Mikkel Staberg

The impact of bevacizumab treatment on survival and quality of life in newly diagnosed glioblastoma patients.

Glioblastoma multiforme (GBM) remains one of the most devastating tumors, and patients have a median survival of 15 months despite aggressive local and systemic therapy, including maximal surgical resection, radiation therapy, and concomitant and adjuvant temozolomide. The purpose of antineoplastic treatment is therefore to prolong life, with a maintenance or improvement of quality of life. GBM is a highly vascular tumor and overexpresses the vascular endothelial growth factor A, which promotes angiogenesis. Preclinical data have suggested that anti-angiogenic treatment efficiently inhibits tumor growth. Bevacizumab is a humanized monoclonal antibody against vascular endothelial growth factor A, and treatment has shown impressive response rates in recurrent GBM. In addition, it has been shown that response is correlated to prolonged survival and improved quality of life. Several investigations in newly diagnosed GBM patients have been performed during recent years to test the hypothesis that newly diagnosed GBM patients should be treated with standard multimodality treatment, in combination with bevacizumab, in order to prolong life and maintain or improve quality of life. The results of these studies along with relevant preclinical data will be described, and pitfalls in clinical and paraclinical endpoints will be discussed.

Inhibition of histone deacetylases sensitizes glioblastoma cells to lomustine

PurposeGlioblastoma (GBM) ranks among the deadliest solid cancers worldwide and its prognosis has remained dismal, despite the use of aggressive chemo-irradiation treatment regimens. Limited drug delivery into the brain parenchyma and frequent resistance to currently available therapies are problems that call for a prompt development of novel therapeutic strategies. While only displaying modest efficacies as mono-therapy in pre-clinical settings, histone deacetylase inhibitors (HDACi) have shown promising sensitizing effects to a number of cytotoxic agents. Here, we sought to investigate the sensitizing effect of the HDACi trichostatin A (TSA) to the alkylating agent lomustine (CCNU), which is used in the clinic for the treatment of GBM.MethodsTwelve primary GBM cell cultures grown as neurospheres were used in this study, as well as one established GBM-derived cell line (U87 MG). Histone deacetylase (HDAC) expression levels were determined using quantitative real-time PCR and Western blotting. The efficacy of either CCNU alone or its combination with TSA was assessed using various assays, i.e., cell viability assays (MTT), cell cycle assays (flow cytometry, FACS), double-strand DNA break (DSB) quantification assays (microscopy/immunofluorescence) and expression profiling assays of proteins involved in apoptosis and cell stress (Western blotting and protein array).ResultsWe found that the HDAC1, 3 and 6 expression levels were significantly increased in GBM samples compared to non-neoplastic brain control samples. Additionally, we found that pre-treatment of GBM cells with TSA resulted in an enhancement of their sensitivity to CCNU, possibly via the accumulation of DSBs, decreased cell proliferation and viability rates, and an increased apoptotic rate.ConclusionFrom our data we conclude that the combined administration of TSA and CCNU eradicates GBM cells with a higher efficacy than either drug alone, thereby opening a novel avenue for the treatment of GBM.

Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro

BackgroundFor Glioblastoma (GBM) patients, a number of anti-neoplastic strategies using specifically targeting drugs have been tested; however, the effects on survival have been limited. One explanation could be treatment resistance due to redundant signaling pathways, which substantiates the need for combination therapies. In GBM, both the epidermal growth factor receptor (EGFR) and the notch signaling pathways are often deregulated and linked to cellular growth, invasion and angiogenesis. Several studies have confirmed cross-talk and co-dependence of these pathways. Therefore, this study aimed at testing a combination treatment strategy using inhibitors targeting the notch and EGFR pathways.MethodsFor evaluation of cell viability a standard MTT assay was used. Western blotting (WB) and Q-RT-PCR were employed in order to assess the protein- and mRNA expression levels, respectively. In order to determine angiogenic processes, we used an endothelial spheroid sprouting assay. For assessment of secreted VEGF from GBM cells we performed a VEGF-quantikine ELISA.ResultsGBM cells were confirmed to express EGFR and Notch and to have the capacity to induce endothelial cell sprouting. Inhibition of EGFR and Notch signaling was achieved using either Iressa (gefitinib) or the gamma-secretase inhibitor DAPT. Our data showed that DAPT combined with Iressa treatment displayed increased inhibitory effect on cell viability and abrogated expression and activation of major pro-survival pathways. Similarly, the combinational treatment significantly increased abrogation of GBM-induced endothelial cell sprouting suggesting reduced GBM angiogenesis.ConclusionThis study finds that simultaneous targeting of notch and EGFR signaling leads to enhanced inhibitory effects on GBM-induced angiogenesis and cell viability, thereby stressing the importance of further evaluation of this targeting approach in a clinical setting.

VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance

Abstract Background Glioblastoma ranks among the most lethal cancers, with current therapies offering only palliation. Paracrine vascular endothelial growth factor (VEGF) signaling has been targeted using anti-angiogenic agents, whereas autocrine VEGF/VEGF receptor 2 (VEGFR2) signaling is poorly understood. Bevacizumab resistance of VEGFR2-expressing glioblastoma cells prompted interrogation of autocrine VEGF-C/VEGFR2 signaling in glioblastoma. Methods Autocrine VEGF-C/VEGFR2 signaling was functionally investigated using RNA interference and exogenous ligands in patient-derived xenograft lines and primary glioblastoma cell cultures in vitro and in vivo. VEGF-C expression and interaction with VEGFR2 in a matched pre- and post-bevacizumab treatment cohort were analyzed by immunohistochemistry and proximity ligation assay. Results VEGF-C was expressed by patient-derived xenograft glioblastoma lines, primary cells, and matched surgical specimens before and after bevacizumab treatment. VEGF-C activated autocrine VEGFR2 signaling to promote cell survival, whereas targeting VEGF-C expression reprogrammed cellular transcription to attenuate survival and cell cycle progression. Supporting potential translational significance, targeting VEGF-C impaired tumor growth in vivo, with superiority to bevacizumab treatment. Conclusions Our results demonstrate VEGF-C serves as both a paracrine and an autocrine pro-survival cytokine in glioblastoma, promoting tumor cell survival and tumorigenesis. VEGF-C permits sustained VEGFR2 activation and tumor growth, where its inhibition appears superior to bevacizumab therapy in improving tumor control.

Targeting glioma stem‐like cell survival and chemoresistance through inhibition of lysine‐specific histone demethylase KDM2B

Glioblastoma (GBM) ranks among the most lethal cancers, with current therapies offering only palliation. Inter‐ and intrapatient heterogeneity is a hallmark of GBM, with epigenetically distinct cancer stem‐like cells (CSCs) at the apex. Targeting GSCs remains a challenging task because of their unique biology, resemblance to normal neural stem/progenitor cells, and resistance to standard cytotoxic therapy. Here, we find that the chromatin regulator, JmjC domain histone H3K36me2/me1 demethylase KDM2B, is highly expressed in glioblastoma surgical specimens compared to normal brain. Targeting KDM2B function genetically or pharmacologically impaired the survival of patient‐derived primary glioblastoma cells through the induction of DNA damage and apoptosis, sensitizing them to chemotherapy. KDM2B loss decreased the GSC pool, which was potentiated by coadministration of chemotherapy. Collectively, our results demonstrate KDM2B is crucial for glioblastoma maintenance, with inhibition causing loss of GSC survival, genomic stability, and chemoresistance.

Abstract 1369: Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The high-grade glioma, Glioblastoma Multiforme (GBM), is the most prevalent and aggressive brain tumour in adults with a median survival of fifteen months. Thus new therapeutic targets are urgently needed. GBM is characterized by extensive vascularisation why targeting angiogenesis has promising potential in improving the treatment of GBM patients. Aberrant EGFR signaling is another characteristic of GBM, and increased EGFR downstream signaling is believed to correlate with poor prognosis. However, little is known of the involvement of EGFR signaling in GBM angiogenesis. Notch signaling is known for its role in angiogenesis during foetal development and in other cancers, but the importance of Notch signaling in GBM angiogenesis is yet to be elucidated. Recently, it has been shown, that crosstalk between Notch- and EGFR signaling occurs in cancer cells, and aberrant EGFR signaling may therefore play a functional role in GBM angiogenesis through crosstalk with Notch signaling. The aim of this study was to investigate the effect of combined Notch- and EGFR inhibition on GBM cell viability and on ability of GBM cells to induce angiogenesis in endothelial cells. In vitro assays of GBM angiogenesis were established using two patient derived GBM cell cultures, one expressing the mutated EGFR, the EGFRvIII, and one expressing wild type EGFR. The functional relevance of EGFR- and Notch signaling in GBM cells was tested using the tyrosine kinase inhibitor Iressa for EGFR inhibition, and the gamma-secretase inhibitor DAPT for Notch inhibition. We show that combined inhibition of EGFR- and Notch signaling caused additive inhibition of cell viability of both cell cultures in vitro. Abrogated EGFR/Notch signaling in GBM cells further reduced angiogenesis in endothelial cells exposed to conditioned media from treated GBM cells. Moreover, when GBM cells were treated with the inhibitors, this inhibited phosphorylation and activation of the two survival signalling pathways ERK and Akt, and abrogated secretion and expression of the most prominent angiogenic factor - VEGF. The presented results indicate that EGFR- and Notch signaling play a functional role in GBM angiogenesis and that combined treatment with drugs targeting both the EGFR- and Notch pathway could prove beneficial over single drug therapy in targeting both the tumorigenic and angiogenic potential of GBM cells. Citation Format: Mikkel Staberg, Signe Regner Michaelsen, Louise Stobbe Olsen, Mette Kjolhede Nedergaard, Mette Villingshoj, Hans Skovgaard Poulsen. Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1369. doi:10.1158/1538-7445.AM2015-1369