Lukas Bunse

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.

mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy

Significance N-myc downstream regulated gene 1 (NDRG1) is a central and druggable molecular hub integrating diverse therapy-induced microenvironmental factors to promote resistance toward alkylating chemotherapy. We suggest that NDRG1-mediated chemoprotection is achieved via binding and stabilizing methyltransferases, such as O6-methylguanine-DNA methyltransferase. A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O6-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.

Proximity ligation assay evaluates IDH1R132H presentation in gliomas

For a targeted cancer vaccine to be effective, the antigen of interest needs to be naturally processed and presented on MHC by the target cell or an antigen-presenting cell (APC) in the tumor stroma. The presence of these characteristics is often assumed based on animal models, evaluation of antigen-overexpressing APCs in vitro, or assays of material-consuming immune precipitation from fresh solid tissue. Here, we evaluated the use of an alternative approach that uses the proximity ligation assay (PLA) to identify the presentation of an MHC class II-restricted antigen in paraffin-embedded tissue sections from patients with brain tumors. This approach required a specific antibody directed against the epitope that was presented. We used an antibody that specifically binds an epitope of mutated isocitrate dehydrogenase type 1 (IDH1R132H), which is frequently expressed in gliomas and other types of tumors. In situ PLA showed that the IDH1R132H epitope colocalizes with MHC class II in IDH1R132H-mutated glioma tissue. Moreover, PLA demonstrated colocalization between the class II epitope-containing melanoma antigen New York esophageal 1 and MHC class II. Collectively, our data suggest that PLA may be a useful tool to acquire information on whether an antigen is presented in situ, and this technique has potential to guide clinical studies that use antigen-specific cancer immunotherapy.

Concepts in glioma immunotherapy

Immunotherapeutic concepts in neurooncology have been developed for many decades but have mainly been hampered by poor definition of relevant antigens and selective measures to target the central nervous system. Independent of the recent remarkable successes in clinical immunooncology with checkpoint inhibitors and vaccines, immunotherapy of brain tumors in general and gliomas in particular has evolved with novel neurooncology-specific concepts over the past years providing new phase 1 approaches of individualized immunotherapy to first phase three clinical trials. These concepts are driven by a high medical need in the absence of approved targeted therapies and refute the classic dogma that the central nervous system is immune-privileged and hence inaccessible to potent antitumor immunity. Instead, measures have been taken to improve the odds for successful immunotherapies, including rational targeting of relevant antigens and integration of immunotherapies into standard of care primary radiochemotherapy to increase the efficacy of antitumor immunity in a meaningful time window. This review highlights concepts and challenges associated with epitope discovery and selection and trial design.

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

Mutant IDH1: An immunotherapeutic target in tumors

The discovery of driver mutations in cancers has raised interest in their suitability as immunotherapeutic targets. A recent study demonstrates that a point mutation in isocitrate dehydrogenase 1 (IDH1R132H), expressed in gliomas and other tumors, is presented on human MHC class II and induces a mutation-specific CD4+ antitumor T cell response in patients and a syngeneic tumor model in MHC-humanized mice.

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.

The promises of immunotherapy in gliomas

Purpose of review Also owing to the limited efficacy of targeted therapies, there has been a renewed interest in targeting gliomas with immunotherapy. But despite considerable efforts using sophisticated approaches, proof of efficacy beyond case studies is still lacking. The purpose of this review is to summarize and discuss current immunotherapeutic approaches and efforts to understand mechanisms of response and resistance. Recent findings The recent failure of large randomized clinical trials using targeted vaccines and checkpoint inhibitors to improve clinical outcome have underlined the grand challenges in this therapeutic arena and illustrated the necessity to understand the biology of immunotherapeutic interventions before conducting large randomized studies. However, these failures should not distract us from continuing to optimize immunotherapeutic concepts. The recent developments in transgenic T cell technologies and personalized vaccines but also rational combinatorial approaches offer tremendous opportunities and should be exploited carefully in early scientifically driven clinical trials. Summary A profound understanding of the cellular and molecular mechanisms of response and resistance to immunotherapy to be gained from these thoroughly designed clinical trials will be essential to carve out successful strategies in selected patient populations.

Vaccine Strategies in Gliomas

Purpose of reviewTo discuss the current state of glioma vaccine development and highlight the challenges associated with clinical implementation of these approaches.Recent findingsVaccination strategies against gliomas have matured considerably during the past years, although proof-of efficacy from controlled clinical trials is still lacking. Advances in antigen discovery, including the definition of neoepitopes including epidermal growth factor receptor variant III (EGFRvIII), isocitrate dehydrogenase (IDH)1R132H and Histone (H)3.3K27M, using multi-omic approaches and computational algorithms allow targeting single antigens, but also implementing truly personalized approaches. In addition, new concepts of vaccine manufacturing including RNA and DNA vaccines improve immunogenicity and applicability in personalized settings.SummaryAs an increasing amount of clinical data defy the concept of the central nervous system (CNS) as a strictly immunoprivileged site, novel vaccine approaches enter the clinic including critical efforts to identify biomarkers of response and resistance and strategies to overcome the immunosuppressive glioma microenvironment.

In situ proximity ligation assay to evaluate presentation of a mutated CD4 epitope in human glioma tissue

astrocytic end feet that form, togetherwith the endothelial cells, pericytes and the basal lamina, the blood brain barrier (BBB). The close interaction of these components, which is necessary for maintaining the integrity of the BBB, is still poorly understood. In this study we investigated the time course and structural correlates of BBB-damage in a model of NMO and the impact of astrocyte loss on the breakdown and restoration of the BBB. Using an in vivo model of focal NMO in rats based on injection of a recombinant human anti-AQP4 antibody and complement, we found a prominent opening of the BBB to endogenous albumin, immunoglobulin G and fibrinogen and the exogenousmarkermolecules FITC-albumin and Texas Red Cadaverine at early stages (6 h) of lesion development. The extravasation of these molecules was on protein level accompanied by the loss of the tight junction (TJ)molecule occludin from the blood vessels in the lesions, while the TJ molecules claudin5 and claudin3 were largely preserved. On mRNA level a compensatory upregulation of these TJ molecules was observed at the same time. Interestingly, the BBB integrity was rapidly restored (within 24 h), although astrocyte loss was highest at this time point and occludin was still lacking from the lesion, thus indicating compensatory mechanisms at the BBB independent of astrocyte signaling. In conclusion, there is a prominent breakdown of the BBB in early stages of experimental NMO associated with loss of astrocytes and tight junction molecules. Several hours later, the BBB is restored again, even though astrocytes are still absent from the lesion.