Promote Tumor Immune Evasion Research Articles

Targeting Tumor-Associated Macrophages to Increase the Efficacy of Immune Checkpoint Inhibitors: A Glimpse into Novel Therapeutic Approaches for Metastatic Melanoma.

Simple SummaryStimulation of the host immune responses, through the use of biotech drugs that remove a brake on the immune system (immune checkpoint inhibitors), is a current widely used strategy to treat a variety of advanced-stage tumors with impressive outcomes also in patients refractory to standard chemotherapy. However, as in the case of metastatic melanoma, many patients fail to achieve a long-lasting clinical benefit. The aim of this article is to provide an overview of the current scientific evidence concerning the role played by cells of the tumor micro-environment, and in particular tumor-associated M2 macrophages, on the innate or acquired resistance of melanoma to immune checkpoint inhibitors. A special focus will be given to potential therapeutic interventions capable of counteracting tumor ability to evade the control of the immune system in order to enhance the efficacy of immune checkpoint inhibitors.Immune checkpoint inhibitors (ICIs) represent a promising therapeutic intervention for a variety of advanced/metastatic solid tumors, including melanoma, but in a large number of cases, patients fail to establish a sustained anti-tumor immunity and to achieve a long-lasting clinical benefit. Cells of the tumor micro-environment such as tumor-associated M2 macrophages (M2-TAMs) have been reported to limit the efficacy of immunotherapy, promoting tumor immune evasion and progression. Thus, strategies targeting M2-TAMs have been suggested to synergize with immune checkpoint blockade. This review recapitulates the molecular mechanisms by which M2-TAMs promote cancer immune evasion, with focus on the potential cross-talk between pharmacological interventions targeting M2-TAMs and ICIs for melanoma treatment.

Abstract PO-057: Host cell-derived TXA2-mediated Gq signaling in tumor cells promotes tumor immune evasion

Abstract Tumor-derived prostaglandin E2 (PGE2) promotes tumor progression through evasion of anti-tumor immunity. However, little is known about how PGE2 secretion is regulated within tumor tissues. Here, we show that host cell-derived thromboxane A2 (TXA2) triggers tumor cells to secrete PGE2, resulting in the evasion of anti-tumor immunity. An induction of Ca2+ transients in BrafV600E mouse melanoma cells is sufficient to trigger PGE2 secretion. The frequency of Ca2+ transients is markedly higher in melanoma cells implanted into mice than those in vitro. We show that t he melanoma cells deficient in TXA2 receptor or Gq subunit α do not exhibit Ca2+ transients in vivo nor evade anti-tumor immunity, as do not melanoma cells deficient in cyclooxygenases (COX). Motesanib, a VEGF receptor antagonist, suppresses Ca2+ transients, suggesting the involvement of tumor endothelial cells in the TXA2 production within tumor tissue. These resultsresults identifyresults identifyidentify TXA2 as a critical promotertargetof PGE2-dependent tumorigenesis. Citation Format: Kenta Terai, Yoshinobu Konishi, Hiroshi Ichise, Tetsuya Watabe, Yukari Sando, Takefumi Kondo, Choji Oki, Shinya Tsukiji, Yoko Hamazaki, Yasuhiro Murakawa, Akifumi Takaori-Kondo, Michiyuki Matsuda. Host cell-derived TXA2-mediated Gq signaling in tumor cells promotes tumor immune evasion [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-057.

Transcriptomic Analyses of Myeloid-Derived Suppressor Cell Subsets in the Circulation of Colorectal Cancer Patients

Myeloid-derived suppressor cells (MDSCs) promote tumor immune evasion and favor tumorigenesis by activating various tumor-promoting downstream signals. MDSC expansion is evident in the circulation and tumor microenvironment of many solid tumors including colorectal cancer (CRC). We have recently reported the transcriptomic profiles of tumor-infiltrating MDSCs in CRC patients and uncovered pathways, which could potentially assist tumor progression. In this study, we sorted different subsets of circulating MDSCs in CRC patients and investigated their transcriptomic profiles in order to disclose pathways, which could potentially contribute to disease progression. The sorted subsets included polymorphonuclear/granulocytic MDSCs (PMN-MDSCs), immature MDSCs (I-MDSCs), and monocytic MDSCs (M-MDSCs). Our functional annotation analyses revealed that multiple pathways including DNA damage-, chemotaxis-, apoptosis-, mitogen-activated protein kinase-, transforming growth factor β-, and myeloid differentiation–related transcripts were higher in PMN-MDSCs, compared with monocytic antigen-presenting cells (APCs) or I-MDSCs. Furthermore, genes related to Janus kinase (JAK)–signal transducer and activator of transcription (STAT) were also elevated in PMN-MDSCs. These data suggest that upregulation of JAK-STAT pathway could trigger multiple downstream targets in PMN-MDSCs, which favor tumor progression. Additionally, we found that pathways including phosphatidyl inositol 3-kinase (PI3K), interleukin 6, and TGF-β in M-MDSCs and cell cycle–related pathways in I-MDSCs were upregulated, compared with monocytic APCs. Moreover, acetylation-related genes were upregulated in both PMN-MDSCs and M-MDSCs. This latter finding implicates that epigenetic modifications could also play a role in the regulation of multiple tumor-promoting genes in PMN-MDSCs and M-MDSCs. Taken together, this study reveals various signaling pathways, which regulate the function of MDSC subsets in the circulation of CRC patients. However, functional studies are warranted to support these findings.

Abstract 2278: CTLA-4 targeted engineered toxin bodies designed to deplete regulatory T cells (Tregs)

Abstract Tumor resident regulatory T cells (Tregs) are important mediators of an immunosuppressive tumor microenvironment (TME) promoting tumor immune evasion. The presence of Tregs, and a higher ratio of Tregs to effector T cells in the TME, are associated with poor prognosis. The depletion of Tregs in the TME is expected to re-expose the tumor to the immune system to allow for tumor control. CLTA-4 is expressed on the cell surface of Tregs and is a clinically validated target. Better responses to CTLA-4 monoclonal antibody (mAb) treatment are correlated with stronger ADCC-mediated Treg depletion in preclinical models, and patient FcγR polymorphism has been reported to correlate with response to CTLA-4 mAb therapy. Towards improving on current therapies, many efforts to increase effective depletion of Tregs (such as by Fc effector modification) are being pursued. Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. Targeted ETBs are being developed to specifically target and destroy CTLA-4 expressing cells. In vitro, cells expressing CTLA-4 are effectively and specifically killed by targeted ETBs. This direct cell kill activity of the ETB has the potential to deplete Tregs in the TME without a requirement for ADCC mechanisms. ETBs have been designed to bind various CTLA-4 epitopes, and to comprise of different targeting domains formats, including monomeric and diabody single chain variable fragments (scFvs) as well as single domain and biparatopic antibody fragments. The development of a CTLA-4 targeted ETB is ongoing. The entry of a protein with direct cell kill properties into the therapeutic space represents a differentiated mechanism of action to deplete Tregs for ultimate re-invigoration of the anti-tumor immune response, and has the potential to provide benefit to patients, including in the relapsed or refractory setting. Citation Format: Aimee Iberg, Edith Acquaye-Seedah, Lilia A. Rabia, Garrett L. Robinson, Hilario J. Ramos, Joseph D. Dekker, Jay Zhao, Erin K. Willert. CTLA-4 targeted engineered toxin bodies designed to deplete regulatory T cells (Tregs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2278.

Abstract 3259: CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy controlling damaged and tumor cells phagocytosis

Abstract Myeloid cells represent one of the most abundant immune cell types in solid tumors and are generally associated with poor clinical outcome. We previously identified the orphan C-type lectin receptors (CLRs) CLEC-1 as over-expressed in situation of established immune tolerance and reported that CLEC-1 expression by dendritic cells (DCs) and macrophages is enhanced by TGFβ and tempers downstream T cells responses. Recent literature demonstrates that tumors could hijack physiological mechanisms of CLRs that normally restrain immune cell-mediated tissue damage to suppress myeloid cell activation and promote tumor immune evasion. Here we report that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment (e.g. ovarian cancer) and significantly more expressed by in vitro generated M2 macrophages compared to monocytes or M1 macrophages. RNAseq public data indicates that Clec1a is highly expressed in XCR1-expressing DC including CD8α+ and CD103+ DCs from lymphoid and peripheral tissues respectively. As these DCs are specialized in cross-presentation of dying/dead cell associated antigens, we evaluated whether CLEC-1 could be a receptor of damaged cells. We found that both human and mouse CLEC-1 fusion protein, but not irrelevant CLRs or negative controls, binds specifically to late apoptotic and secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray) or culture stress conditions. No binding was observed with viable, primary necrotic or with early apoptotic cells suggesting that CLEC-1 ligand(s) (CLEC-1L) correspond(s) to DAMP modified during the cell death process. Using a CLEC-1 reporter NFAT-lacZ BWZ.36 cell line, we show that this interaction is functional. Importantly, we observed in vivo that dendritic cells from Clec1a deficient mice more efficiently cross-present antigen (OVA-loaded dead cells) to CD8+ T cells (OT-1) and that CLEC-1 deficient mice, but not wild-type, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy (eg. cyclophosphamide). Treatment of wild-type mice with a Fc-CLEC-1 fusion protein also synergized with chemotherapy and induced complete response in 37% of mice versus 0% in chemotherapy group. We then generated, screened and identified different anti-human Clec-1 antagonist monoclonal antibodies (mAbs) with the capacity to block the CLEC-1/CLEC-1L interaction. We discovered that antagonist CLEC-1 mAbs, but not non-antagonist CLEC-1 control mAbs, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs but also of CLEC-1L-positive opsonized tumor cells (eg: Raji cells opsonized with anti-CD20 Rituximab) by human macrophages. Altogether, these data indicate that tumor cells inhibit myeloid cells phagocytosis through CLEC-1 and that antagonists of the CLEC-1/CLEC-1L novel myeloid checkpoint pathway constitute a novel cancer immunotherapy approach synergistic with chemotherapy. Citation Format: Vanessa Gauttier, Marion Drouin, Javier Saenz, Bérangère Evrard, Caroline Mary, Géraldine Teppaz, Ariane Desalle, Virginie Thépénier, Emmanuelle Wilhelm, Nicolas Poirier, Elise Chiffoleau. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy controlling damaged and tumor cells phagocytosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3259.

Identification of a Six-Immune-Related Long Non-coding RNA Signature for Predicting Survival and Immune Infiltrating Status in Breast Cancer.

Long non-coding RNAs (lncRNAs) play critical roles in tumor immunity; however, the functional roles of immune-related lncRNAs in breast cancer (BC) remain elusive. To further explore the immune−related lncRNAs in BC, whole genomic expression data and corresponding clinical information were obtained from multiple BC datasets. Based on correlation with the immune-related genes within the training set, we screened out the most promising immune-related lncRNAs. Subsequently, Lasso penalized Cox regression analysis followed by stepwise multivariate Cox regression analysis identified six survival-related lncRNAs (AC116366.1, AC244502.1, AC100810.1, MIAT, AC093297.2, and AL356417.2) and constructed a prognostic signature. The cohorts in the high−risk group had significantly poor survival time compared to those in the low−risk group. In addition, a nomogram integrated with clinical features and the prognostic signature was developed on the basis of the training set. Importantly, all the findings had a similar performance in three validated datasets. In the following studies, our integrative analyses indicated that the infiltration of CD8-positive (CD8) T cells associated with a good prognosis was strikingly activated in the low−risk group. To further provide an interpretation of biological mechanisms for the prognostic signature, we performed weighted gene co−expression network analysis (WGCNA) followed by KEGG pathway-enrichment analysis. Our results showed that the antigen presentation pathway involved in protein processing in endoplasmic reticulum and antigen processing and presentation was markedly altered in the high-risk group, which might promote tumor immune evasion and associate with poor clinical outcomes in BC patients with high risk scores. In conclusion, we aimed to take advantage of bioinformatics analyses to explore immune−related lncRNAs, which could function as prognostic indicators and promising therapeutic targets for BC patients.

Formyl peptide receptor (FPR) agonists promote immune evasion independent of functional Ncf1 derived ROS

Abstract Previous studies have shown that in both the absence of the Ncf1 gene or in the presence of a loss-of-function SNP, mice have a lower tumor burden when compared to wildtype animals. Here, we investigate a formyl peptide receptor (FPR) agonist in this setting and demonstrate that it promotes tumor immune evasion through an immune-Ncf1/ROS-dependent mechanism. We observed an increased colonization of B16F10 tumor cells in the lungs of FPR-agonist-treated wildtype C57BL6N mice compared to vehicle-control-treated mice. A significant reduction in tumor burden was expectedly seen in the Ncf1*/* mutant strain compared to the wildtype. However, FPR-agonist treatment did not promote tumor growth in Ncf1*/* treated mice when compared to vehicle-control-treated Ncf1*/* mice. These results suggest that FPR agonists can promote tumor evasion by acting through FPRs on immune cells in a NOX2/ROS-independent fashion and in a fashion independent of FPRs on the tumor.

ADORA1 Inhibition Promotes Tumor Immune Evasion by Regulating the ATF3-PD-L1 Axis

Here, we show that tumor ADORA1 deletion suppresses cell growth in human melanoma cell lines invitro and tumor development invivo in immune-deficient xenografts. However, this deletion induces the upregulation of PD-L1 levels, which inactivates cocultured Tcells invitro, compromises anti-tumor immunity invivo, and reduces anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of ADORA1-deficient or ADORA1 antagonist-treated melanoma and NSCLC immune-competent mouse models. Mechanistically, we identify ATF3 as the factor transcriptionally upregulating PD-L1 expression. Tumor ATF3 deletion improves the effect of ADORA1 antagonist treatment of melanoma and NSCLC xenografts. We observe higher ADORA1, lower ATF3, and lower PD-L1 expression levels in tumor tissues from nonresponders among PD-1 mAb-treated NSCLC patients.

CCL21 Programs Immune Activity in Tumor Microenvironment.

CCL21 promotes immune activity in the tumor microenvironment (TME) by colocalizing dendritic cells (DC) and T cells programing ectopic lymph node architectural structures that correlate with cancer prognosis. Innovative strategies to deliver CCL21 in cancer patients will reactivate the downregulated immune activity in the TME. Immune escape mechanisms are upregulated in the TME that promote tumor immune evasion. CCL21 combined with inhibition of dominant pathways of immune evasion will aid in the development of effective immunotherapy for cancer.

Myeloid cell-synthesized coagulation factor X dampens antitumor immunity.

Immune evasion in the tumor microenvironment (TME) is a crucial barrier for effective cancer therapy, and plasticity of innate immune cells may contribute to failures of targeted immunotherapies. Here, we show that rivaroxaban, a direct inhibitor of activated coagulation factor X (FX), promotes antitumor immunity by enhancing infiltration of dendritic cells and cytotoxic T cells at the tumor site. Profiling FX expression in the TME identifies monocytes and macrophages as crucial sources of extravascular FX. By generating mice with immune cells lacking the ability to produce FX, we show that myeloid cell-derived FX plays a pivotal role in promoting tumor immune evasion. In mouse models of cancer, we report that the efficacy of rivaroxaban is comparable with anti-programmed cell death ligand 1 (PD-L1) therapy and that rivaroxaban synergizes with anti-PD-L1 in improving antitumor immunity. Mechanistically, we demonstrate that FXa promotes immune evasion by signaling through protease-activated receptor 2 and that rivaroxaban specifically targets this cell-autonomous signaling pathway to reprogram tumor-associated macrophages. Collectively, our results have uncovered the importance of FX produced in the TME as a regulator of immune cell activation and suggest translational potential of direct oral anticoagulants to remove persisting roadblocks for immunotherapy and provide extravascular benefits in other diseases.

Abstract 5118: TIDE-E: an online evaluator of tumor immune-suppressive function of gene sets

Abstract Recent immunotherapy studies have generated a diverse collection of datasets, covering different aspects of cancer immune evasion. However, to the best of our knowledge, there are no public tools that can systematically rank genes regarding their function on tumor immune evasion and comprehensively evaluate biomarkers across multiple clinical cohorts. Therefore, we presented the TIDE-E (evaluator) website for users to evaluate their gene sets across a large panel of data cohorts (http://tide.dfci.harvard.edu). The website integrated two categories of data resources, human clinical datasets from public immunotherapy studies, TCGA, PRECOG, and METABRIC databases, as well as functional genomics datasets from CRISPR screens in preclinical models. For a user-defined gene set, the web application can test its performance as immunotherapy response biomarkers across multiple clinical studies, and rank the gene members according to their relevance in promoting tumor immune evasion. Citation Format: Jingxin Fu, Wubing Zhang, Peng Jiang, Xiaole S. Liu. TIDE-E: an online evaluator of tumor immune-suppressive function of gene sets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5118.

Abstract 4154: EOS100850 inhibits A2A receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies

Abstract High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant, orally administered agent to treat a wide range of tumor types. To monitor A2A receptor engagement by EOS100850 in clinical studies, we developed two pharmacodynamic assays that allow to quantify the inhibition of adenosine signaling in cancer patients dosed with this compound. The assays were developed and optimized to: 1) minimize the amount of patient blood required for each test; 2) minimize sample manipulation; 3) allow marker quantification in cryopreserved samples. The first assay is based on the quantification of phosphorylated cAMP response-element binding protein (pCREB) as a proximal readout of A2A receptor signaling. Whole blood is ex vivo stimulated with the A2A receptor agonist CGS-21608, or with dimethyl sulfoxide or phorbol-12-myristate-13 acetate and ionomycin as controls, and pCREB in CD4+ and CD8+ cells is analyzed by flow cytometry. After having confirmed that pCREB was rapidly induced after incubation with CGS-21680 (median fold-induction in 24 healthy donors was 5.4 and 4.0 in CD4+ and CD8+ cells, respectively), we showed that EOS100850 inhibited CGS-21608-induced pCREB in a dose-dependent manner, with an IC50 of 9.7 nM and 11.2 nM, respectively, for CD4+ and CD8+ T cells, and 83-87% signal inhibition in the presence of 40 nM EOS100850 (data from 10 donors). The second assay is based on the quantification of soluble mediators as a distal readout of A2A receptor signaling. Stimulation of whole blood with lipopolysaccharide (LPS) normally induces the release of innate cytokines and chemokines, but in the presence of the A2A receptor agonist CGS-21680, LPS activity is impaired. In this assay, whole blood is ex vivo stimulated with LPS in the presence or in the absence of CGS-21608 using TruCulture tubes (Myriad RBM), and secreted mediators are quantified using the human multianalyte profile immunoassay platform. After having confirmed that CGS-21680 induced a profound (>50%) alteration of cytokine secretion in LPS-stimulated blood, we showed that EOS100850 restored IFN-γ, IL-8, IL-10 and TNF-α secretion in a dose-dependent manner, with a complete rescue at 40-100 nM of compound (data from 3 donors). Both the assays were qualified in terms of precision, robustness and stability using blood of healthy volunteers, applied to blood of five cancer donors and are now ready to be used for the clinics. Citation Format: Chiara Martinoli, Margreet Brouwer, Erica Houthuys, Marjorie Mercier, Noemie Wald, Bruno Gomes, Stefano Crosignani, Veronique Bodo. EOS100850 inhibits A2A receptor signaling in human whole blood: Two pharmacodynamic assays to monitor EOS100850 activity in clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4154.

Abstract 3261: EOS100850, a non-brain penetrant highly selective A2Areceptor antagonist, uniquely maintains high potency within the adenosine rich tumor microenvironment

Abstract High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor, suppresses innate and adaptive immune cell responses leading to suppression of antitumor immunity. We demonstrated that A2A receptor antagonists initially designed for Parkinson’s disease but repurposed for immuno-oncology dramatically loose potency in a high adenosine environment. We therefore developed EOS100850, an A2A receptor antagonist specifically designed as a potent, highly selective, non-brain penetrant orally administered agent to treat a wide range of tumor types. EOS100850 has been discovered via structure-based drug design followed by rational medicinal chemistry to improve drug-like properties. EOS100850 demonstrated nanomolar affinity and high selectivity for human A2A receptors, with pico- to nanomolar potency on human and mouse A2A receptors. Assays were performed on A2A-overexpressing HEK293 cells and primary human T cells, using conditions mimicking normal physiological and tumor-like microenvironments. In these assays, EOS100850 potency was not affected by increasing extracellular adenosine concentrations. A unique feature of this compound is the remarkable long residence time on human A2A receptors after binding, resulting in a prolonged inhibition of A2A receptor signaling at a wide range of adenosine concentrations (up to 1000 µM). Furthermore, unlike competitor A2A receptor antagonists, EOS100850 reversed with nanomolar potency A2A receptor-dependent inhibition of cytokine secretion by T cells in tumor-like assay conditions. EOS100850 potency to inhibit A2A receptor-mediated CREB phosphorylation in T cells in human and mouse whole blood was in the low nanomolar range.One of the major metabolites of EOS100850, EOS100612, identified both in vitro and in vivo, has been synthesized and was found to be active on A2A receptors. Similar to EOS100850, EOS100612 displayed pico- to nanomolar potency, high selectivity and long residence time on A2A receptors in various in vitro assays.In conclusion, EOS100850 represents a novel non-brain penetrant best-in-class A2A receptor antagonist. It remains highly potent in environments characterized by high adenosine concentrations, such as the TME, maximizing the potential intra-tumor effect and further minimizing the risk of collateral CNS toxicity. Citation Format: Erica Houthuys, Paola Basilico, Margreet Brouwer, Theo Deregnaucourt, Michel Detheux, Gregory Driessens, Bruno Gomes, Xavier Leroy, Joao Marchante, Reece Marillier, Jakub Swiercz, Stefano Crosignani. EOS100850, a non-brain penetrant highly selective A2Areceptor antagonist, uniquely maintains high potency within the adenosine rich tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3261.

Combination immunotherapy with interleukin-2 surface-modified tumor cell vaccine and programmed death receptor-1 blockade against renal cell carcinoma.

Immunotherapy may be an effective way to prevent postoperative recurrence of renal cell carcinoma. Streptavidin‐interleukin‐2 (SA‐IL‐2) surface‐modified tumor cell vaccine developed through our protein‐anchor technology could induce specific antitumor T‐cell responses, but this immunotherapy cannot completely eradicate the tumor. These effector T cells highly expressed programmed death receptor‐1 (PD‐1), and the expression of programmed death ligand‐1 (PD‐L1) in the tumor environment also was upregulated after SA‐IL‐2‐modified vaccine therapy. PD‐1/PD‐L1 interaction promotes tumor immune evasion. Adding PD‐1 blockade to SA‐IL‐2‐modified vaccine therapy increased the number of CD4+, CD8+ and CD8+interferon‐γ+ but not CD4+Foxp3+ T cells. PD‐1 blockade could rescue the activity of tumor‐specific T lymphocytes induced by the SA‐IL‐2‐modified vaccine. Combination therapy delayed tumor growth and protected mice against a second Renca cells but not melanoma cells challenge. Taken together, PD‐1 blockade could reverse immune evasion in the treatment with SA‐IL‐2‐modified vaccine, and eventually induce a stronger specific antitumor immune response against renal cell carcinoma.

NKILA lncRNA promotes tumor immune evasion by sensitizing T cells to activation-induced cell death.

Activation-induced cell death (AICD) of T lymphocytes can be exploited by cancers to escape immunological destruction. We demonstrated that tumor-specific cytotoxic T lymphocytes (CTLs) and type 1 helper T (TH1) cells, rather than type 2 helper T cells and regulatory T cells, were sensitive to AICD in breast and lung cancer microenvironments. NKILA, an NF-κB-interacting long noncoding RNA (lncRNA), regulates T cell sensitivity to AICD by inhibiting NF-κB activity. Mechanistically, calcium influx in stimulated T cells via T cell-receptor signaling activates calmodulin, thereby removing deacetylase from the NKILA promoter and enhancing STAT1-mediated transcription. Administering CTLs with NKILA knockdown effectively inhibited growth of breast cancer patient-derived xenografts in mice by increasing CTL infiltration. Clinically, NKILA overexpression in tumor-specific CTLs and TH1 cells correlated with their apoptosis and shorter patient survival. Our findings underscore the importance of lncRNAs in determining tumor-mediated T cell AICD and suggest that engineering lncRNAs in adoptively transferred T cells might provide a novel antitumor immunotherapy.

Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma

AbstractMature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

Abstract 128: NKILA lncRNA promotes tumor immune evasion by mediating activation-induced cell death in tumor-specific CTLs

Abstract Activation-induced cell death (AICD) of T lymphocytes may be exploited by cancers to eliminate cytotoxic T cells (CTLs). However, the mechanisms underlying AICD in tumor specific CTLs remain unknown. Here, we demonstrated excessive apoptosis of tumor antigen-specific CTLs in breast and lung cancers. Interestingly, NKILA, an NFκB-interacting lncRNA, mediates AICD of CTLs by inhibiting NFκB activities after their activation. Mechanistically, NKILA promoter is closed for transcription by histone deacetylation in resting T cells, while calcium influx by TCR signaling activates calmodulin to remove the deacetylase from NKILA promoter and enhances its transcription. In vivo, administering CTLs with NKILA silencing to immunocompromised mice with breast cancer patient-derived xenografts (PDXs) effectively inhibits PDX growth by increasing CTL infiltration. Clinically, NKILA was overexpressed in the tumor-specific CTLs of breast and lung cancers, which was associated with less CTL infiltration in the tumors and shorter patient survival. Our findings present the first evidence that AICD in tumor-specific CTLs is crucial to cancer immune evasion, and targeting NKILA in CTLs emerges as a novel antitumor immunotherapy. Citation Format: Erwei Song. NKILA lncRNA promotes tumor immune evasion by mediating activation-induced cell death in tumor-specific CTLs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 128.

Abstract LB-291: EOS100850, an insurmountable and non-brain penetrant A2A receptor antagonist, inhibits adenosine-mediated T cell suppression, demonstrates anti-tumor activity and exhibits best-in class characteristics

Abstract High levels of extracellular adenosine in the tumor microenvironment promote tumor immune evasion. We and others have shown that adenosine, predominantly through the A2A receptor (A2AR), suppresses the Th1 cytokine production of T cells and monocytes and cytolytic activity of T and NK cells. We demonstrated that A2AR antagonists initially designed for Parkinson's disease but repurposed for immuno-oncology dramatically lost potency in a high adenosine environment. We therefore developed EOS100850, a novel, non-brain penetrant and highly selective inhibitor of A2AR with sub-nanomolar Ki. Using experimental conditions that mimic tumor environment, we have shown that EOS100850 potently inhibited A2AR signalling in human T lymphocytes independently of adenosine concentrations, and rescued cytokine production, even in the presence of high concentrations of A2AR agonists. iTeos A2AR antagonist potently rescued Th1 cytokine production in human whole blood treated by A2AR agonists, and increased CD8+ T cell cytotoxicity in a co-culture assay of effector CD8+ T cells and target cancer cells. An in vivo pharmacodynamic assay based on phosphorylation of CREB (pCREB) in mouse peripheral CD8+ and CD4+ T was developed and validated as a readout for A2AR activation. EOS100850, 30 minutes after oral gavage at doses ranging from 0,03 to 1mg/kg demonstrated 80 to 100% inhibition of pCREB induced by the ex-vivo addition of A2AR agonist. Remarkably, 12 hours after gavage at 1 and 3mg/kg, when the EOS100850 antagonist was no longer detectable in the plasma, more than 50% of inhibition of pCREB was still observed. These results demonstrate that EOS100850 has a PD activity that extends well beyond its PK based on a long residence time. iTeos's A2AR antagonist, uniquely designed to address the challenge of counteracting elevated adenosine concentrations in tumors, was tested for the first time in a mouse A20 lymphoma model. iTeos's A2AR antagonist in combination with anti-PD-L1 demonstrated significant tumor growth suppression (p=0.0008) compared with anti-PD-L1 alone, with a 6 fold decrease in tumor volume compared to anti-PD-L1 alone (median tumor volume =105±79 mm3 vs 696±427mm3 on day 23, respectively). EOS100850 represents a novel, potent, insurmountable and best-in-class A2AR blocker, specifically optimized for immuno-oncology indications, that deserves studies in Human. Citation Format: Erica Houthuys, Reece Marillier, Théo Deregnaucourt, margreet Brouwer, Paola Basilico, Romain Pirson, Joao Marchante, Shruthi Prasad, Annelise Hermant, Florence Nyawouame, Julie Preillon, Kim Frederix, Anne Catherine Michaux, Florence Lambolez, Jakub Swiercz, Gregory Driessens, Noemie Wald, Chiara Martinoli, Veronique Bodo, Michel Detheux, Xavier Leroy, Stefano Crosignani. EOS100850, an insurmountable and non-brain penetrant A2A receptor antagonist, inhibits adenosine-mediated T cell suppression, demonstrates anti-tumor activity and exhibits best-in class characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-291.

Abstract 1447: Aberrant tryptophan catabolism marked by high kynureninase expression contributes to immunosuppression and poor outcome in lung adenocarcinoma

Abstract Low tryptophan levels promote tumor immune evasion by suppressing immune function. Elucidation of the immunomodulatory effects of altered tryptophan catabolism has relevance to cancer immunotherapy. Previous studies have largely focused on the role of the rate-limiting enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan depletion in the microenvironment. However, IDO1 levels are highly heterogeneous in different cancers, and numerous other downstream tryptophan catabolites have also been shown to have immunomodulatory activity. We surveyed the proteomes of 123 cancer cell lines for regulators of tryptophan metabolism which revealed overexpression of kynureninase (KYNU) in KEAP1 mutant lung adenocarcinomas. Functional analysis indicated that KYNU expression is regulated through nuclear factor, erythroid 2-like (Nrf2) activation. KYNU-high cell lines exhibited increased secretion of KYNU-derived anthranilate/3-hydroxyanthranilate. Ex-vivo studies using isolated PBMCs demonstrated that 3-hydroxyanthranilate reduced CD8+ T-cell viability. KYNU mRNA expression in three independent datasets of lung adenocarcinomas and additionally KYNU protein expression in lung adenocarcinoma tissue microarrays revealed that high KYNU expression predicts poor survival and disease recurrence. Our findings indicate that KYNU promotes immune suppression and is an independent prognostic marker for lung adenocarcinoma. Citation Format: Edwin J. Ostrin, Johannes F. Fahrmann, Ichidai Tanaka, Muge Celiktas, Clemente Aguilar, Mitzi Aguilar, Jennifer B. Dennison, Eunice Murage, Satyendra C. Tripathi, Oliver Delgado, Hong Wang, Jaime Rodriguez-Canales, Carmen Behrens, Ignacio I. Wistuba, Ayumu Taguchi, Samir M. Hanash. Aberrant tryptophan catabolism marked by high kynureninase expression contributes to immunosuppression and poor outcome in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1447.

Focal adhesion kinase (FAK) controls programmed death-ligand-1 (PD-L1) expression and promotes tumor immune evasion in a syngeneic murine model of epithelial ovarian cancer

Focal adhesion kinase (FAK) controls programmed death-ligand-1 (PD-L1) expression and promotes tumor immune evasion in a syngeneic murine model of epithelial ovarian cancer