Early and progressive colonization of the healthy brain is one hallmark of diffuse gliomas, including glioblastomas. We recently discovered ultralong (Ͼ10 to hundreds of microns) membrane protrusions [tumor microtubes (TMs)] extended by glioma cells. TMs have been associated with the capacity of glioma cells to effectively invade the brain and proliferate. Moreover, TMs are also used by some tumor cells to interconnect to one large, resistant multicellular network. Here, we performed a correlative gene-expression microarray and in vivo imaging analysis, and identified novel molecular candidates for TM formation and function. Interestingly, these genes were previously linked to normal CNS development. One of the genes scoring highest in tests related to the outgrowth of TMs was tweety-homolog 1 (TTYH1), which was highly expressed in a fraction of TMs in mice and patients. Ttyh1 was confirmed to be a potent regulator of normal TM morphology and of TM-mediated tumor-cell invasion and proliferation. Glioma cells with one or two TMs were mainly responsible for effective brain colonization, and Ttyh1 downregulation particularly affected this cellular subtype, resulting in reduced tumor progression and prolonged survival of mice. The remaining Ttyh1-deficient tumor cells, however, had more interconnecting TMs, which were associated with increased radioresistance in those small tumors. These findings imply a cellular and molecular heterogeneity in gliomas regarding formation and function of distinct TM subtypes, with multiple parallels to neuronal development, and suggest that Ttyh1 might be a promising target to specifically reduce TM-associated brain colonization by glioma cells in patients.

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 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 did not differentially affect GCN2-proficient vs. GCN2-deficient T cells . 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.

Purpose: Resistance is an obstacle of glioma therapy. Despite targeted interventions, tumors harbor primary resistance or become resistant over short course of treatment. This study examined the mouse double minute 2 (MDM2) inhibitor RG7388 together with radiotherapy and analyzed strategies to overcome acquired MDM2 inhibitor resistance in glioblastoma.Experimental Design: Effects of RG7388 and radiotherapy were analyzed in p53 wild-type glioblastoma cell lines and glioma-initiating cells. RG7388 resistant cells were generated by increasing RG7388 doses over 3 months. Regulated pathways were investigated by microarray, qRT-PCR, and immunoblot analysis and specifically inhibited to evaluate rational salvage therapies at RG7388 resistance. Effects of RG7388 and trametinib treatment were challenged in an orthotopical mouse model with RG7388 resistant U87MG glioblastoma cells.Results: MDM2 inhibition required functional p53 and showed synergistic activity with radiotherapy in first-line treatment. Long-term exposure to RG7388 induced resistance by activation of the extracellular signal-regulated kinases 1/2 (ERK1/2)-insulin growth factor binding protein 1 (IGFBP1) signaling cascade, which was specifically overcome by ERK1/2 pathway inhibition with trametinib and knockdown of IGFBP1. Combining trametinib with continued RG7388 treatment enhanced antitumor effects at RG7388 resistance in vitro and in vivo.Conclusions: These data provide a rationale for combining RG7388 and radiotherapy as first-line therapy with a specific relevance for tumors insensitive to alkylating standard chemotherapy and for the addition of trametinib to continued RG7388 treatment as salvage therapy after acquired resistance against RG7388 for clinical practice.

Pituitary carcinomas are rare and neurosurgically challenging lesions, as they commonly relapse after surgical removal. Their prognosis is dismal due to their limited response to radiotherapy and chemotherapy. In recent studies, temozolomide was administered in very few patients with partial effects. We report a patient with an ACTH-secreting pituitary carcinoma and widespread intracranial, spinal and systemic metastases despite repeated surgical treatment, bilateral adrenalectomy, medical treatment and radiotherapy. Additionally to chemotherapy with temozolomide, the patient received SOM230 as salvage therapy with an improvement of the patient's clinical status, and a reduction of ACTH levels. After 12 months of combination therapy a sustained tumor control was achieved and persisted upon monotherapy with SOM230 for more than 9 months thereafter. Thus, temozolomide in combination with SOM230 seems to be promising in patients with ACTH-secreting metastasized pituitary carcinoma.

The objective of this study was to investigate the effects of modulating brain amyloid-β (Aβ) levels at different stages of amyloid pathology on synaptic function, inflammatory cell changes and hippocampal neurogenesis, i.e. processes perturbed in Alzheimer’s disease (AD). Young (4- to 6-month-old) and older (15- to 18-month-old) APPSWE transgenic (Tg2576) mice were treated with the AD candidate drug (+)-phenserine for 16 consecutive days. We found significant reductions in insoluble Aβ1-42 levels in the cortices of both young and older transgenic mice, while significant reductions in soluble Aβ1-42 levels and insoluble Aβ1-40 levels were only found in animals aged 15–18 months. Autoradiography binding with the amyloid ligand Pittsburgh Compound B (3H-PIB) revealed a trend for reduced fibrillar Aβ deposition in the brains of older phenserine-treated Tg2576 mice. Phenserine treatment increased cortical synaptophysin levels in younger mice, while decreased interleukin-1β and increased monocyte chemoattractant protein-1 and tumor necrosis factor-alpha levels were detected in the cortices of older mice. The reduction in Aβ1-42 levels was associated with an increased number of bromodeoxyuridine-positive proliferating cells in the hippocampi of both young and older Tg2576 mice. To determine whether the increased cell proliferation was accompanied by increased neuronal production, the endogenous early neuronal marker doublecortin (DCX) was examined in the dentate gyrus (DG) using immunohistochemical detection. Although no changes in the total number of DCX+-expressing neurons were detected in the DG in Tg2576 mice at either age following (+)-phenserine treatment, dendritic arborization was increased in differentiating neurons in young Tg2576 mice. Collectively, these findings indicate that reducing Aβ1-42 levels in Tg2576 mice at an early pathological stage affects synaptic function by modulating the maturation and plasticity of newborn neurons in the brain. In contrast, lowering Aβ levels in Tg2576 mice when Aβ plaque pathology is prominent mainly alters the levels of proinflammatory cytokines and chemokines.

CD95 (Fas/APO-1), a death receptor family member, activity has been linked to tumorigenicity in multiple cancers, including glioblastoma multiforme (GBM). A phase II clinical trial on relapsed glioblastoma patients demonstrated that targeted inhibition of CD95 signaling via the CD95 ligand (CD95L) binding and neutralizing Fc-fusion protein APG101 (asunercept) prolonged patient survival. Although CD95 signaling may be relevant for multiple aspects of tumor growth, the mechanism of action of APG101 in glioblastoma is not clear. APG101 action was examined by proliferation, apoptosis, and invasion assays with human and murine glioma and human microglial cells, as well as therapy studies with orthotopic gliomas and clinical data. APG101 inhibits CD95L-mediated invasion of glioma cells. APG101 treatment was effective in glioma-bearing mice, independently of the presence or absence of CD4 and CD8 T lymphocytes, which should be sensitive to CD95L. Combined with radiotherapy, APG101 demonstrated a reduction of tumor growth, fewer tumor satellites, reduced activity of matrix metalloproteinases (MMP) as well as prolonged survival of tumor-bearing mice compared with radiotherapy alone. Inhibiting rather than inducing CD95 activity is a break-of-paradigm therapeutic approach for malignant gliomas. Evidence, both and, is provided that CD95L-binding fusion protein treatment enhanced the efficacy of radiotherapy and reduced unwanted proinfiltrative effects by reducing metalloproteinase activity by directly affecting the tumor cells. APG101 (asunercept) successfully used in a controlled phase II glioblastoma trial (NCT01071837) acts anti-invasively by inhibiting matrix metalloproteinase signaling, resulting in additive effects together with radiotherapy and helping to further develop a treatment for this devastating disease. .

There is a lack of relevant prognostic and predictive factors in neurooncology besides mutation of isocitrate dehydrogenase 1, codeletion of 1p/19q and promoter hypermethylation of O (6) -methylguanine-DNA-methyltransferase. More importantly, there is limited translation of these factors into clinical practice. The cancer genome atlas data and also clinical correlative analyses suggest a pivotal role for the epidermal growth factor receptor /protein kinase B/mammalian target of rapamycin (mTOR) pathway in both biology and the clinical course of gliomas. However, attempts to stratify gliomas by activating alterations in this pathway have failed thus far. The tumors of 40 patients with WHO grade II gliomas without immediate postoperative genotoxic treatment and known progression and survival status at a median follow-up of 12.2 years were analyzed for expression of the mTOR complex 2 downstream target N-myc downstream regulated gene (NDRG)1 using immunohistochemistry. Baseline characteristics for NDRG1 absent/low versus moderate/high patients were similar. Time to reintervention was significantly longer in the NDRG1 group (P = 0.026). NDRG1 may become a novel biomarker to guide the decision which WHO°II glioma patients may be followed without postsurgical intervention and which patients should receive genotoxic treatment early on. Validation of this hypothesis will be possible with the observational arm of the RTOG 9802 and the pretreatment step of the EORTC 22033/26032 trials.

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