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Cited by 105Cancer Immunotherapy and BiomarkersProtein Tyrosine PhosphatasesImmune cells in cancer

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Combined Inhibition of SHP2 and CXCR1/2 Promotes Antitumor T-cell Response in NSCLC

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Tang, Kwan Ho, Li, Shuai, Khodadadi-Jamayran, Alireza, Jen, Jayu, Han, Han, Guidry, Kayla, Chen, Ting, Hao, Yuan, Fedele, Carmine, Zebala, John A., Maeda, Dean Y., Christensen, James G., Olson, Peter, Athanas, Argus, Loomis, Cynthia A., Tsirigos, Aristotelis, Wong, Kwok-Kin, Neel, Benjamin G.·2022·Cancer Discovery
DOI 10.1158/2159-8290.cd-21-0369PMC PMC8758507

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Abstract

SHP2 inhibitors (SHP2i) alone and in various combinations are being tested in multiple tumors with overactivation of the RAS/ERK pathway. SHP2 plays critical roles in normal cell signaling; hence, SHP2is could influence the tumor microenvironment. We found that SHP2i treatment depleted alveolar and M2-like macrophages, induced tumor-intrinsic CCL5/CXCL10 secretion, and promoted B and T lymphocyte infiltration in Kras- and Egfr-mutant non–small cell lung cancer (NSCLC). However, treatment also increased intratumor granulocytic myeloid-derived suppressor cells (gMDSC) via tumor-intrinsic, NFκB-dependent production of CXCR2 ligands. Other RAS/ERK pathway inhibitors also induced CXCR2 ligands and gMDSC influx in mice, and CXCR2 ligands were induced in tumors from patients on KRASG12C inhibitor trials. Combined SHP2 (SHP099)/CXCR1/2 (SX682) inhibition depleted a specific cluster of S100a8/9hi gMDSCs, generated Klrg1+ CD8+ effector T cells with a strong cytotoxic phenotype but expressing the checkpoint receptor NKG2A, and enhanced survival in Kras- and Egfr-mutant models. Our results argue for testing RAS/ERK pathway/CXCR1/2/NKG2A inhibitor combinations in patients with NSCLC. Significance: Our study shows that inhibiting the SHP2/RAS/ERK pathway triggers NFκB-dependent upregulation of CXCR2 ligands and recruitment of S100A8hi gMDSCs, which suppress T cells. Combining SHP2/CXCR2 inhibitors blocks gMDSC immigration, resulting in enhanced Th1 polarization, induced CD8+KLRG1+ effector T cells with high cytotoxic activity, and improved survival in multiple NSCLC models. This article is highlighted in the In This Issue feature, p. 1

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Figure 1. SHP099 treatment promotes T lymphocyte infiltration but not activation. A, KP NSCLC cells were injected intravenously into C57BL/6 mice. When tumors reached 100 mm3 (assessed by MRI), treatment was initiated with SHP099 (75 mg/kg) or vehicle daily by oral gavage. B, KP allografts were treated with vehicle or SHP099 for 11 days, and immune profiling was performed on tumor nodules. C, Formalin-fixed, paraffin-embedded (FFPE) sections of representative tumor nodules from mice treated as indicated were stained with anti-CD3 antibody and visualized with fluorophore-conjugated secondary antibody. D, Immune profiling of tumor nodules from vehicle- or SHP099-treated mice after 11 days of treatment. E, CD4+ or CD8+ T cells were depleted using rat-anti-mouse antibodies (clones GK1.5 and 2.43, respectively), and tumor growth with/without SHP099 treatment for 7 days was assessed by MRI. F, CD4+ or CD8+ T-cell depletion shortens overall survival of tumor-bearing mice, with/without SHP099 treatment. G, Phenotypic characterization of infiltrating CD8+ and CD4+ T-cell subsets from vehicle- or SHP099-treated mice. H, Scheme showing gMDSC depletion experiments. I, Depletion of gMDSCs promotes greater T-cell infiltration, when combined with SHP099 treatment. J, Effect of gMDSC depletion on phenotypes of infiltrating T lymphocytes. A–B, D–E, Student t test. F, Log-rank test. I–J, One-way ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Figure 1. SHP099 treatment promotes T lymphocyte infiltration but not activation. A, KP NSCLC cells were injected intravenously into C57BL/6 mice. When tumors reached 100 mm3 (assessed by MRI), treatment was initiated with SHP099 (75 mg/kg) or vehicle daily by oral gavage. B, KP allografts were treated with vehicle or SHP099 for 11 days, and immune profiling was performed on tumor nodules. C, Formalin-fixed, paraffin-embedded (FFPE) sections of representative tumor nodules from mice treated as indicated were stained with anti-CD3 antibody and visualized with fluorophore-conjugated secondary antibody. D, Immune profiling of tumor nodules from vehicle- or SHP099-treated mice after 11 days of treatment. E, CD4+ or CD8+ T cells were depleted using rat-anti-mouse antibodies (clones GK1.5 and 2.43, respectively), and tumor growth with/without SHP099 treatment for 7 days was assessed by MRI. F, CD4+ or CD8+ T-cell depletion shortens overall survival of tumor-bearing mice, with/without SHP099 treatment. G, Phenotypic characterization of infiltrating CD8+ and CD4+ T-cell subsets from vehicle- or SHP099-treated mice. H, Scheme showing gMDSC depletion experiments. I, Depletion of gMDSCs promotes greater T-cell infiltration, when combined with SHP099 treatment. J, Effect of gMDSC depletion on phenotypes of infiltrating T lymphocytes. A–B, D–E, Student t test. F, Log-rank test. I–J, One-way ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.CC BY-NC-ND · PubMed Central
Figure 2. SHP2 inhibition results in NFκB-evoked Cxcl1/Cxcl5 upregulation. A, RNA-seq of KP tumors identifies Cxcl1 and Cxcl5 as the most upregulated chemokines after SHP099 treatment. B, Expressing SHP099-resistant mutant (PTPN11TM/QL) resistant in KP cells prevents SHP099-mediated ERK inhibition. C, SHP099 treatment significantly induces Cxcl1/5 expression in WT but not SHP099-resistant mutant KP cells. D, Tumor cell–dependent and –independent effects of SHP099 on immune cells of the tumor microenvironment. E, Promoter–enrichment analysis by Enrichr (GhEA 2016) for Cxcl1 and Cxcl5 suggests RELA is active and binds to Cxcl1 and Cxcl5 promoters. F, SHP2 or MEK inhibition increases NFκB transcriptional activity. An NFκB-GFP reporter was transduced stably into KP cells, and GFP fluorescence was assessed after 72-hour treatment with SHP099 (10 μmol/L), trametinib (25 nmol/L), or DMSO control. G, Immunoblots showing activation of NFκB signaling on KP cells treated with 10 μmol/L SHP099. H, The IKK inhibitor ML120B (10 μmol/L) prevents Cxcl1 and Cxcl5 upregulation induced by SHP099 or trametinib in KP cells (72 hours). I, Luminex analysis of supernatants from KP cells treated with SHP099 (10 μmol/L), trametinib (25 nmol/L), or DMSO control. J, Luminex analysis of lysates prepared from tumor nodules harvested from KP allograft–bearing mice treated as indicated. K, FFPE sections of representative WT and SHP099-resistant mutant tumor nodules from mice treated as indicated were stained with anti-p65 and anti-CXCL1 antibodies and visualized with fluorophore-conjugated secondary antibody. Red arrows indicate cells with nuclear p65. C–D, H, J, Student t test.*, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2. SHP2 inhibition results in NFκB-evoked Cxcl1/Cxcl5 upregulation. A, RNA-seq of KP tumors identifies Cxcl1 and Cxcl5 as the most upregulated chemokines after SHP099 treatment. B, Expressing SHP099-resistant mutant (PTPN11TM/QL) resistant in KP cells prevents SHP099-mediated ERK inhibition. C, SHP099 treatment significantly induces Cxcl1/5 expression in WT but not SHP099-resistant mutant KP cells. D, Tumor cell–dependent and –independent effects of SHP099 on immune cells of the tumor microenvironment. E, Promoter–enrichment analysis by Enrichr (GhEA 2016) for Cxcl1 and Cxcl5 suggests RELA is active and binds to Cxcl1 and Cxcl5 promoters. F, SHP2 or MEK inhibition increases NFκB transcriptional activity. An NFκB-GFP reporter was transduced stably into KP cells, and GFP fluorescence was assessed after 72-hour treatment with SHP099 (10 μmol/L), trametinib (25 nmol/L), or DMSO control. G, Immunoblots showing activation of NFκB signaling on KP cells treated with 10 μmol/L SHP099. H, The IKK inhibitor ML120B (10 μmol/L) prevents Cxcl1 and Cxcl5 upregulation induced by SHP099 or trametinib in KP cells (72 hours). I, Luminex analysis of supernatants from KP cells treated with SHP099 (10 μmol/L), trametinib (25 nmol/L), or DMSO control. J, Luminex analysis of lysates prepared from tumor nodules harvested from KP allograft–bearing mice treated as indicated. K, FFPE sections of representative WT and SHP099-resistant mutant tumor nodules from mice treated as indicated were stained with anti-p65 and anti-CXCL1 antibodies and visualized with fluorophore-conjugated secondary antibody. Red arrows indicate cells with nuclear p65. C–D, H, J, Student t test.*, P < 0.05; **, P < 0.01; ***, P < 0.001.CC BY-NC-ND · PubMed Central
Figure 3. Combined inhibition of SHP2 and CXCR2 promotes antitumor T-cell response. A, SX682 significantly suppresses gMDSC infiltration induced by SHP099 and further increases T-cell infiltration and proliferation, whereas SX682/SHP099 combination enhances CD8+ and CD4+ T-cell activation and induces CD8 CTLs and Th1 polarization of CD4 T cells. B, SX682/SHP099 effectively blocks KP tumor growth at 2 weeks of treatment (as measured by MRI). C, SX682/SHP099 prolongs overall survival compared with vehicle- or single-agent-treated mice. D, scRNA-seq was performed on cells isolated from treated tumors (n = 3 per treatment group) and analyzed by t-SNE or KNetL map. Note the additional clusters revealed by the latter. DC, dendritic cells; NK, natural killer. E,Cxcl1 and Cxcl5 expression are largely restricted to tumor cells and CAFs. F and G, Identification of six clusters of S100a8/9+ gMDSC with distinct transcriptional profiles. H, SHP099/SX682 specifically depletes cells in cluster N4, which is also induced by SHP099 treatment. I and J, Identification of 9 clusters of Cd3e+ T cells with distinct transcriptional profiles. K, SHP099/SX682 specifically induces cells in cluster T2 and T5. A and B, One-way ANOVA. C, Log-rank test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Figure 3. Combined inhibition of SHP2 and CXCR2 promotes antitumor T-cell response. A, SX682 significantly suppresses gMDSC infiltration induced by SHP099 and further increases T-cell infiltration and proliferation, whereas SX682/SHP099 combination enhances CD8+ and CD4+ T-cell activation and induces CD8 CTLs and Th1 polarization of CD4 T cells. B, SX682/SHP099 effectively blocks KP tumor growth at 2 weeks of treatment (as measured by MRI). C, SX682/SHP099 prolongs overall survival compared with vehicle- or single-agent-treated mice. D, scRNA-seq was performed on cells isolated from treated tumors (n = 3 per treatment group) and analyzed by t-SNE or KNetL map. Note the additional clusters revealed by the latter. DC, dendritic cells; NK, natural killer. E,Cxcl1 and Cxcl5 expression are largely restricted to tumor cells and CAFs. F and G, Identification of six clusters of S100a8/9+ gMDSC with distinct transcriptional profiles. H, SHP099/SX682 specifically depletes cells in cluster N4, which is also induced by SHP099 treatment. I and J, Identification of 9 clusters of Cd3e+ T cells with distinct transcriptional profiles. K, SHP099/SX682 specifically induces cells in cluster T2 and T5. A and B, One-way ANOVA. C, Log-rank test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.CC BY-NC-ND · PubMed Central
Figure 4. Combined inhibition of SHP2 and CXCR2 is also efficacious in EGFR-mutant NSCLC. A, SHP099 (10 μmol/L, 1 hour) inhibits MEK/ERK signaling in EGFR-mutant NSCLC cell lines. B, SHP099 (10 μmol/L, 3 days) suppresses proliferation of EGFR-mutant NSCLC cell lines. C, SHP099 (10 μmol/L), trametinib (25 nmol/L), or osimertinib (250 nmol/L) treatment induces GRO family chemokine expression in H1975 cells (72 hours treatment), and induction is blocked by IKK inhibitor ML120b. D, Schematic of osimertinib-resistant GEMM (EGFR-TLC). E and F, Effects of SHP099, SX682, and SHP099/SX682 on the indicated immune cell populations in EGFR-TLC tumors. G and H, SHP099/SX682 significantly enhances antitumor efficacy compared with either single agent, quantified after 4 weeks of treatment by MRI (G) and with representative image shown in H. I, Schematic of osimertinib-sensitive GEMM (EGFR-TL). J, Osimertinib/SHP099/SX682 (5, 75, and 100 mg/kg daily) induces significantly stronger T-cell response in EGFR-TL GEMM, compared with single-agent osimertinib (5 mg/kg qd) or osimertinib/SHP099 (5 and 75 mg/kg qd). K, Osimertinib/SHP099/SX682 delays disease relapse following drug withdrawal in EGFR-TL mice, compared with mice treated with osimertinib alone or osimertinib/SHP099 (doses as above). L,CXCL1 and CXCL6 expression (by RNA-seq) in paired specimens from patients with NSCLC before and after MRTX849 treatment; RECIST response in each patient is indicated. M, Correlation of CXCL1 and CXCL6 induction and duration of response in biopsies from MRTX849-treated patients. N, MCP-Counter analysis of RNA-seq data reveals increased neutrophil signature in all MRTX849-treated tumors. O, scRNA-seq performed on cells isolated from fresh tumor biopsies from patients with NSCLC with/without G12Ci treatment. P, Increased expression of CXCL1 and CXCL6 in tumor cells from G12Ci-treated patients with NSCLC, compared with those from G12Ci-naïve patients. CPM, counts per million.Q, Significantly higher proportions of neutrophils were observed in tumors from G12Ci-treated patients, compared with those not receiving G12Ci.B, C, K, Q, Student t test. E–G and J, one-way ANOVA. *, P < 0.05; *, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Figure 4. Combined inhibition of SHP2 and CXCR2 is also efficacious in EGFR-mutant NSCLC. A, SHP099 (10 μmol/L, 1 hour) inhibits MEK/ERK signaling in EGFR-mutant NSCLC cell lines. B, SHP099 (10 μmol/L, 3 days) suppresses proliferation of EGFR-mutant NSCLC cell lines. C, SHP099 (10 μmol/L), trametinib (25 nmol/L), or osimertinib (250 nmol/L) treatment induces GRO family chemokine expression in H1975 cells (72 hours treatment), and induction is blocked by IKK inhibitor ML120b. D, Schematic of osimertinib-resistant GEMM (EGFR-TLC). E and F, Effects of SHP099, SX682, and SHP099/SX682 on the indicated immune cell populations in EGFR-TLC tumors. G and H, SHP099/SX682 significantly enhances antitumor efficacy compared with either single agent, quantified after 4 weeks of treatment by MRI (G) and with representative image shown in H. I, Schematic of osimertinib-sensitive GEMM (EGFR-TL). J, Osimertinib/SHP099/SX682 (5, 75, and 100 mg/kg daily) induces significantly stronger T-cell response in EGFR-TL GEMM, compared with single-agent osimertinib (5 mg/kg qd) or osimertinib/SHP099 (5 and 75 mg/kg qd). K, Osimertinib/SHP099/SX682 delays disease relapse following drug withdrawal in EGFR-TL mice, compared with mice treated with osimertinib alone or osimertinib/SHP099 (doses as above). L,CXCL1 and CXCL6 expression (by RNA-seq) in paired specimens from patients with NSCLC before and after MRTX849 treatment; RECIST response in each patient is indicated. M, Correlation of CXCL1 and CXCL6 induction and duration of response in biopsies from MRTX849-treated patients. N, MCP-Counter analysis of RNA-seq data reveals increased neutrophil signature in all MRTX849-treated tumors. O, scRNA-seq performed on cells isolated from fresh tumor biopsies from patients with NSCLC with/without G12Ci treatment. P, Increased expression of CXCL1 and CXCL6 in tumor cells from G12Ci-treated patients with NSCLC, compared with those from G12Ci-naïve patients. CPM, counts per million.Q, Significantly higher proportions of neutrophils were observed in tumors from G12Ci-treated patients, compared with those not receiving G12Ci.B, C, K, Q, Student t test. E–G and J, one-way ANOVA. *, P < 0.05; *, P < 0.01; ***, P < 0.001; ****, P < 0.0001.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Tang, K. H., Li, S., Khodadadi-Jamayran, A., Jen, J., Han, H., Guidry, K., Chen, T., Hao, Y., Fedele, C., Zebala, J. A., Maeda, D. Y., Christensen, J. G., Olson, P., Athanas, A., Loomis, C. A., Tsirigos, A., Wong, K.-K., & Neel, B. G.
Affiliations
  • NYU Langone Health (US)
  • 2Applied Bioinformatics Laboratories, Office of Science and Research, New York University Grossman School of Medicine, New York, New York.
  • New York University (US)
  • 3Syntrix Pharmaceuticals, Auburn, Washington.
  • Mirati Therapeutics (United States) (US)
  • 5Monoceros Biosystems, Inc., San Diego, California.
Corresponding author
  • Tang, Kwan Ho
  • Wong, Kwok-Kin
  • Neel, Benjamin G.
ORCID
Venue
Cancer Discovery
Publisher
American Association for Cancer Research (AACR)
Published
2021-08-05
Volume
12
Issue
1
Pages
47-61
ISSN
  • 2159-8274 (ISSN-L)
  • 2159-8290
DOI
10.1158/2159-8290.cd-21-0369
PMC
PMC8758507
OpenAlex
W3191958191
Cited by
105
References
74
Citations by year
2026: 11 · 2025: 25 · 2024: 21 · 2023: 28 · 2022: 18 · 2021: 2
FWCI
6.28
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancer Discovery (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
National Institutes of Health (CA49152, CA248896, CA016087)
Topics
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
Keywords
SHP2 inhibition, non-small cell lung cancer, Ras/ERK pathway, M2 macrophages, granulocytic myeloid-derived suppressor cells, S100A8/S100A9, NKG2A, NF-κB signaling, KRAS mutation, EGFR mutation, combination therapy, antitumor T-cell responses, Th1 polarization
MeSH terms
Animals, Antineoplastic Agents, Carcinoma, Non-Small-Cell Lung, Enzyme Inhibitors, Humans, Lung Neoplasms, Male, Mice, Inbred C57BL, Xenograft Model Antitumor Assays, Receptors, Interleukin-8B, Cell Line, Tumor, Mice, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Tumor Microenvironment

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