Martina Ott

An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor

Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liver- and neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO–AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology.

A vaccine targeting mutant IDH1 induces antitumour immunity

Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas and other types of tumour. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG), genomic hypermethylation, genetic instability and malignant transformation. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4+ T-helper-1 (TH1) responses. CD4+ TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4+ T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

Protein kinase Cβ as a therapeutic target stabilizing blood–brain barrier disruption in experimental autoimmune encephalomyelitis

Disruption of the blood–brain barrier (BBB) is a hallmark of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis. This disruption may precede and facilitate the infiltration of encephalitogenic T cells. The signaling events that lead to this BBB disruption are incompletely understood but appear to involve dysregulation of tight-junction proteins such as claudins. Pharmacological interventions aiming at stabilizing the BBB in MS might have therapeutic potential. Here, we show that the orally available small molecule LY-317615, a synthetic bisindolylmaleimide and inhibitor of protein kinase Cβ, which is clinically under investigation for the treatment of cancer, suppresses the transmigration of activated T cells through an inflamed endothelial cell barrier, where it leads to the induction of the tight-junction molecules zona occludens-1, claudin 3, and claudin 5 and other pathways critically involved in transendothelial leukocyte migration. Treatment of mice with ongoing experimental autoimmune encephalomyelitis with LY-317615 ameliorates inflammation, demyelination, axonal damage, and clinical symptoms. Although LY-317615 dose-dependently suppresses T-cell proliferation and cytokine production independent of antigen specificity, its therapeutic effect is abrogated in a mouse model requiring pertussis toxin. This abrogation indicates that the anti-inflammatory and clinical efficacy is mainly mediated by stabilization of the BBB, thus suppressing the transmigration of encephalitogenic T cells. Collectively, our data suggest the involvement of endothelial protein kinase Cβ in stabilizing the BBB in autoimmune neuroinflammation and imply a therapeutic potential of BBB-targeting agents such as LY-317615 as therapeutic approaches for MS.

Promotion of Glioblastoma Cell Motility by Enhancer of Zeste Homolog 2 (EZH2) Is Mediated by AXL Receptor Kinase

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that epigenetically silences gene transcription through histone H3 lysine trimethylation (H3K27me3). EZH2 has been implicated in stem cell maintenance and is overexpressed in hematological and solid malignancie`s including malignant glioma. EZH2 is thought to promote tumor progression by silencing tumor suppressor genes. Hence pharmacological disruption of the PRC2 is an attractive therapeutic strategy for cancer treatment. Here we show that EZH2 is expressed in human glioma and correlates with malignancy. Silencing of EZH2 reduced glioma cell proliferation and invasiveness. While we did not observe induction of cell cycle-associated tumor suppressor genes by silencing or pharmacological inhibition of EZH2, microarray analyses demonstrated a strong transcriptional reduction of the AXL receptor kinase. Neither histone nor DNA methylation appeared to be involved in the positive regulation of AXL by EZH2. Silencing AXL mimicked the antiinvasive effects of EZH2 knockdown. Finally, AXL expression is found in human gliomas with high EZH2 expression. Collectively these data suggest that EZH2 drives glioma invasiveness via transcriptional control of AXL independent of histone or DNA methylation.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular d-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Suppression of TDO-mediated tryptophan catabolism in glioblastoma cells by a steroid-responsive FKBP52-dependent pathway

Tryptophan catabolism is increasingly recognized as a key and druggable molecular mechanism active in cancer, immune, and glioneural cells and involved in the modulation of antitumor immunity, autoimmunity and glioneural function. In addition to the pivotal rate limiting enzyme indoleamine‐2,3‐dioxygenase, expression of tryptophan‐2,3‐dioxygenase (TDO) has recently been described as an alternative pathway responsible for constitutive tryptophan degradation in malignant gliomas and other types of cancer. In addition, TDO has been implicated as a key regulator of neurotoxicity involved in neurodegenerative diseases and ageing. The pathways regulating TDO expression, however, are largely unknown. Here, a siRNA‐based transcription factor profiling in human glioblastoma cells revealed that the expression of human TDO is suppressed by endogenous glucocorticoid signaling. Similarly, treatment of glioblastoma cells with the synthetic glucocorticoid dexamethasone led to a reduction of TDO expression and activity in vitro and in vivo. TDO inhibition was dependent on the immunophilin FKBP52, whose FK1 domain physically interacted with the glucocorticoid receptor as demonstrated by bimolecular fluorescence complementation and in situ proximity ligation assays. Accordingly, gene expression profile analyses revealed negative correlation of FKBP52 and TDO in glial and neural tumors and in normal brain. Knockdown of FKBP52 and treatment with the FK‐binding immunosuppressant FK506 enhanced TDO expression and activity in glioblastoma cells. In summary, we identify a novel steroid‐responsive FKBP52‐dependent pathway suppressing the expression and activity of TDO, a central and rate‐limiting enzyme in tryptophan metabolism, in human gliomas. GLIA 2015;63:78–90

K27M-mutant histone-3 as a novel target for glioma immunotherapy

ABSTRACT Mutation-specific vaccines have become increasingly important in glioma immunotherapy; however, shared neoepitopes are rare. For diffuse gliomas, a driver mutation in the gene for isocitrate dehydrogenase type-1 has been shown to produce an immunogenic epitope currently targeted in clinical trials. For highly aggressive midline gliomas, a recurrent point mutation in the histone-3 gene (H3F3A) causes an amino acid change from lysine to methionine at position 27 (K27M). Here, we demonstrate that a peptide vaccine against K27M-mutant histone-3 is capable of inducing effective, mutation-specific, cytotoxic T-cell- and T-helper-1-cell-mediated immune responses in a major histocompatibility complex (MHC)-humanized mouse model. By proving an immunologically effective presentation of the driver mutation H3K27M on MHC class II in human H3K27M-mutant gliomas, our data provide a basis for the further clinical development of vaccine-based or cell-based immunotherapeutic approaches targeting H3K27M.

Correlated magnetic resonance imaging and ultramicroscopy (MR-UM) is a tool kit to assess the dynamics of glioma angiogenesis

Neoangiogenesis is a pivotal therapeutic target in glioblastoma. Tumor monitoring requires imaging methods to assess treatment effects and disease progression. Until now mapping of the tumor vasculature has been difficult. We have developed a combined magnetic resonance and optical toolkit to study neoangiogenesis in glioma models. We use in vivo magnetic resonance imaging (MRI) and correlative ultramicroscopy (UM) of ex vivo cleared whole brains to track neovascularization. T2* imaging allows the identification of single vessels in glioma development and the quantification of neovessels over time. Pharmacological VEGF inhibition leads to partial vascular normalization with decreased vessel caliber, density, and permeability. To further resolve the tumor microvasculature, we performed correlated UM of fluorescently labeled microvessels in cleared brains. UM resolved typical features of neoangiogenesis and tumor cell invasion with a spatial resolution of ~5 µm. MR-UM can be used as a platform for three-dimensional mapping and high-resolution quantification of tumor angiogenesis. DOI: http://dx.doi.org/10.7554/eLife.11712.001

The stress kinase GCN2 does not mediate suppression of antitumor T cell responses by tryptophan catabolism in experimental melanomas

ABSTRACT Tryptophan metabolism is a key process that shapes the immunosuppressive tumor microenvironment. The two rate-limiting enzymes that mediate tryptophan depletion, indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), have moved into the focus of research and inhibitors targeting IDO and TDO have entered clinical trials. Local tryptophan depletion is generally viewed as the crucial immunosuppressive mechanism. In T cells, the kinase general control non-derepressible 2 (GCN2) has been identified as a molecular sensor of tryptophan deprivation. GCN2 activation by tryptophan depletion induces apoptosis and mitigates T cell proliferation. Here, we investigated whether GCN2 attenuates tumor rejection in experimental B16 melanoma using T cell-specific Gcn2 knockout mice. Our data demonstrate that GCN2 in T cells did not affect immunity to B16 tumors even when animals were treated with antibodies targeting cytotoxic T lymphocyte antigen-4 (CTLA4). GCN2-deficient gp100 TCR-transgenic T cells were equally effective as wild-type pmel T cells against gp100-expressing B16 melanomas after adoptive transfer and gp100 peptide vaccination. Even augmentation of tumoral tryptophan metabolism in B16 tumors by lentiviral overexpression of Tdo did not differentially affect GCN2-proficient vs. GCN2-deficient T cells in vivo. Importantly, GCN2 target genes were not upregulated in tumor-infiltrating T cells. MALDI-TOF MS imaging of B16 melanomas demonstrated maintenance of intratumoral tryptophan levels despite high tryptophan turnover, which prohibits a drop in tryptophan sufficient to activate GCN2 in tumor-infiltrating T cells. In conclusion, our results do not suggest that suppression of antitumor immune responses by tryptophan metabolism is driven by local tryptophan depletion and subsequent GCN2-mediated T cell anergy.

Tryptophan‐2,3‐dioxygenase is regulated by prostaglandin E2 in malignant glioma via a positive signaling loop involving prostaglandin E receptor‐4

Malignant gliomas and other types of tumors generate a local immunosuppressive microenvironment, which prohibits an effective anti‐tumor immune response and promotes tumor growth. Along with others, we have recently demonstrated that catabolism of the essential amino acid tryptophan via tryptophan‐2,3‐dioxygenase (TDO) is an important mechanism mediating tumor‐associated immunosuppression particularly in gliomas. The pathways regulating TDO in tumors, however, are poorly understood. Here, we show that prostaglandins enhance TDO expression and enzymatic activity in malignant gliomas via activation of prostaglandin E receptor‐4 (EP4). Stimulation with prostaglandin E2 (PGE2) up‐regulated TDO‐mediated kynurenine release in human glioma cell lines, whereas knockdown of the PGE2 receptor EP4 inhibited TDO expression and activity. In human malignant glioma tissue expression of the PGE2‐producing enzyme cyclooxygenase‐2 (COX2) and its receptor EP4 were associated with TDO expression both on transcript and protein level. High expression of EP4 correlated with poor survival in malignant glioma patients WHO III‐IV. Importantly, treatment of glioma cells with an EP4 inhibitor decreased TDO expression and activity. Moreover, TDO‐over‐expressing murine gliomas showed increased COX2 and EP4 expression suggesting a positive feedback mechanism in vivo. In summary, targeting EP4 may inhibit – in addition to immunosuppressive COX2 signaling – tryptophan degradation as another important immunosuppressive pathway and thus, could provide a dual clinically relevant immunotherapeutic avenue for the treatment of malignant gliomas.