Lotte M. E. Berghauser Pont

The HDAC Inhibitors Scriptaid and LBH589 Combined with the Oncolytic Virus Delta24-RGD Exert Enhanced Anti-Tumor Efficacy in Patient-Derived Glioblastoma Cells.

Background A phase I/II trial for glioblastoma with the oncolytic adenovirus Delta24-RGD was recently completed. Delta24-RGD conditionally replicates in cells with a disrupted retinoblastoma-pathway and enters cells via αvβ3/5 integrins. Glioblastomas are differentially sensitive to Delta24-RGD. HDAC inhibitors (HDACi) affect integrins and share common cell death pathways with Delta24-RGD. We studied the combination treatment effects of HDACi and Delta24-RGD in patient-derived glioblastoma stem-like cells (GSC), and we determined the most effective HDACi. Methods SAHA, Valproic Acid, Scriptaid, MS275 and LBH589 were combined with Delta24-RGD in fourteen distinct GSCs. Synergy was determined by Chou Talalay method. Viral infection and replication were assessed using luciferase and GFP encoding vectors and hexon-titration assays. Coxsackie adenovirus receptor and αvβ3 integrin levels were determined by flow cytometry. Oncolysis and mechanisms of cell death were studied by viability, caspase-3/7, LDH and LC3B/p62, phospho-p70S6K. Toxicity was studied on normal human astrocytes. MGMT promotor methylation status, TCGA classification, Rb-pathway and integrin gene expression levels were assessed as markers of responsiveness. Results Scriptaid and LBH589 acted synergistically with Delta24-RGD in approximately 50% of the GSCs. Both drugs moderately increased αvβ3 integrin levels and viral infection in responding but not in non-responding GSCs. LBH589 moderately increased late viral gene expression, however, virus titration revealed diminished viral progeny production by both HDACi, Scriptaid augmented caspase-3/7 activity, LC3B conversion, p62 and phospho-p70S6K consumption, as well as LDH levels. LBH589 increased LDH and phospho-p70S6K consumption. Responsiveness correlated with expression of various Rb-pathway genes and integrins. Combination treatments induced limited toxicity to human astrocytes. Conclusion LBH589 and Scriptaid combined with Delta24-RGD revealed synergistic anti-tumor activity in a subset of GSCs. Both HDACi moderately augmented viral infection and late gene expression, but slightly reduced progeny production. The drugs differentially activated multiple cell death pathways. The limited toxicity on astrocytes supports further evaluation of the proposed combination therapies.

DNA damage response and anti-apoptotic proteins predict radiosensitization efficacy of HDAC inhibitors SAHA and LBH589 in patient-derived glioblastoma cells

HDAC inhibitors have radiosensitizing effects in established cancer cell lines. This study was conducted to compare the efficacy of SAHA, LBH589, Valproic Acid (VPA), MS275 and Scriptaid in the patient-derived glioblastoma model. In more detail, SAHA and LBH589 were evaluated to determine predictors of response. Acetylated-histone-H3, γH2AX/53BP1, (p)Chek2/ATM, Bcl-2/Bcl-XL, p21(CIP1/WAF1) and caspase-3/7 were studied in relation to response. SAHA sensitized 50% of cultures, LBH589 45%, VPA and Scriptaid 40% and MS275 60%. Differences after treatment with SAHA/RTx or LBH589/RTx in a sensitive and resistant culture were increased acetylated-H3, caspase-3/7 and prolonged DNA damage repair γH2AX/53BP1 foci. pChek2 was found to be associated with both SAHA/RTx and LBH589/RTx response with a positive predictive value (PPV) of 90%. Bcl-XL had a PPV of 100% for LBH589/RTx response. Incubation with HDACi 24 and 48 hours pre-RTx resulted in the best efficacy of combination treatment. In conclusion a subset of patient-derived glioblastoma cultures were sensitive to HDACi/RTx. For SAHA and LBH589 responses were strongly associated with pChek2 and Bcl-XL, which warrant further clinical exploration. Additional information on responsiveness was obtained by DNA damage response markers and apoptosis related proteins.

ABT-888 enhances cytotoxic effects of temozolomide independent of MGMT status in serum free cultured glioma cells

BackgroundThe current standard of care for Glioblastoma Multiforme (GBM) consists of fractionated focal irradiation with concomitant temozolomide (TMZ) chemotherapy. A promising strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). The objective of the present study was to investigate the therapeutic benefit of combination therapy in patient-derived glioma stem-like cells (GSC).MethodsCombination therapy feasibility was tested on established GBM cell lines U373 and T98. We developed an in vitro drug-screening assay based on GSC cultures derived from a panel of primary patient tissue samples (n = 20) to evaluate the effect of PARPi (ABT-888) monotherapy and combination therapy with TMZ. Therapeutic effect was assessed by viability, double stranded breaks, apoptosis and autophagy assays and longitudinal microscopic cell monitoring was performed. O-6-methylguanine-DNA methyltransferase (MGMT) status was determined by methylation assay and protein expression by western blots.ResultsPARPi monotherapy was found to decrease viability by more than 25% in 4 of the 20 GSCs (20%) at 10 μM. TMZ monotherapy at 50 μM and 100 μM was effective in 12 and 14 of the 20 GSCs, respectively. TMZ resistance to 100 μM was found in 7 of 8 MGMT protein positive cultures. Potentiation of TMZ therapy through PARPi was found in 90% (n = 20) of GSCs, of which 6 were initially resistant and 7 were sensitive to TMZ monotherapy. Increased induction of double stranded breaks and apoptosis were noted in responsive GSCs. There was a trend noted, albeit statistically insignificant, of increased autophagy both in western blots and accumulation of autophagosomes.ConclusionPARPi mediated potentiation of TMZ is independent of TMZ sensitivity and can override MGMT(-) mediated resistance when administered simultaneously. Response to combination therapy was associated with increased double strand breaks induction, and coincided by increased apoptosis and autophagy. PARPi addition potentiates TMZ treatment in primary GSCs. PARPi could potentially enhance the therapeutic efficacy of the standard of care in GBM.

Therapeutic concentrations of anti-epileptic drugs do not inhibit the activity of the oncolytic adenovirus Delta24-RGD in malignant glioma

The oncolytic adenovirus Delta24‐RGD is currently being tested in phase I trials for the treatment of glioblastoma (GBM). Literature suggests that frequently prescribed anticonvulsants for these patients, phenytoin (PHE), valproic acid (VPA) and levetiracetam (LEV), may interfere with cellular mechanisms of cancer or oncolytic virus activity. We therefore investigated the direct effects of these drugs on Delta24‐RGD infection and oncolytic activity.

Currarino’s triad diagnosed in an adult woman

PurposeTo report on a female patient diagnosed with Currarino’s triad in adulthood.Case reportThis case presents an adult patient with a medical history of a congenital anal atresia, a partial sacral agenesis, and a surgically treated ectopic anus. After a coincidentally observed presacral mass by MRI, due to unexplained constipation later in adulthood, Currarino’s triad was suspected in this patient. This triad consists of anorectal malformation(s), sacrococcygeal defects and a presacral mass of various origin. Further investigation confirmed the mass to be a meningocele, and showed a tethered cord and a syrinx.ConclusionsIn (young) patients with anorectal malformations, although having no other symptoms, further examination might be required to exclude Currarino’s triad. Importance of early diagnosis and multidisciplinary assessment is recommended to establish adequate treatment if needed.

TP53 mutated glioblastoma stem-like cell cultures are sensitive to dual mTORC1/2 inhibition while resistance in TP53 wild type cultures can be overcome by combined inhibition of mTORC1/2 and Bcl-2

Background Glioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs). Results We found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged. Conclusion Our data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored.

Management of Chronic Subdural Hematoma: Part I—Pathogenesis and Diagnosis

Chronic subdural hematoma (CSDH) is a common condition in neurosurgical practice. A CSDH is defi ned as an “old” collection of blood and blood breakdown products in the subdural space, developing over a period of longer than 14 days. CSDH has a high prevalence, ranging from 1.72 per 100,000 in the general population to 58 per 100,000 in the elderly (≥65 years). Because of an aging global population, the prevalence of CSDH is expected to increase. Moreover, the use of anticoagulants and antiplatelet treatment for cardiovascular problems, which is an important risk factor for developing CSDH, is increasing. Although CSDH is well treated with burr-hole craniostomy or conservative modalities in selected cases, the mortality rate after 1 year is as high as 32%. Therefore, early diagnosis and adequate treatment are essential. In part I of this review, we discuss the pathophysiology and diagnosis of CSDH. In part II, we present treatment options and the most important determinants of disease outcome in patients with CSDH.

Abstract 299: In vitro compound screening identifies enhancers of adenoviral oncolysis with Delta24-RGD in patient-derived glioblastoma stem cells

The tumor targeted oncolytic adenovirus Delta24-RGD is currently under phase I/II investigation for the malignant brain tumor glioblastoma. Despite encouraging results, the efficacy of oncolytic virotherapy still requires improvements due to heterogeneous or poor responses. In this study, we performed a screen of 446 clinically applied drugs to identify those that enhance Delta24-RGD oncolysis in glioblastoma. Cell viability was determined five days post-infection in Delta24-RGD resistant patient-derived glioblastoma stem cell (GSCs) cultures. Potential ‘hits’ were tested for synergistic viral sensitization using the Chou-Talalay method. Effects on viral infection and replication were investigated using Ad-Luc-RGD and Delta24-RGD-GFP viruses, and apoptosis and necrosis were evaluated using caspase-3/7 and lactate dehydrogenase (LDH) assays. Selection based on the efficacy of combination treatment led to the identification of ten drugs as potential Delta24-RGD sensitizers from the initial screen. Further analysis of effects on viral replication, synergistic interactions and ability to penetrate the blood-brain barrier narrowed this down to four remaining compounds, fluphenazine, indirubin, lofepramine and ranolazine. These four agents increased caspase-3/7 activity and fluphenazine also increased LDH levels in combination with Delta24-RGD. Fluphenazine, indirubin, lofepramine and ranolazine sensitized 12/12, 11/12, 9/12 and 11/12 distinct GSC cultures to Delta24-RGD, respectively. In conclusion, a clinical compound screen on glioblastoma stem cells in combination with in vitro mechanistic studies, revealed four highly effective compounds that sensitize GSCs to Delta24-RGD oncolytic therapy. Citation Format: Lotte ME Berghauser Pont, Rutger Balvers, Jenneke Kloezeman, Michal O. Nowicki, Andreas Kremer, E. Antonio Chiocca, Sieger Leenstra, Clemens MF Dirven, Sean Lawler, Martine LM Lamfers. In vitro compound screening identifies enhancers of adenoviral oncolysis with Delta24-RGD in patient-derived glioblastoma stem cells. [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 299. doi:10.1158/1538-7445.AM2015-299

Abstract 5543: DNA damage response and anti-apoptotic proteins are predictors of histone deacetylase inhibitors-mediated radiosensitization of glioblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background. Histone deacytelase inhibitors (HDACi) have radiosensitizing properties in established cancer cell lines. This study was conducted in the context of ‘precision medicine’, to determine the response of various HDACi as radiosensitizers in a variety of primary glioblastoma stem cell (GSC) cultures. The objective was to assess the response rate, the predictors of outcome and response markers related to DNA repair and apoptosis. Further, the optimal timing and sequence of treatments was investigated of combined treatment as well as effects of fractionation. Methods. SAHA, LBH589, Valproic Acid (VPA), MS-275 and Scriptaid were tested alone and in combination with radiation on twenty-two primary serum-free GSC cultures. SAHA and LBH589 were studied in more detail, with regard to acetylated-H3, p21Cip1/WAF1, Bcl-2, LC3I/II, caspase-3/7, 53BP1, and Chek2/ATM activation in various primary GSC cultures with different sensitivity to treatment. A variety of timing schedules and dose fractions were tested, the latter compared with single radiation dose. Results. The various HDACi sensitized (p 0.05). Conclusion. This study shows that HDACi as radiosensitizers have a wide range of effects in patient-derived GSC cultures, and tumour sensitivity is associated with protein levels of untreated GSC cultures of DNA damage response markers Chek2, ATM, and apoptotic markers Bcl-XL, and Bcl-2. The Chek2 activation upon radiation was associated with response in vitro. Candidates to measure treatment effects are enhanced DNA damage response foci, and caspase-3/7 activation. In the light of precision medicine, these markers need to be studied to assess their in vivo specificity. Furthermore, timing of the treatments is essential to obtain the sensitizing effects. Citation Format: Lotte M. Berghauser Pont, Jenneke Kloezeman, Kishan Naipail, Martin van den Bent, Dik van Gent, Clemens Dirven, Roland Kanaar, Martine Lamfers, Sieger Leenstra. DNA damage response and anti-apoptotic proteins are predictors of histone deacetylase inhibitors-mediated radiosensitization of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5543. doi:10.1158/1538-7445.AM2014-5543