Georg Karpel-Massler

Therapeutic Inhibition of the Epidermal Growth Factor Receptor in High-Grade Gliomas: Where Do We Stand?

High-grade gliomas account for the majority of intra-axial brain tumors. Despite abundant therapeutic efforts, clinical outcome is still poor. Thus, new therapeutic approaches are intensely being investigated. Overexpression of the epidermal growth factor receptor (HER1/EGFR) is found in various epithelial tumors and represents one of the most common molecular abnormalities seen in high-grade gliomas. Dysregulated HER1/EGFR is found in 40% to 50% of glioblastoma, the most malignant subtype of glioma. Several agents such as tyrosine kinase (TK) inhibitors, antibodies, radio-immuno conjugates, ligand-toxin conjugates, or RNA-based agents have been developed to target HER1/EGFR or its mutant form, EGFRvIII. To date, most agents are in various stages of clinical development. Clinical data are sparse but most advanced for TK inhibitors. Although data from experimental studies seem promising, proof of a significant clinical benefit is still missing. Among the problems that have to be further addressed is the prediction of the individual patients response to HER1/EGFR-targeted therapeutics based on molecular determinants. It is quite possible that blocking HER1/EGFR alone will not sufficiently translate into a clinical benefit. Therefore, a multiple target approach concomitantly aimed at different molecular sites might be a favorable concept. This review focuses on current HER1/EGFR-targeted therapeutics and their development for high-grade gliomas. (Mol Cancer Res 2009;7(7):1000–12)

Impaired Cerebral Vasomotor Activity in Spontaneous Intracerebral Hemorrhage

Background and Purpose— Impairment of cerebrovascular autoregulation may promote secondary brain injury in acute brain insults. Until now, only limited data are available on autoregulation in patients with spontaneous intracerebral hemorrhage. In the current study, we aimed to investigate cerebrovascular reactivity and its significance for outcome in spontaneous intracerebral hemorrhage. Methods— We continuously recorded mean arterial pressure, intracranial pressure, and cerebral perfusion pressure for mean 95 hours in 20 patients with spontaneous intracerebral hemorrhage. The moving correlation coefficient between mean arterial pressure and intracranial pressure (pressure reactivity index), an index of cerebral vasoreactivity, was calculated from the available artifact-free monitoring time (mean, 50.4 hours). Results— In the univariate analysis pressure reactivity index (r=0.66; P=0.002), hemorrhage volume (r=0.62; P=0.007), cerebral perfusion pressure (r=−0.71; P=0.001), mean arterial pressure (r=−0.61; P=0.005), and hematoma growth (r=0.53; P=0.02) significantly correlated with National Institutes of Health Stroke Scale Score at discharge. In a multivariate stepwise linear regression model, pressure reactivity index remained the only independent predictor of outcome (&bgr;=0.659; P=0.004). In the subgroup of patients with pressure reactivity index greater than a functional threshold of >0.2, the correlation between mean cerebral perfusion pressure and outcome remained significant (r=−0.73; P=0.0102), whereas National Institutes of Health Stroke Scale Score at discharge did not correlate with cerebral perfusion pressure in patients with pressure reactivity index <0.2 (r=−0.05; P=0.9078). Conclusions— We found evidence for impaired cerebral vasomotor activity as measured by pressure reactivity index in patients with spontaneous intracerebral hemorrhage. We suggest that impaired cerebrovascular reactivity contributes to poor outcome in intracerebral hemorrhage patients. This effect may be mediated by fluctuations in cerebral perfusion.

Bortezomib primes glioblastoma including glioblastoma stem cells for TRAIL by increasing tBid stability and mitochondrial apoptosis

Purpose: Searching for novel approaches to sensitize glioblastoma for cell death, we investigated the proteasome inhibitor bortezomib. Experimental Design: The effect of bortezomib on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)–induced apoptosis signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures, and in an in vivo model. Results: Bortezomib and TRAIL synergistically trigger cell death and reduce colony formation of glioblastoma cells (combination index < 0.1). Investigations into the underlying molecular mechanisms reveal that bortezomib and TRAIL act in concert to cause accumulation of tBid, the active cleavage product of Bid. Also, the stability of TRAIL-derived tBid markedly increases on proteasome inhibition. Notably, knockdown of Bid significantly decreases bortezomib- and TRAIL-mediated cell death. By comparison, silencing of Noxa, which is also upregulated by bortezomib, does not confer protection. Coinciding with tBid accumulation, the activation of Bax/Bak and loss of mitochondrial membrane potential are strongly increased in cotreated cells. Overexpression of Bcl-2 significantly reduces mitochondrial perturbations and cell death, underscoring the functional relevance of the mitochondrial pathway. In addition, bortezomib cooperates with TRAIL to reduce colony formation of glioblastoma cells, showing an effect on long-term survival. Of note, bortezomib profoundly enhances TRAIL-triggered cell death in primary cultured glioblastoma cells and in patient-derived glioblastoma stem cells, underlining the clinical relevance. Importantly, bortezomib cooperates with TRAIL to suppress tumor growth in an in vivo glioblastoma model. Conclusion: These findings provide compelling evidence that the combination of bortezomib and TRAIL presents a promising novel strategy to trigger cell death in glioblastoma, including glioblastoma stem cells, which warrants further investigation. Clin Cancer Res; 17(12); 4019–30. ©2011 AACR.

Inhibition of NF-κB Signaling Ablates the Invasive Phenotype of Glioblastoma

Glioblastoma multiforme, the most common primary brain tumor, is highly refractory to therapy, mainly due to its ability to form micrometastases, which are small clusters or individual cells that rapidly transverse the brain and make full surgical resection impossible. Here, it is demonstrated that the invasive phenotype of glioblastoma multiforme is orchestrated by the transcription factor NF-κB which, via metalloproteinases (MMP), regulates fibronectin processing. Both, cell lines and tumor stem cells from primary glioblastoma multiforme, secrete high levels of fibronectin which when cleaved by MMPs forms an extracellular substrate. Subsequently, forming and interacting with their own microenvironment, glioblastoma multiforme cells are licensed to invade their surroundings. Mechanistic study revealed that NF-κB inhibition, either genetically or pharmacologically, by treatment with Disulfiram, significantly abolished the invasive phenotype in the chick chorioallantoic membrane assay. Furthermore, having delineated the underlying molecular mechanism of glioblastoma multiforme invasion, the potential of a disulfiram-based therapy was revealed in a highly invasive orthotrophic glioblastoma multiforme mouse model. Implications: This study defines a novel therapeutic approach that inhibits micrometastases invasion and reverts lethal glioblastoma into a less aggressive disease. Mol Cancer Res; 11(12); 1611–23. ©2013 AACR.

A critical evaluation of PI3K inhibition in Glioblastoma and Neuroblastoma therapy

Members of the PI3K/Akt/mTor signaling cascade are among the most frequently altered proteins in cancer, yet the therapeutic application of pharmacological inhibitors of this signaling network, either as monotherapy or in combination therapy (CT) has so far not been particularly successful. In this review we will focus on the role of PI3K/Akt/mTOR in two distinct tumors, Glioblastoma multiforme (GBM), an adult brain tumor which frequently exhibits PTEN inactivation, and Neuroblastoma (NB), a childhood malignancy that affects the central nervous system and does not harbor any classic alterations in PI3K/Akt signaling. We will argue that inhibitors of PI3K/Akt signaling can be components for potentially promising new CTs in both tumor entities, but further understanding of the signal cascade’s complexity is essential for successful implementation of these CTs. Importantly, failure to do this might lead to severe adverse effects, such as treatment failure and enhanced therapy resistance.

Phosphoinositide 3-Kinases Upregulate System xc− via Eukaryotic Initiation Factor 2α and Activating Transcription Factor 4 – A Pathway Active in Glioblastomas and Epilepsy

AIMS Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc(-) imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc(-) and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. RESULTS PI3Ks induce system xc(-) through glycogen synthase kinase 3β (GSK-3β) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. INNOVATION Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3β, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc(-). Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. CONCLUSION PI3K-regulated system xc(-) activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate.

TIC10/ONC201 synergizes with Bcl-2/Bcl-xL inhibition in glioblastoma by suppression of Mcl-1 and its binding partners in vitro and in vivo

Glioblastoma is the most frequent primary brain tumor in adults. Current therapeutic options are sparse and the prognosis of patients suffering from this disease is grim. Abundance in intratumoral heterogeneity among different deregulated signaling pathways is a hallmark of glioblastoma and likely accounts for its recurrence and resistance to treatment. Glioblastomas harbor a plethora of deregulated pathways driving tumor formation and growth. In this study, we show that TIC10/ONC201, a promising compound that is currently in planned clinical development, along with Bcl-2/Bcl-xL inhibition by ABT263 yields a strong synergistic antiproliferative effect on pediatric, adult, proneural glioblastoma and glioma stem-like cells. On the molecular level, treatment with TIC10/ONC201 results in a posttranslational decrease of the anti-apoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), through modulation of the chaperone Bag3 and the deubiquitinase Usp9X. Consistently, the combination treatment of TIC10/ONC201 and ABT263 required the presence of functional BAX and BAK to drive intrinsic apoptosis, but is surprisingly independent of the extrinsic apoptotic pathway. Moreover, the expression of Noxa protein was required for efficient apoptosis induction by TIC10/ONC201 and ABT263. Importantly, the drug combination of TIC10/ONC201 and the BH3-mimetic, ABT263, led to a regression of tumors in vivo, without any notable toxicity and side effects. Overall, TIC10/ONC201 along with Bcl-2/Bcl-xL inhibition holds significant promise as a novel potential approach for the treatment of recalcitrant tumors such as glioblastoma.

Artesunate enhances the antiproliferative effect of temozolomide on U87MG and A172 glioblastoma cell lines.

As chemotherapy with temozolomide is far from providing satisfactory clinical outcomes for patients with glioblastoma, more efficient drugs and drug combinations are urgently needed. The anti-malarial artesunate was previously shown to exert a profound cytotoxic effect on various tumor cell lines including those derived from glioblastoma. In the current study, we sought to examine the antiproliferative effect of a combination of temozolomide and artesunate on two different established human glioblastoma cell lines. The IC50 and IC25 were determined for temozolomide and artesunate in U87MG and A172 glioblastoma cell lines after 144 h of continuous drug exposure. The antiproliferative effect of combining both agents at IC50/IC50 and IC25/IC25 was determined by a cell viability assay. Moreover, necrosis and apoptosis were analyzed by annexin V/PI staining and flow cytometric analysis. In addition, cytostatic effects were examined by carboxyfluorescein diacetate succinimidyl ester staining and subsequent flow cytometry. In both glioblastoma cell lines, artesunate was found to enhance the antiproliferative effect exerted by temozolomide. Moreover, artesunate acted in concert with temozolomide in terms of cytostatic and necrotizing effects. These observations suggest that a combination of artesunate and temozolomide might result in increased cytotoxicity in glioblastoma.

RIST: A potent new combination therapy for glioblastoma

Glioblastoma is a highly aggressive, common brain tumor with poor prognosis. Therefore, this study examines a new therapeutic approach targeting oncogenic and survival pathways combined with common chemotherapeutics. The RIST (rapamycin, irinotecan, sunitinib, temozolomide) and the variant aRIST (alternative to rapamycin, GDC‐0941) therapy delineate growth inhibiting effects in established glioblastoma cell lines and primary cultured patient material. These combinations significantly decreased cell numbers and viability compared to inhibitors and chemotherapeutics alone with aRIST being superior to RIST. Notably, RIST/aRIST appeared to be apoptogenic evoked by reduction of anti‐apoptotic protein levels of XIAP and BCL‐2, with concomitant up‐regulation of pro‐apoptotic protein levels of p53 and BAX. The treatment success of RIST therapy was confirmed in an orthotopic mouse model. This combination treatment revealed significantly prolonged survival time and drastically reduced the tumor burden by acting anti‐proliferative and pro‐apoptotic. Surprisingly, in vivo, aRIST only marginally extended survival time with tumor volumes comparable to controls. We found that aRIST down‐regulates the microvessel density suggesting an insufficient distribution of chemotherapy. Further, alterations in different molecular modes of action in vivo than in vitro suggest, that in vivo RIST therapy may mimic the superior aRIST protocols pro‐apoptotic inhibition of pAKT in vitro. Of note, all substances were administered in therapeutically relevant low doses with no adverse side effects observed. We also provide evidence of the potential benefits of the RIST therapy in a clinical setting. Our data indicates RIST therapy as a novel treatment strategy for glioblastoma achieving significant anti‐tumorigenic activity avoiding high‐dose chemotherapy.

Combined Inhibition of HER1/EGFR and RAC1 Results in a Synergistic Antiproliferative Effect on Established and Primary Cultured Human Glioblastoma Cells

Glioblastoma is the most frequent brain tumor of glial origin in adults. With the best available standard-of-care, patients with this disease have a life expectancy of only approximately 15 months after diagnosis. Because the EGF receptor (HER1/EGFR) is one of the most commonly dysregulated oncogenes in glioblastoma, HER1/EGFR–targeted agents, such as erlotinib, were expected to provide a therapeutic benefit. However, their application in the clinical setting failed. Seeking an explanation for this finding, we previously identified several candidate genes for resistance of human glioblastoma cell lines toward erlotinib. On the basis of this panel of genes, we aimed at identifying drugs that synergistically enhance the antiproliferative effect of erlotinib on established and primary glioblastoma cell lines. We found that NSC23766, an inhibitor of RAC1, enhanced the antineoplastic effects of erlotinib in U87MG, T98MG, and A172MG glioblastoma cell lines for the most part in a synergistic or at least in an additive manner. In addition, the synergistic antiproliferative effect of erlotinib and NSC23766 was confirmed in primary cultured cells, indicating a common underlying cellular and molecular mechanism in glioblastoma. Therefore, agents that suppress RAC1 activation may be useful therapeutic partners for erlotinib in a combined targeted treatment of glioblastoma. Mol Cancer Ther; 12(9); 1783–95. ©2013 AACR.