM of three different experiments. *P < 0.05, **P < 0.01 vs. PBS. †P < 0.05 vs. SHP099+anti-PD-1. Prf, perforin; GzmB, granzyme B. Scale bar, 10 μm.
Figure 5 SHP2 inhibition synergizes with PD-1 blockade in MC-38 xenograft mice. The mice were subcutaneously inoculated with 1×106 MC-38 colon cancer cells, and treated with anti-PD-1 (5 mg/kg i.p. once every three days) and/or SHP099 (5 mg/kg i.p. every day) from day 3 onwards. All results presented are of day 13 post-inoculation. (A) Representative images of tumors. (B) Mean tumor volumes of different groups. (C) Mean tumor weights of different groups. (D) Tumor volume of individual mice. (E) Mean body weight of mice. (F) H & E staining of tumor sections. (G) TUNEL assay for apoptotic cells in tumor sections. (H) qPCR assay for the Ifn-γ, GzmB, Prf and FasL mRNA levels in tumor tissues. Data in (B), (C), (E), and (H) are presented as mean ± SEM of 6 mice per group. (I) IHC for IFN-γ and GZMB in tumor sections. (J) Intracellular staining for IFN-γ production in tumor infiltrating CD8+ T cells. Data in (J) are presented as mean ± SEM of 3 mice per group. (K) MTT viability assay for MC-38 colon cancer cells. Data are presented as mean ± SEM of three different experiments. *P < 0.05, **P < 0.01 vs. PBS. †P < 0.05 vs. SHP099+anti-PD-1. Prf, perforin; GzmB, granzyme B. Scale bar, 10 μm.CC BY-NC-ND · PubMed Central
Figure 6 SHP2 inhibition enhances the activation of CD8+ cytotoxic T lymphocytes in vitro. (A)–(C) Murine splenic CD8+ T cells were incubated with various concentrations of SHP099 for 24 h in the presence of plate-bound 0.5 μg/mL murine anti-CD3 and 0.5 μg/mL murine anti-CD28. (A) and (B) Flow cytomytry assay for detecting GZMB and PRF in CD8+ T cells. (C) qPCR assay for mRNA expressions of Ifn-γ, Prf, GzmB, FasL and Tnf-α in CD8+ T cells. (D) Human peripheral blood mononuclear cells (PBMCs) were incubated with 10 μmol/L SHP099 for 24 h in the presence of plate-bound 0.25 μg/mL human anti-CD3 and 1 μg/mL human anti-CD28. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. (E) MTT assay for murine splenic CD8+ T cell viability. (F)–(I) Murine splenic cells were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA expressions of Ifn-γ, Prf, Tnf-α and FasL were measured by qPCR assay. (J) Human PBMCs were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. Data are presented as mean ± SEM of three different experiments. *P < 0.05 vs. Medium. †P < 0.05 vs. anti-CD3/CD28 or Con A. NS represents no significance. Prf, perforin; GzmB, granzyme B.
Figure 6 SHP2 inhibition enhances the activation of CD8+ cytotoxic T lymphocytes in vitro. (A)–(C) Murine splenic CD8+ T cells were incubated with various concentrations of SHP099 for 24 h in the presence of plate-bound 0.5 μg/mL murine anti-CD3 and 0.5 μg/mL murine anti-CD28. (A) and (B) Flow cytomytry assay for detecting GZMB and PRF in CD8+ T cells. (C) qPCR assay for mRNA expressions of Ifn-γ, Prf, GzmB, FasL and Tnf-α in CD8+ T cells. (D) Human peripheral blood mononuclear cells (PBMCs) were incubated with 10 μmol/L SHP099 for 24 h in the presence of plate-bound 0.25 μg/mL human anti-CD3 and 1 μg/mL human anti-CD28. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. (E) MTT assay for murine splenic CD8+ T cell viability. (F)–(I) Murine splenic cells were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA expressions of Ifn-γ, Prf, Tnf-α and FasL were measured by qPCR assay. (J) Human PBMCs were incubated with 10 μmol/L SHP099 for 24 h in the presence of 10 μg/mL Con A. Relative mRNA levels of Ifn-γ, GzmB and Prf were measured by qPCR assay. Data are presented as mean ± SEM of three different experiments. *P < 0.05 vs. Medium. †P < 0.05 vs. anti-CD3/CD28 or Con A. NS represents no significance. Prf, perforin; GzmB, granzyme B.CC BY-NC-ND · PubMed Central
Figure 7 The graphic illustration of the mechanism of SHP2 inhibition by SHP099 in triggering anti-tumor immunity. On one hand, SHP2 inhibition by its allosteric inhibitor SHP099 reverses the inhibitory effect on T cells triggered by PD-L1-expressing cancer cells, which is called “normalization” of anti-tumor immunity (Green). On the other hand, SHP2 inhibition also contributes to T cell activation, which is called “enhancement” of anti-tumor immunity (Red). Both “normalization” and “enhancement” of anti-tumor immunity by SHP2 inhibition are responsible for anti-tumor effect of SHP099 in vivo.
Figure 7 The graphic illustration of the mechanism of SHP2 inhibition by SHP099 in triggering anti-tumor immunity. On one hand, SHP2 inhibition by its allosteric inhibitor SHP099 reverses the inhibitory effect on T cells triggered by PD-L1-expressing cancer cells, which is called “normalization” of anti-tumor immunity (Green). On the other hand, SHP2 inhibition also contributes to T cell activation, which is called “enhancement” of anti-tumor immunity (Red). Both “normalization” and “enhancement” of anti-tumor immunity by SHP2 inhibition are responsible for anti-tumor effect of SHP099 in vivo.CC BY-NC-ND · PubMed Central

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Authors
Zhao, M., Guo, W., Wu, Y., Yang, C., Zhong, L., Deng, G., Zhu, Y., Liu, W., Gu, Y., Lu, Y., Kong, L., Meng, X., Xu, Q., & Sun, Y.
Affiliations
  • State Key Laboratory of Pharmaceutical Biotechnology (CN)
  • Nanjing University of Chinese Medicine (CN)
  • Peking University (CN)
  • Jiangsu Province Hospital (CN)
Corresponding author
  • Meng, Xiangbao
  • Xu, Qiang
  • Sun, Yang
ORCID
Venue
Acta Pharmaceutica Sinica B
Publisher
Elsevier BV
Published
2019-03
Volume
9
Issue
2
Pages
304-315
ISSN
2211-3835 (ISSN-L)
DOI
10.1016/j.apsb.2018.08.009
PMC
PMC6437555
OpenAlex
W2891517856
Cited by
190
References
38
Citations by year
2026: 15 · 2025: 25 · 2024: 25 · 2023: 18 · 2022: 37 · 2021: 27 · 2020: 33 · 2019: 10
FWCI
6.23
Type
journal article
Language
en
Update notices
erratum (2025-05-01)
Open access
Licence
Locations
  • Acta Pharmaceutica Sinica B (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (81673436, 81673437, 21472091, 81872877)
  • Nanjing University
  • Priority Academic Program Development of Jiangsu Higher Education Institutions
  • State Key Laboratory of Pharmaceutical Biotechnology (KF-GN-201703)
  • Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica (JKLPSE201802)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
SHP2, SHP099, protein tyrosine phosphatase inhibition, antitumor immunity, anti-PD-1 therapy, combination immunotherapy, colon cancer, CD8+ T cells, cytotoxic T lymphocytes, T cell exhaustion, allosteric inhibitors

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