Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IkB kinase e (IKKe) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKe substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKe activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNb. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKe in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic.Significance: These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.
Neisseria gonorrhoeae, the human obligate pathogen responsible for the sexually transmitted disease gonorrhea, has evolved several mechanisms to evade the host immune response. One such mechanism is the modulation of host cell death pathways. In this study, we defined cell death pathways induced by N gonorrhoeae in human monocyte-derived macrophages (MDMs). In a dose-dependent manner, N gonorrhoeae stimulation of MDMs resulted in caspase 1 and 4–dependent cell deaths, indicative of canonical and noncanonical pyroptosis, respectively. Internalization of bacteria or stimulation with lipooligosaccharide (LOS) specifically induced pyroptosis in MDMs and increased secretion of IL-1β. Collectively, our results demonstrate that N gonorrhoeae induces inflammatory pyroptosis in human macrophages due in part to intracellular LOS. We propose that this in turn may exacerbate inflammatory outcomes observed during mucosal infection.
BackgroundVSIG4 (V-set immunoglobulin-domain-containing 4) is a B7 family related protein with known roles as a complement receptor involved in pathogen clearance as well as a negative regulator of T cell activation by an undetermined mechanism.1–3 VSIG4 is expressed in tumor associated macrophages (TAMs) with exquisite specificity. In cancer, increased expression of VSIG4 has been associated with worse survival in multiple indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, and glioma, suggesting an important role in tumor immune evasion.3–6 Based upon computational analysis of transcript data across thousands of primary cancer and normal tissue samples, we hypothesized that VSIG4 has an important regulatory role in promoting M2-like immune suppressive macrophages in the tumor microenvironment, and that targeting VSIG4 via a monoclonal antibody could relieve VSIG4-mediated macrophage suppression by repolarizing TAMs to an inflammatory phenotype capable of coordinating an anti-tumor immune response.MethodsThe ability of anti-VSIG4 antibodies to repolarize M2-like macrophages and induce T cell activation was assessed in vitro and ex vivo, by measuring production of inflammatory mediators. In vitro assays were performed primarily with M-CSF plus IL-10 driven monocyte-derived M2c macrophages from healthy donors. Ex vivo assays were performed with fresh, patient-derived tumor samples in culture. To determine whether targeting VSIG4 can lead to an anti-tumor effect in vivo, syngeneic mouse models were dosed with anti-mouse VSIG4 antibodies and characterized for changes in tumor volume and immune cell populations.ResultsIn in vitro and ex vivo assays anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Targeting VSIG4 upregulates pro-inflammatory cytokines in M2c macrophages, as well as upregulates pro-inflammatory myeloid-derived cytokines and T cell-derived cytokines in M2c macrophages co-cultured with autologous T cells in the presence of staphylococcal enterotoxin B (SEB) activation. To assess targeting VSIG4 in a relevant translational model, fresh, patient-derived tumor samples were treated ex vivo with anti-VSIG4. Across multiple tumor types, anti-VSIG4 treatment resulted in a significant upregulation of cytokines involved in TAM repolarization and T cell activation, and chemokines involved in immune cell recruitment, at levels greater than observed by treatment with anti-PD-1 or a clinical macrophage repolarizing agent (anti-ILT-4). In vivo, tumor growth inhibition is observed in syngeneic mouse models dosed with anti-mouse-VSIG4 alone and in combination with anti-PD-1.ConclusionsTaken together, these data suggest that VSIG4 represents a promising new target capable of stimulating an anti-cancer response via multiple key immune mechanisms.Referencesvan Lookeren Campagne M, Verschoor A. Pathogen clearance and immune adherence “revisited”: immuno-regulatory roles for CRIg. Semin Immunol 2018;37:4–11.Xu S, Sun Z, Li L, Liu J, He J, Song D, Shan G, Liu H, Wu X. Induction of T cells suppression by dendritic cells transfected with VSIG4 recombinant adenovirus. Immunol Lett 2010;128(1):46–50.Liao Y, Guo S, Chen Y, Cao D, Xu H, Yang C, Fei L, Ni B, Ruan Z. VSIG4 expression on macrophages facilitates lung cancer development. Lab Invest 2014;94(7):706–715.Roh J, Jeon Y, Lee A, Lee S, Kim Y, Sung C, Park C, Hong J, Yoon D, Suh C, Huh J, Choi I, Park C. The immune checkpoint molecule V-set Ig domain-containing 4 is an independent prognostic factor for multiple myeloma. Oncotarget 2017;8(35):58122–58132.Xu T, Jiang Y, Yan Y, Wang H, Lu C, Xu H, Li W, Fu D, Lu Y, Chen J. VSIG4 is highly expressed and correlated with poor prognosis of high-grade glioma patients. Am J Transl Res 2015;7(6):1172–1180.Byun J, Jeong D, Choi I, Lee D, Kang M, Jung K, Jeon Y, Kim Y, Jung E, Lee K, Sung M, Kim K. The significance of VSIG4 expression in ovarian cancer. Int J Gynecol Cancer 2017;27(5):872–878.Ethics ApprovalAll legal and ethical requirements were met with regards to the humane treatment of animals described in the study. The animal study was conducted in compliance with CRL IACUC under IACUC No. I033.
BackgroundSuppressive myeloid cell populations in the tumor microenvironment (TME) are associated with worse survival of cancer patients and low effectiveness of T cell checkpoint inhibitors. Recently, several early clinical trials have produced positive data for therapies aimed at repolarizing immuno-suppressive myeloid populations in the TME. One new macrophage repolarizing target, PSGL-1 (P-selectin glycoprotein ligand-1), is expressed at high levels on suppressive tumor-associated macrophages (TAMs) and in vitro differentiated M2 macrophages. PSGL-1 has been shown to have an immune-modulatory activity, which includes its role in maintaining an immuno-suppressive macrophage state.MethodsTo assess the ability of PSGL-1 antibodies to convert macrophages and the tumor microenvironment from an immuno-suppressive toward a pro-inflammatory state, we employed in vitro primary macrophage and multi-cellular assays, ex vivo patient-derived tumor cultures, and a humanized mouse PDX model.ResultsWe have determined that our lead anti-PSGL-1 antibody repolarized M2-like macrophages to a more M1-like state both phenotypically and functionally as assessed in primary in vitro macrophage assays. Transcriptomics profiling of M2c macrophages showed that the anti-PSGL-1 antibody upregulated TNF-alpha/NF-kB and chemokine-mediated signaling, while downregulating oxidative phosphorylation, fatty acid metabolism and Myc signaling pathways, consistent with a broad M2-to-M1 shift of the macrophage state. Furthermore, these repolarized M1-like macrophages enhanced the inflammatory response in complex multi-cellular assays.Pre-clinical efficacy of the anti-PSGL-1 antibody was demonstrated using ex vivo cultures of fresh patient-derived tumors that preserve the cellular heterogeneity of the TME. Anti-PSGL-1 increased production of inflammatory cytokines and chemokines involved in immune activation of the TME and T cell recruitment.Lastly, our lead anti-PSGL-1 antibody also showed in vivo anti-tumor effect in a humanized mouse PDX model of melanoma. The antibody suppressed tumor growth to a significantly greater degree compared to anti-PD-1. At the cellular and molecular levels, the anti-PSGL-1 treatment led to a more enhanced inflammatory microenvironment, including a reduced M2:M1 macrophage ratio, and an increase in systemic pro-inflammatory mediators. Compared to anti-PD-1 monotherapy, anti-PSGL-1 alone and in combination with anti-PD-1 increased the fraction of effector CD8+ T cells among the infiltrating T cells. Significant combination effects of anti-PSGL-1 plus anti-PD-1 were seen at the cellular and molecular levels within the tumor tissue, the spleen, and peripheral blood.ConclusionsThe data presented here provide biological and mechanistic support for clinical testing of antibodies targeting PSGL-1 for the treatment of cancer.Ethics ApprovalAll legal and ethical requirements were met with regards to the humane treatment of animals described in the study. The animal study was conducted in compliance with IACUC PROTO202000042 and the institutional assurance certification of the University of Massachusetts Medical School. The University of Massachusetts Medical School is fully accredited by AAALAC and has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW).
Suppressive myeloid cells in the tumor microenvironment (TME) are associated with poor survival of cancer patients and resistance to T cell checkpoint inhibitors. These myeloid cells shield cancer cells from the sentinel immune response and create a niche for tumor growth. Repolarizing immuno-suppressive myeloid populations in the TME attracted considerable interest from scientific community and several companies, and has produced early positive clinical data. One of the targets that potently sustains macrophage suppressive phenotypes and is highly expressed on tumor associated macrophages is PSGL-1. Blocking a previously unknown epitope on PSGL-1 triggers macrophage repolarization to a pro inflammatory state. We have determined that our therapeutic candidate VTX-0811, anti-PSGL-1 monoclonal antibody (mAb), repolarizes M2-like macrophages to a more M1-like state both phenotypically and functionally as assessed by primary in vitro macrophage assays. Transcriptomics profiling of M2c macrophages showed that VTX-0811 upregulated TNF-α/NF-κB and chemokine-mediated signaling, while downregulating oxidative phosphorylation, fatty acid metabolism, and Myc signaling pathways, consistent with a broad M2-to-M1 shift. Furthermore, repolarized macrophages enhanced inflammatory responses in complex primary multi-cellular assays.VTX-0811 also showed efficacy in a humanized mouse PDX model of melanoma. VTX-0811 suppressed tumor growth to a significantly greater degree compared with an anti-PD-1 mAb. At the cellular and molecular levels, the treatment led to an inflammatory microenvironment, including a reduced MDSC population, and increased systemic pro-inflammatory mediators. Compared with anti-PD-1 monotherapy, VTX-0811 alone and in combination with anti-PD-1 increased the fraction of CD8+ T cells among the infiltrating T cells. Significant combination effects of VTX-0811 plus anti-PD-1 were seen within the tumor tissue, spleen, and peripheral blood.Additionally, pre-clinical efficacy of VTX-0811 was demonstrated using ex vivo cultures of fresh patient-derived tumors that preserve cellular and molecular composition of the TME. VTX-0811 increased secretion of inflammatory cytokines and chemokines known to be involved in immune activation of the TME and fresh leukocyte recruitment, as well as linked to clinical response to T cell checkpoint inhibitors.VTX-0811 is a humanized high affinity IgG4/κ mAb that demonstrated safety in NHP primates up to 200mg/kg dosed weekly for 5 total doses. VTX-0811 does not induce activation of unstimulated PBMC or unstimulated blood-derived neutrophil or T cell cultures. These data provide biological and mechanistic support for advancing this program into the clinic with a first in human trial planned for early 2022. Citation Format: Igor Feldman, Tatiana Novobrantseva, Ani Nguyen, Jessica Ritter, Mohammad Zafari, Denise Manfra, Susan Low, Steve Sazinsky, Michael Brehm, Boris Klebanov. VTX-0811, a first-in-class PSGL-1 blocking monoclonal antibody, repolarizes tumor associated macrophages and induces inflammation in the tumor microenvironment, leading to suppression of tumor growth in pre-clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5602.
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