The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma
© The Author(s). 2018
Received: 21 November 2017
Accepted: 14 February 2018
Published: 27 February 2018
The Correction to this article has been published in Journal of Hematology & Oncology 2018 11:38
The use of alkylating agents such as temozolomide in association with radiotherapy (RT) is the therapeutic standard of glioblastoma (GBM). This regimen modestly prolongs overall survival, also if, in light of the still dismal prognosis, further improvements are desperately needed, especially in the patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors, in which the benefit of standard treatment is less. Tinostamustine (EDO-S101) is a first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule that fuses the DNA damaging effect of bendamustine with the fully functional pan-histone deacetylase (HDAC) inhibitor, vorinostat, in a completely new chemical entity.
Tinostamustine has been tested in models of GBM by using 13 GBM cell lines and seven patient-derived GBM proliferating/stem cell lines in vitro. U87MG and U251MG (MGMT negative), as well as T98G (MGMT positive), were subcutaneously injected in nude mice, whereas luciferase positive U251MG cells and patient-derived GBM stem cell line (CSCs-5) were evaluated the orthotopic intra-brain in vivo experiments.
We demonstrated that tinostamustine possesses stronger antiproliferative and pro-apoptotic effects than those observed for vorinostat and bendamustine alone and similar to their combination and irrespective of MGMT expression. In addition, we observed a stronger radio-sensitization of single treatment and temozolomide used as control due to reduced expression and increased time of disappearance of γH2AX indicative of reduced signal and DNA repair. This was associated with higher caspase-3 activation and reduction of RT-mediated autophagy. In vivo, tinostamustine increased time-to-progression (TTP) and this was additive/synergistic to RT. Tinostamustine had significant therapeutic activity with suppression of tumor growth and prolongation of DFS (disease-free survival) and OS (overall survival) in orthotopic intra-brain models that was superior to bendamustine, RT and temozolomide and showing stronger radio sensitivity.
Our data suggest that tinostamustine deserves further investigation in patients with glioblastoma.
High-grade gliomas are the most frequent and aggressive primary brain tumor in the adults. Grade IV gliomas named also glioblastoma (GBM) show a devastating clinical evolution, a short survival, and a poor quality of life. Currently, therapeutic standards show 27 and 10% of survival when considered the analyses at 2 and 5 years from diagnosis . Although the benefits are modest and of limited duration, temozolomide-based chemotherapy is part of the standard treatment for GBM in the first-line and recurrent settings . Temozolomide is an alkylating agent that triggers DNA damage . Molecular pathways (direct DNA damage reversal, base excision repair [BER], and mismatch repair [MMR]) are activated after alkylation DNA damage as mechanisms of defense towards cell death or large mutations. Temozolomide (TMZ) is a second-generation imidazotetrazine lipophilic prodrug that crosses the blood-brain barrier (BBB) and induces GBM cell death by introducing alkyl groups into DNA cross links . This drug forms O6-methylguanine, N3 methyl adenine, and N7 methylguanine adducts leading to the formation of single- and double-strand DNA breaks associated with both apoptosis and senescence of tumor cells . Temozolomide resistance involves, indeed, both O6-methylguanine-DNA-methyltransferase (MGMT)-dependent and MGMT-independent mechanisms. MGMT protects the cellular genome from the mutagenic effects of TMZ by removing the O6-alkylguanine DNA adducts , thereby reducing the effect of TMZ. MGMT promoter methylation status is responsible for regulating MGMT expression and has been statistically significantly correlated with increased survival in GBM patients receiving the standard treatment (49 versus 15%, 2-year survival rate) [5, 6]. The MMR system is critical for the maintenance of replication fidelity and for inducing appropriate cellular responses to DNA damage . In mismatch repair, MMR-deficient cells may become tolerant to the mispairing of O6-methylguanine with thymine. N3 methyl adenine and N7 methylguanine may be repaired by BER enzymes including MPG, 3-methylpurine-DNA glycosylase. Maximal safe surgical resection followed by RT with concomitant TMZ administration indicates that patients have a post-operative expected survival of 12–15 months [1, 6]. Tumor recurrence is mediated by the recruitment of glioma stem-like cells named also as tumor-initiating cells (TIC). This event is often associated also with resistance to standard therapies [7, 8].
Bendamustine (BDM) is an alkylating chemotherapeutic agent displaying a unique pattern of cytotoxicity compared with TMZ . This is a bifunctional mechlorethamine derivative with properties similar to those seen with cyclophosphamide, chlorambucil, and melphalan, containing a purine-like benzimidazole ring similar to cladribine. This molecule was designed to have both alkylating and anti-metabolite properties. Differences with other alkylating agents have been observed in regard to its effects on DNA repair and cell cycle progression. Moreover, BDM can induce cell death through both apoptotic and non-apoptotic pathways, thereby retaining activity even in cells without a functional apoptotic pathway. BDN appears to have only a partial cross resistance to other alkylating agents [9–11] and was used as a salvage therapy monotherapy for recurrent GBM . Histone deacetylases (HDAC) are frequently overexpressed in tumors including GBMs  and control the gene expression, cell proliferation, and drug resistance of tumor cells . Recently, we observed that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome in patients with GBM treated with standard chemo-radiotherapy . In vitro experiments demonstrated also that silencing of HDAC4 or HDAC6 was able to radio-sensitize resistant U87MG and U251MG GBM cell lines through the promotion of DNA double-strand break (DSB) accumulation and down-modulation of DSB repair molecular machinery activity. HDAC inhibitors (HDACi) represent anti-cancer drugs that alter both the epigenome (and this is the gene expression) and the function of crucial non-histone protein. It has been widely demonstrated that administration of HDACi modulates the differentiation status [14–17] of glioma cells through modification of expression levels of differentiation or stemness proteins  as for example GFAP, β-catenin, or nestin. The detection of these proteins together to stem cell markers (CD133, CD44, and Sox2) may help to discriminate glioma cells to glioma stem-like cells [16, 17]. They also exert a synergistic effect with, or additive to, other anticancer treatments, including RT [18, 19]. While vorinostat (suberoylanilide hydroxamic acid, SAHA) and panobinostat have undergone equal evaluation as combination therapies for TMZ in glioblastoma [20, 21], of the potential for HDACi may be as a radio-sensitizer inducing chromatin relaxation, altering transcription of DNA damage repair genes and common cell death pathway synergisms . Furthermore, despite increasing the efficacy of chemotherapeutic agents such as TMZ [22, 23], HDAC inhibitors may potentiate the evolution of acquired TMZ resistance linked to MGMT upregulation in glioblastoma xenografts . Tinostamustine (EDO-S101; TINO) is the first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule which was designed to create a very potent cytotoxic agent for systemic use, with the aim of increasing the efficacy of the alkylating DNA damage through deacetylase-mediated chromatin relaxation. In TINO, the active structures of SAHA and BDM were fused together to create a completely new chemical entity. Preclinical experiments and biochemical characteristics are shown in references [25–27] and demonstrated that TINO exerts significant activity against several hematological malignancies. In addition, the molecule is active in primary resistant cells as well as in cells that have acquired resistance .
The aim of this study is to investigate the antitumor effects of TINO in association with radiotherapy in preclinical models and to compare activity with standard of care.
Reagents and drugs preparation
Plasticware and materials for tissue culture were purchased from Euroclone (EuroClone S.p.A, Milan, Italy). Antibodies for β-actin [sc-130065], acetylated-histone H3 (Lys 24) [sc-34262], and total histone H3 (Ser 28) [sc-24516], GFAP (2E1) [sc-33673], nestin [sc-23927], β-catenin [sc-7199], LC3-II [sc-271625], beclin1 [sc-48341], and p62 [sc-28359] were purchased from Santa Cruz (Santa Cruz, CA, USA). Survivin antibody was purchased from Biorbyt (Cambridge, UK). Antibodies against E-cadherin and MIB1 (Ki67) were purchased from Dako (Carpenteria, CA). We used the ApopTag® peroxidase in situ apoptosis detection kit purchased from Merck Millipore (Merck, Milan, Italy). Vessel count was detected by using anti-mouse CD34 from eBioscience, Inc. (Prodotti Gianni SpA, Milan Italy). Bendamustine HCl, TMZ, and vorinostat (SAHA) were purchased from Selleckchem (Aurogene, Rome Italy). Tinostamustine (TINO) was provided by Mundipharma-EDO GmbH. For in vitro cell viability assays, TINO was dissolved in dimethyl sulfoxide (DMSO) and used at final concentrations of < 0.5% DMSO. For in vivo test, TINO was prepared as an intravenous injection solution containing 15% HPBCD, 1.5% acetic acid, and 1.25% NaHCO3. Since TINO undergoes hydrolysis in aqueous solution, it is very important to immediately dose the animals after each fresh formulation is made.
AT101, a small Bcl2 inhibition molecule, was kindly provided from Jeffrey M. Brill (Ascenta Therapeutics, Inc., Malvern, PA). AT101 was dissolved in DMSO to obtain a stock solution of 100 mM. and used at 5 μM in culture.
Twelve human glioma cell lines (U251MG, U373, U118, U138, A172, U87MG, LN19, SW1783, SNB19, LN229, T98G, and D54) were cultured at 37 °C in 5% CO2 and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum, 4 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 1% nonessential amino acid (Thermo Fisher/Life Technologies, Inc., Carlsbad, CA, USA). To minimize the risk of working with misidentified and/or contaminated cell lines, we stocked the cells used in this report at very low passages and used < 20 subcultures. Periodically, a DNA profiling by GenePrint® 10 System (Promega Corporation, Madison, WI) was carried out to authenticate cell cultures. Karyotypically distinct U251MG, SNB19, and U373 cell lines were defined to be the same origin but have different drug treatment sensitivities. Similarly, U138-MG cells show strong similarity to U118-MG cells, sharing at least six derivative marker chromosomes. Luciferase-transfected U87MG cells were kindly provided by Jari E. Heikkila, Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland. Six GBM patient-derived stem cell lines (BT12M, BT25M, BT48EF, BT50EF, BT53M), kindly provided by J. Gregory Cairncross and Samuel Weiss (Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada)  and GSCs-5 and CSCs-7  from Marta Izquierdo (Departamento de Biología Molecular, Universidad Autónoma de Madrid, Spain) were maintained as neurosphere cultures in Neurocult medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with epidermal growth factor (20 ng/ml) and fibroblast growth factor (10 ng/ml). GSCs-5 cells were transfected with pGL4.13 vector (Promega Italia, Milan) using jetPEI (Polyplus) to create a stable luciferase expression clone selected by limited dilution.
Cell viability assay
The cytotoxicity of TINO, BDM, and TMZ was measured by the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies Inc., Tokyo, Japan) as suggested by its producer. The optical density (OD) values were averaged and normalized against the controls to generate dose-response curves to calculate the IC50 values using Grafit software.
Cell cycle and apoptosis analysis
SubG1 cell percentage (resulting in a mixture of necrosis, autophagy, and apoptosis) was detected with Propidium Iodide (PI) by using Tali™ instrument (Thermo Fisher Scientifics, Carlsbad, CA, USA). Apoptosis was analyzed using Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Life Technologies Europe BV, Monza, Italy). All cells were then measured on a Tali® Image-Based Cytometer measuring the fluorescence emission at 530 nm (e.g., FL1) and > 575 nm. The results were expressed as the percentage of cell death by apoptosis in controls and in treated cultures.
HDAC activity assay
HDAC activity was evaluated by a colorimetric HDAC activity assay kit (Enzo Life Sciences GmbH) in nuclear extracts of cells treated as previously described .
Clonogenic survival assay
For clonogenic survival, exponentially growing cells (70% confluence) were cultured in regular media and treated with TINO, at the appropriate concentrations, or vehicle (final DMSO concentration of 0.1%) for 24 h. Tumor cells were then irradiated at room temperature with increasing doses of radiation (0–6 Gy) using an X-ray linear accelerator (dose rate of 200 cGy/min). Non-irradiated controls were handled identically to the irradiated cells, with the exception of the radiation exposure. After treatment, cells were diluted at the appropriate concentration (1000 cells) and re-seeded into a new 100-mm tissue culture dish (in triplicate) and incubated for 14 days. At day 14, the media was removed and colonies were fixed with methanol:acetic acid (10:1, v/v) and stained with crystal violet. Colonies containing > 50 cells were counted. The plating efficiency (PE) was calculated as the number of colonies observed/the number of cells plated; the surviving fraction (SF) was calculated as follows: colonies counted/cells seeded X (PE/100). The mean inactivation dose was calculated according to the method of Fertil , and the cell survival enhancement ratio (ER) was calculated as the ratio of the mean inactivation dose under controlled conditions, divided by the mean inactivation dose after drug exposure, as described by Morgan . We also used the “dose enhancement factor” as the ratio between “dose with radiation alone and dose with radiation + drug”. A value significantly > 1 indicates radio-sensitization.
The comet assay was carried out by using the OxiSelect™ 96-well Comet Assay Kit (Cell Biolabs Inc., San Diego CA) and measured as suggested by its producer. Analyses were performed in triplicate and presented data represent mean ± standard error (SE) and consider three replicated experiments.
AVO staining was used to detect the presence of acidic vesicles after treatments. Cells were treated with a final concentration of 1 μM of acridine orange solution at 37 °C for 15 min, washed in phosphate-buffered saline (PBS), and observed under a fluorescence microscope (excitation = 488 nm, emission = 520 nm) (Carl Zeiss, Germany).
Following treatments, cells, grown in 90 mm diameter Petri dishes, were washed with cold PBS and immediately lysed with 1 ml lysis buffer containing a proteinase and phosphatase inhibitor cocktail. Total lysates were electrophoresed in 7% SDS-PAGE, and separated proteins were transferred to nitrocellulose and probed with the appropriate antibodies using the conditions recommended by the suppliers. Total extracts were normalized by using an anti-β-actin antibody.
After appropriate treatments, floating and adherent cells (obtained from the medium and a PBS wash, or after trypsinization, respectively) were pooled and pelleted by centrifugation. Cell pellets were washed with PBS, lysed with RIPA buffer, and assayed by ELISA determinations for (i) active human Caspase-3 (CBA045, Merck Chemicals Ltd. Nottingham, UK), Beclin-1 protein expression (E98557Hu, USCN life sciences, Houston, TX, USA) and DNA damage assay (EpiQuik in situ DNA Damage Assay Kit; Epigentek, Farmingdale, NY, USA). All experiments were performed following the manufacturer’s protocols. Analyses were performed in triplicate and presented data represent mean ± standard error (SE) and consider three replicated experiments. APOSTRAND™ ELISA apoptosis detection kit (code BML-AK120-0001) and p62 ELISA kit (code ADI-900-212-0001) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Beclin 1 ELISA (code SEJ557Hu) was purchased from 2BScientific (Heyford Park, UK). Human MAP1LC3B / LC3B ELISA Kit was purchased from LifeSpan BioSciences (Seattle, WA, USA).
Mouse glioblastoma xenograft model
Female CD1-nu/nu mice, at 6 weeks of age, were purchased from Charles River (Milan, Italy) under the guidelines established by our Institution (University of L’Aquila, Medical School and Science and Technology School Board Regulations, complying with the Italian government regulation no. 116 January 27, 1992 for the use of laboratory animals). All mice received subcutaneous flank injections (two each) of 1 × 106 U251, U87MG and T98G cells representing models for MGMT negative, MGMT gene methylated and MGMT positive cells. Tumor growth was assessed bi-weekly by measuring tumor diameters with a Vernier caliper. If we considered a xenograft as equivalent to an ovoid having three diameters: the formula used was ‘TW (mg) = tumor volume (mm3) = 4/3πR1 × R2 × R3 in which R1/R2/R3 are the 1/2 diameters (rays), shorter diameter is the thickness/height of tumor, larger diameters are the length and width of tumor [33–35]. At about 10 days after the tumor injection, 30 mice with tumor volumes of 0.5~ 0.8 cm3 were retained and randomly divided into six groups (five mice per group with two tumors each) named (1) control (vehicle), (2) TINO (60 mg/kg at days 1, 8, and 15 q28 days, iv), (3) radiotherapy (RT, 4 Gy delivered in a single fraction ), and (4) TINO plus RT. These treatments were compared with standard therapies consisting of TMZ and TMZ plus RT. Therefore, two additional groups were included: (5) TMZ (16 mg/kg/5 consecutive days) and (6) TMZ plus RT. Anesthetized tumor-bearing mice received a focal irradiation. All mice were shielded with a specially designed lead apparatus to allow irradiation to the right hind limb. Mice were kept under these conditions until all irradiation finished. At the end of experiments (35 days after the start of treatments), animals were sacrificed by carbon dioxide inhalation and tumors were subsequently removed surgically. Half of the tumor was directly frozen in liquid nitrogen for protein analysis and the other half fixed in paraformaldehyde overnight for immunohistochemical analyses. Indirect immunoperoxidase staining of tumor xenograft samples was performed on paraffin-embedded tissue sections (4 μm). Tumor microvessels were counted at × 400 in five arbitrarily selected fields, and the data were presented as number of CD34+ mouse microvessels/× 100 microscopic field for each group. Ki67 labeling index was determined by counting 500 cells at 100× and determining the percentage of cells staining positively for Ki67. Apoptosis was measured as the percentage of tunnel positive cells ±standard deviation (SD) measured on five random fields (400×).
Evaluation of treatment response in vivo (xenograft model)
The following parameters were used to quantify the antitumor effects upon different treatments as previously described [33, 34]: (1) tumor volume measured during and at the end of experiments, (2) tumor weight measured at the end of experiment, (3) tumor progression (TP or doubling time) defined as an increase of greater than 100% of tumor volume with respect to baseline, and (4) time to progression (TTP) defined as the time for tumor progression.
Orthotopic intra-brain model
Female CD1 nu/nu mice were inoculated intra-cerebrally as previously described [33, 34] with luciferase-transfected U251 and patient-derived GBM stem cell line (GSCs-5). Just before treatment initiation (5 days after injection), animals were randomized to treatment groups of 10 mice each. In vivo bioluminescence images were obtained using the UVITEC Cambridge Mini HD6 (UVItec Limited, Cambridge, UK). Animals were anesthetized, and luciferin (150 mg/kg) was injected intra-peritoneally (IP) 15 min prior to imaging. The mice were photographed while placed on their front, and the bioluminescence intensity (BLI) was measured in the region of interest. We deliberately inoculated a small number of cells (3 × 103) to simulate a chemo-radio-therapeutic treatment made after surgery in which a low number of tumor cells, remaining the wound bed, and re-grow resulting in a recurrence. Treatments were started 5 days after cell injection when no luciferase activity was intracranially detectable. Mice were euthanized when they displayed neurological signs (e.g., altered gait, tremors/seizures, lethargy) or weight loss of 20% or greater of pre-surgical weight.
Continuous variables were summarized as mean and SD or as median with 95% CI. For continuous variables not normally distributed, statistical comparisons between control and treated groups were established by carrying out the Kruskal-Wallis tests. When Kruskal-Wallis tests revealed a statistical difference, pair-wise comparisons were made by Dwass-Steel-Chritchlow-Fligner method and the probability of each presumed “non-difference” was indicated. For continuous variables normally distributed, statistical comparisons between control and treated groups were established by carrying out the ANOVA test or by Student t test for unpaired data (for two comparisons). When the ANOVA revealed a statistical difference, pair-wise comparisons were made by Tukey’s HSD (honestly significant difference) test and the probability of each presumed “non-difference” was indicated. Dichotomous variables were summarized by absolute and/or relative frequencies. For dichotomous variables, statistical comparisons between control and treated groups were established by carrying out Fisher’s exact test. For multiple comparisons, the level of significance was corrected by multiplying the P value by the number of comparisons performed (n) according to Bonferroni correction. TTP was analyzed by Kaplan-Meier curves and Gehan’s generalized Wilcoxon test. When more than two survival curves were compared, the log-rank test for trend was used. This tests the probability that there is a trend in survival scores across the groups. All tests were two-sided and were determined by Monte Carlo significance. P values < 0.05 were considered statistically significant. SPSS® (statistical analysis software package) version 10.0 and StatDirect (version. 2.3.3., StatDirect Ltd) were used for statistical analysis and graphic presentation.
First, glioma cell models were grouped for MGMT expression levels. As previously described SF268, T98G, U138, U118, LN18, D54, and SW1783 show high levels of MGMT, whereas U251, U87, A172, U373, SNB19, and LN229 show low or absent levels due to complete or hemi-methylation of MGMT gene [36–39]. Seven GBM patient-derived stem cell lines were characterized as MGMT positive (BT12M, BT25M, BT50EF, and CSC-7) and negative (BT48EF BT53M and CSCs-5) .
Antitumor effects of TINO in glioma cell models: comparison with BDM and SAHA alone or in combination
Tinostamustine increases the effects of radiotherapy in glioma models in vitro
In vivo efficacy of TINO alone or in combination with RT (subcutaneous xenograft model)
The combination with RT and TMZ or TINO showed higher radio-sensitizing effects of TINO than TMZ (Fig. 6c, g and Additional file 1: Figure S1C for U251, T98G, and U87 cell xenografts, respectively). Tinostamustine showed similar effects to the gold standard in all cell models. However, a forest plot graphical representation of this meta-analysis demonstrates greater effects (higher hazard ratios (HR)) with TINO than RT and TMZ as single treatments. Similarly, TINO showed greater RT sensitizing effects with higher HRs when combined with RT compared with the RT plus TMZ combination (Fig. 6c, 6d; Fig. 6h–6l; Additional file 1: Figure S1C, D and E, in U251, T98G, and U87MG cell xenografts, respectively). The combination TINO plus RT was more effective than the gold standard (RT plus TMZ) in all cell xenografts with HR = 0.94, 2.35, and 3.39 in U251, U87MG, and T98G xenografts, respectively, suggesting a higher effects in MGMT+/TMZ resistant T98G cells. Additional file 2: Table S1 summarized the statistical analysis for the Kaplan-Meier curves showed in Additional file 1: Figure S1 and Fig. 6.
Histopathological and immunohistochemical effects of treatment with RT and/or tinostamustine
Orthotopic models: luciferase-transfected GBM cells
High-grade gliomas represent the most aggressive primary brain tumors showing high resistance to classic cytotoxic therapies. DNA damage triggers a series of signaling cascades promoting cell death as well as cellular survival, including DNA repair, cell cycle arrest, and autophagy. The elevated basal and/or stressful levels of both DNA repair and autophagy observed in tumor cells, in contrast to normal cells, have been identified as the most important drug-responsive programs that impact the outcome of anticancer therapy. For over a decade, RT and TMZ combination therapy has been the gold standard with clinical trial of alternatives being unsuccessful [1, 3]. However, the prognosis remains poor and some subgroups of patients, like those with MGMT unmethylated tumors do not benefit from this regimen [1, 8, 9] and therapeutic resistance in MGMT-unmethylated tumors has emerged as an important clinical issue. It is necessary to consider that 35 μM is the blood peak of concentration of TMZ when this compound was administered to humans [44, 45] and this concentration was active in 5 out 12 GBM cells lines and 2 out 7 patient-derived GBM proliferating/stem cell lines. BDM is distributed freely in blood, with a concentration at the peak ranging between 10 and 100 μg/ml (corresponding to 28–280 μM) [46, 47] and that plasma concentrations were approximately 100-fold higher than in mouse brain . So in vitro concentrations of BDM are higher when compared with those observed in vivo. In addition, T98G and SW1783 cell lines, which are characterized by high levels of MGMT, showed a pronounced resistance to TMZ (TMZ IC50 of 147.2 ± 2.1 μM and 234.6 ± 2.3 μM, respectively), whereas A172 and LN229 cell lines, which exhibit no or low MGMT expression, had low TMZ IC50 values (14.1 ± 1.1 μM and 14.5 ± 1.1 μM, respectively). MGMT expression status had little effect on the activity of BDM, which demonstrated an antitumor effect in TMZ-resistant GBM cells (Fig. 1). The potential of SAHA [15, 49] and BDM [10, 11] as a target for radio-sensitization has been previously suggested in studies with other agents in experimental tumor models. In our report we test a new therapeutic strategy to improve the therapeutic outcome of combined radiotherapy with alkylating agents such as BDM by using tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule. This molecule fuses the DNA damaging effect of BDM with the fully functional pan-histone deacetylase (HDAC) inhibitor, vorinostat in a completely new chemical entity [25–27, 49]. We hypothesized that tinostamustine may induce radio-sensitization through different mechanisms associated with enhanced apoptosis despite a RT-induced autophagy. Target cells for these effects are both glioma cells and the glioma stem like cells (GSCs). One of the factors underlying tumor recurrence and poor long-term survival is, indeed, the presence of a cancer stem-like cell population GSCs which contribute to cancer invasion, angiogenesis, immune evasion, therapeutic resistance, and may drive recurrence [50–52]. The HDAC inhibitor vorinostat (SAHA) has received significant attention in recent years as an “epigenetic” drug used to treat solid tumors  which down-modulate cancer stem markers inducing differentiation of GSCs. The data presented here indicate that TINO increases DNA damage induced after RT, based on comet assay analyses and γH2Ax detection. Tinostamustine was able to reduce the expression of autophagy marker (LC3B and beclin1) increasing those of p62 suggesting a shift of cell death from autophagy to apoptosis. A further prove of this event is the reduction of levels of Bcl2 by TINO as well as the pharmacological inhibition of Bcl2 by AT101 (Gossypol). Reduced Bcl2 levels after co-administration of TINO to RT were translated both into an increased activity of caspase 3 (and an increased percentage of annexin V positive apoptotic cells. To further elucidate the role of Bcl2 in increased radio-sensitivity we performed clonogenic experiments on glioma cell cultures by administration of fixed 5 μM AT101. These experiments indicated a strong radio-sensitization of Bcl2 activity inhibition. The analyses of dose enhancement factors, calculated for the administration of AT101 with RT indicated the stronger effect in T98G (DRE = 1.50) and the lower effect in U251 (DRE = 1.15). A172 showed a DRE = 1.25 and U87MG a DRE = 1.28. This DRE values were compared with those observed for Tinostamustine. We found that the administration of AT101 determined similar DRE of Tinostamustine in U87MG and A172 with an increment of 2.4 and 1.6% in Tinostamustine treated cells, respectively. Tinostamustine was more active compared to AT101 in U251 (+ 13.6%) whereas AT101 was more active compared to tinostamustine in T98G (+ 20%) cells. This could be due to differences in basal Bcl2 expression levels observed in the different cell lines. When AT101 was administered with dual tinostamustine + RT (triple co-administration experiments) we noted an additive effect with increments in DRE values of 4% (U87MG), 5.6% (U251), 6.7% (A172) and 30% (T98G). This suggest that the efficacy of tinostamustine may be influenced by Bcl2 levels also if further studies should be necessary including Bcl2 transfection in low Bcl2 expressing glioma cell lines. Preliminary our data suggest that the single administration of Tinostamustine is more active in glioma cells with lower basal levels of Bcl2 (manuscript in preparation). Although the dose of 5.0 μM AT101, used for this analysis, was too high to evaluate a possible therapeutic approach, this was not a topic of the present report also if should be considered for further studies. Increasing evidence suggests that an inflammatory microenvironment may promote invasion by GBM cells [54, 55] through the activation of pathways that recruit myeloid precursors. One interesting possible mechanism involved in the sensitivity of GBM tumors to TINO and TINO plus RT is the recruitment of monocytes/macrophages. The possible involvement of proliferating monocytes is also invoked due to the presence of close “germinal cores/cluster” dispersed in the necrotic tumor masses. This event could have dual effects: to participate in the elimination of necrotic cells (resolution) or to mediate the awakening of quiescent stem cells (leading to recurrence). The latter effect was not supported by results in the orthotopic models, which demonstrated no increase in recurrence with TINO and the TINO plus RT combination after only 35 days of treatment (one treatment cycle). The rate of recurrence and the survival percentage in combination treatment were significantly better than those observed for the standard treatment, TMZ plus RT, providing evidence against recurrence due to stimulation of cancer stem cells.
These data demonstrate that TINO is a broadly active antitumor agent in vitro and in vivo, with potent radio-sensitizing activity in aggressive and TMZ-resistant glioblastoma tumor models, supporting ongoing clinical evaluation of this compound in combination with RT for the treatment of post-surgery glioblastoma patients.
The cost of this research is partially funding by Mundipharma Edo Gmbh, Basel Switzerland.
CF, YC, and TM have made substantial contributions to conception and design of experiments, interpretation of results and have been involved in drafting the manuscript and revising it critically for important intellectual content. AM, CC, GLG, LC, and FM have made substantial contributions to acquisition of in vivo and in vitro data. FV provided orthotopic intra-brain injection expertise. AV and SP have made substantial contribution to obtaining of immunohistochemical data on tissue harvested from untreated or treated tumors. VT has contributed to radiotherapy procedures analyses. CF contributed to the study supervision. All authors have read and approved the manuscript.
Thomas Mehrling is an employee of Mundipharma Edo Gmbh, Basel Switzerland. Other authors declare that they have no competing interests.
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- Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma N. Engl J Med. 2005;352:987–96.View ArticleGoogle Scholar
- Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO, European Organisation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups; National Cancer Institute of Canada Clinical Trials Group. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10:459–66.View ArticlePubMedGoogle Scholar
- Denny BJ, Wheelhouse RT, Stevens MF, Tsang LL, Slack JA. NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry. 1994;33:9045–51.View ArticlePubMedGoogle Scholar
- Sibghat-Ullah, Day RS 3rd. Incision at O6-methylguanine: thymine mispairs in DNA by extracts of human cells. Biochemistry. 1992;31:7998–8008.View ArticlePubMedGoogle Scholar
- Hegi ME, Diserens AC, Gorlia T, Hamou MF, De Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352:997–1003.View ArticlePubMedGoogle Scholar
- Yin AA, Zhang LH, Cheng JX, Dong Y, Liu BL, Han N, Zhang X. The predictive but not prognostic value of MGMT promoter methylation status in elderly glioblastoma patients: a meta-analysis. PLoS One. 2014;9:e85102.View ArticlePubMedPubMed CentralGoogle Scholar
- Dahlrot RH, Hansen S, Jensen SS, Schrøder HD, Hjelmborg J, Kristensen BW. Clinical value of CD133 and nestin in patients with glioma: a population-based study. Int J Clin Exp Pathol. 2014;7:3739–51.PubMedPubMed CentralGoogle Scholar
- Melguizo C, Prados J, González B, Ortiz R, Concha A, Alvarez PJ, Madeddu R, Perazzoli G, Oliver JA, López R, Rodríguez-Serrano F, Aránega A. MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy. J Transl Med. 2012;10:250.View ArticlePubMedPubMed CentralGoogle Scholar
- Blumel S, Goodrich A, Martin C, Dang NH. Bendamustine: a novel cytotoxic agent for hematologic malignancies. Clin J Oncol Nurs. 2008;12:799–806.View ArticlePubMedGoogle Scholar
- Hartmann JT, Mayer F, Schleicher J, Horger M, Huober J, Meisinger I, Pintoffl J, Käfer G, Kanz L, Grünwald V, German sarcoma group. Bendamustine hydrochloride in patients with refractory soft tissue sarcoma: a noncomparative multicenter phase 2 study of the German sarcoma group (AIO-001). Cancer. 2007;110:861–6.View ArticlePubMedGoogle Scholar
- Pan E, Yu D, Zhao X, Neuger A, Smith P, Chinnaiyan P, Yu HH. Phase I study of bendamustine with concurrent whole brain radiation therapy in patients with brain metastases from solid tumors. J Neuro-Oncol. 2014;119:413–20.View ArticleGoogle Scholar
- Chamberlain MC, Johnston SK. Salvage therapy with single agent bendamustine for recurrent glioblastoma. J Neuro-Oncol. 2011;105:523–30.View ArticleGoogle Scholar
- Marampon F, Megiorni F, Camero S, Crescioli C, McDowell HP, Sferra R, Vetuschi A, Pompili S, Ventura L, De Felice F, Tombolini V, Dominici C, Maggio R, Festuccia C, Gravina GL. HDAC4 and HDAC6 sustain DNA double strand break repair and stem-like phenotype by promoting radioresistance in glioblastoma cells. Cancer Lett. 2017;397:1–11.View ArticlePubMedGoogle Scholar
- Restrepo A, Smith CA, Agnihotri S, Shekarforoush M, Kongkham PN, Seol HJ, Northcott P, Rutka JT. Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas. Neuro-Oncology. 2011;13:42–50.View ArticlePubMedGoogle Scholar
- Hsu CC, Chang WC, Hsu TI, Liu JJ, Yeh SH, Wang JY, Liou JP, Ko CY, Chang KY, Chuang JY. Suberoylanilide hydroxamic acid represses glioma stem-like cells. J Biomed Sci. 2016;23:81.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhu T, Li X, Luo L, Wang X, Li Z, Xie P, Gao X, Song Z, Su J, Liang G. Reversion of malignant phenotypes of human glioblastoma cells by β-elemene through β-catenin-mediated regulation of stemness-, differentiation- and epithelial-to-mesenchymal transition-related molecules. J Transl Med. 2015;13:356.View ArticlePubMedPubMed CentralGoogle Scholar
- Svechnikova I, Almqvist PM, Ekström TJ. HDAC inhibitors effectively induce cell type-specific differentiation in human glioblastoma cell lines of different origin. Int J Oncol. 2008;32:821–7.PubMedGoogle Scholar
- Gatti L, Zuco V, Zaffaroni N, Perego P. Drug combinations with proteasome inhibitors in antitumor therapy. Curr Pharm Des. 2013;19:4094–114. ReviewView ArticlePubMedGoogle Scholar
- Smits KM, Melotte V, Niessen HE, Dubois L, Oberije C, Troost EG, Starmans MH, Boutros PC, Vooijs M, van Engeland M, Lambin P. Epigenetics in radiotherapy: where are we heading? Radiother Oncol. 2014;111:168–77. ReviewView ArticlePubMedGoogle Scholar
- Lee EQ, Puduvalli VK, Reid JM, Kuhn JG, Lamborn KR, Cloughesy TF, Chang SM, Drappatz J, Yung WK, Gilbert MR, Robins HI, Lieberman FS, Lassman AB, McGovern RM, Xu J, Desideri S, Ye X, Ames MM, Espinoza-Delgado I, Prados MD, Wen PY. Phase I study of vorinostat in combination with temozolomide in patients with high-grade gliomas: North American brain tumor consortium study 04-03. Clin Cancer Res. 2012;18:6032–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee EQ, Reardon DA, Schiff D, Drappatz J, Muzikansky A, Grimm SA, Norden AD, Nayak L, Beroukhim R, Rinne ML, Chi AS, Batchelor TT, Hempfling K, McCluskey C, Smith KH, Gaffey SC, Wrigley B, Ligon KL, Raizer JJ, Wen PY. Phase II study of panobinostat in combination with bevacizumab for recurrent glioblastoma and anaplastic glioma. Neuro-Oncology. 2015;17:862–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Groselj B, Sharma NL, Hamdy FC, Kerr M, Kiltie AE. Histone deacetylase inhibitors as radiosensitisers: effects on DNA damage signalling and repair. Br J Cancer. 2013;108:748–54. ReviewView ArticlePubMedPubMed CentralGoogle Scholar
- Choi EJ, Cho BJ, Lee DJ, Hwang YH, Chun SH, Kim HH, Kim IA. Enhanced cytotoxic effect of radiation and temozolomide in malignant glioma cells: targeting PI3K-AKT-mTOR signaling, HSP90 and histone deacetylases. BMC Cancer. 2014;14:17.View ArticlePubMedPubMed CentralGoogle Scholar
- Hoja S, Schulze M, Rehli M, Proescholdt M, Herold-Mende C, Hau P, Riemenschneider MJ. Molecular dissection of the valproic acid effects on glioma cells. Oncotarget. 2016;7:62989–3002.View ArticlePubMedPubMed CentralGoogle Scholar
- López-Iglesias AA, Herrero AB, Chesi M, San-Segundo L, González-Méndez L, Hernández-García S, Misiewicz-Krzeminska I, Quwaider D, Martín-Sánchez M, Primo D, Paíno T, Bergsagel PL, Mehrling T, González-Díaz M, San-Miguel JF, Mateos MV, Gutiérrez NC, Garayoa M, Ocio EM. Preclinical anti-myeloma activity of EDO-S101, a new bendamustine-derived molecule with added HDACi activity, through potent DNA damage induction and impairment of DNA repair. J Hematol Oncol. 2017;10(1):127.View ArticlePubMedPubMed CentralGoogle Scholar
- Besse L, Kraus M, Besse A, Bader J, Silzle T, Mehrling T, Driessen C. The first-in-class alkylating HDAC inhibitor EDO-S101 is highly synergistic with proteasome inhibition against multiple myeloma through activation of multiple pathways. Blood Cancer J. 2017;7(7):e589.View ArticlePubMedPubMed CentralGoogle Scholar
- Mehrling T, Chen Y. The alkylating-HDAC inhibition fusion principle: taking chemotherapy to the next level with the first in class molecule EDO-S101. Anti Cancer Agents Med Chem. 2016;16(1):20–8.View ArticleGoogle Scholar
- Luchman HA, Stechishin OD, Dang NH, Blough MD, Chesnelong C, Kelly JJ, Nguyen SA, Chan JA, Weljie AM, Cairncross JG, Weiss S. An in vivo patient-derived model of endogenous IDH1-mutant glioma. Neuro-Oncology. 2012;14:184–91.View ArticlePubMedGoogle Scholar
- Mendiburu-Eliçabe M, Gil-Ranedo J, Izquierdo M. Efficacy of rapamycin against glioblastoma cancer stem cells. Clin Transl Oncol. 2014;16:495–502.View ArticlePubMedGoogle Scholar
- Gravina GL, Marampon F, Muzi P, Mancini A, Piccolella M, Negri-Cesi P, Motta M, Lenzi A, Di Cesare E, Tombolini V, Jannini EA, Festuccia C. PXD101 potentiates hormonal therapy and prevents the onset of castration-resistant phenotype modulating androgen receptor, HSP90, and CRM1 in preclinical models of prostate cancer. Endocr Relat Cancer. 2013;20:321–37.View ArticlePubMedGoogle Scholar
- Fertil B, Dertinger H, Courdi A, Malaise EP. Mean inactivation dose: a useful concept for intercomparison of human cell survival curves. Radiat Res. 1984;99:73–84.View ArticlePubMedGoogle Scholar
- Morgan MA, Parsels LA, Parsels JD, Mesiwala AK, Maybaum J, Lawrence TS. Role of checkpoint kinase 1 in preventing premature mitosis in response to gemcitabine. Cancer Res. 2005;65:6835–42.View ArticlePubMedGoogle Scholar
- Gravina GL, Mancini A, Marampon F, Colapietro A, Delle Monache S, Sferra R, Vitale F, Richardson PJ, Patient L, Burbidge S, Festuccia C. The brain-penetrating CXCR4 antagonist, PRX177561, increases the antitumor effects of bevacizumab and sunitinib in preclinical models of human glioblastoma. J Hematol Oncol. 2017;10:5.View ArticlePubMedPubMed CentralGoogle Scholar
- Gravina GL, Mancini A, Mattei C, Vitale F, Marampon F, Colapietro A, Rossi G, Ventura L, Vetuschi A, Di Cesare E, Fox JA, Festuccia C. Enhancement of radiosensitivity by the novel anticancer quinolone derivative vosaroxin in preclinical glioblastoma models. Oncotarget. 2017;8:29865–86.PubMedPubMed CentralGoogle Scholar
- Gravina GL, Mancini A, Colapietro A, Vitale F, Vetuschi A, Pompili S, Rossi G, Marampon F, Richardson PJ, Patient L, Patient L, Burbidge S, Festuccia C. The novel CXCR4 antagonist, PRX177561, reduces tumor cell proliferation and accelerates cancer stem cell differentiation in glioblastoma preclinical models. Tumour Biol. 2017;39:1010428317695528.View ArticlePubMedGoogle Scholar
- Perazzoli G, Prados J, Ortiz R, Caba O, Cabeza L, Berdasco M, Gónzalez B, Melguizo C. Temozolomide resistance in glioblastoma cell lines: implication of MGMT, MMR, P-glycoprotein and CD133 expression. PLoS One. 2015;10:e0140131.View ArticlePubMedPubMed CentralGoogle Scholar
- Ryu CH, Yoon WS, Park KY, Kim SM, Lim JY, Woo JS, Jeong CH, Hou Y, Jeun SS. Valproic acid downregulates the expression of MGMT and sensitizes temozolomide-resistant glioma cells. J Biomed Biotechnol. 2012;2012:987495.View ArticlePubMedPubMed CentralGoogle Scholar
- Gielen PR, Aftab Q, Ma N, Chen VC, Hong X, Lozinsky S, Naus CC, Sin WC. Connexin43 confers temozolomide resistance in human glioma cells by modulating the mitochondrial apoptosis pathway. Neuropharmacology. 2013;75:539–48.View ArticlePubMedGoogle Scholar
- Blough MD, Westgate MR, Beauchamp D, Kelly JJ, Stechishin O, Ramirez AL, Weiss S, Cairncross JG. Sensitivity to temozolomide in brain tumor initiating cells. Neuro-Oncology. 2010;12:756–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Wojton J, Meisen WH, Kaur B. How to train glioma cells to die: molecular challenges in cell death. J Neuro-Oncol. 2016;126:377–84.View ArticleGoogle Scholar
- Zhang L, Zhou Y, Chen K, Shi P, Li Y, Deng M, Jiang Z, Wang X, Li P, Xu B. The pan-Bcl2 inhibitor AT101 activates the intrinsic apoptotic pathway and causes DNA damage in acute myeloid leukemia stem-like cells. Target Oncol. 2017;12:677–87.View ArticlePubMedGoogle Scholar
- Festuccia C, Gravina G, Marampon F, Biordi L, Ficorella C, Tombolini V. Gossypol induces apoptosis and synergize with radiotherapy and temzolomide in glioblastoma cells. Poster 8733 European multidisciplinary cancer congress, Stockholm 23-27 September 2011. Eur J Cancer. 2011;41S1:S584.View ArticleGoogle Scholar
- Desmarais G, Charest G, Fortin D, Bujold R, Mathieu D, Paquette B. Cyclooxygenase-2 inhibitor prevents radiation-enhanced infiltration of F98 glioma cells in brain of Fischer rat. Int J Radiat Biol. 2015;91:624–33.View ArticlePubMedGoogle Scholar
- Shen F, Decosterd LA, Gander M, Leyvraz S, Biollax J, Lejeune F. Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction. J Chromatogr B Biomed Appl. 1995;667:291–300.View ArticlePubMedGoogle Scholar
- Darwish M, Bond M, Hellriegel E, Robertson P Jr, Chovan JP. Pharmacokinetic and pharmacodynamic profile of bendamustine and its metabolites. Cancer Chemother Pharmacol. 2015;75:1143–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Chandrashekar DV, Suresh PS, Kumar R, Bhamidipati RK, Mullangi R, Richter W, Srinivas NR. Sensitive LC-MS/MS method for the simultaneous determination of Bendamustine and its active metabolite, γ-hydroxybendamustine in small volume mice and dog plasma and its application to a pharmacokinetic study in mice and dogs. Drug Res (Stuttg). 2017;67(9):497–508.View ArticleGoogle Scholar
- Zimmer P, Mierau A, Bloch W, Strüder HK, Hülsdünker T, Schenk A, Fiebig L, Baumann FT, Hahn M, Reinart N, Hallek M, Elter T. Post-chemotherapy cognitive impairment in patients with B-cell non-Hodgkin lymphoma: a first comprehensive approach to determine cognitive impairments after treatment with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone or rituximab and bendamustine. Leuk Lymphoma. 2015;56(2):347–52.View ArticlePubMedGoogle Scholar
- Chamberlain MC, Colman H, Kim BT, Raizer J. Salvage therapy with bendamustine for temozolomide refractory recurrent anaplastic gliomas: a prospective phase II trial. J Neurooncol. 2017;131:507–16.Google Scholar
- Chiao MT, Cheng WY, Yang YC, Shen CC, Ko JL. Suberoylanilide hydroxamic acid (SAHA) causes tumor growth slowdown and triggers autophagy in glioblastoma stem cells. Autophagy. 2013;9:1509–26.View ArticlePubMedGoogle Scholar
- Davis B, Shen Y, Poon CC, Luchman HA, Stechishin OD, Pontifex CS, Wu W, Kelly JJ, Blough MD, Terry Fox Research Institute Glioblastoma Consortium. Comparative genomic and genetic analysis of glioblastoma-derived brain tumor-initiating cells and their parent tumors. Neuro-Oncology. 2016;18:350–60.View ArticlePubMedGoogle Scholar
- Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, Fiocco R, Foroni C, Dimeco F, Vescovi A. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res. 2004;64:7011–21.View ArticlePubMedGoogle Scholar
- Nervi C, De Marinis E, Codacci-Pisanelli G. Epigenetic treatment of solid tumours: a review of clinical trials. Clin Epigenetics. 2015;7:127.View ArticlePubMedPubMed CentralGoogle Scholar
- Booth L, Roberts JL, Conley A, Cruickshanks N, Ridder T, Grant S, Poklepovic A, Dent P. HDAC inhibitors enhance the lethality of low dose salinomycin in parental and stem-like GBM cells. Cancer Biol Ther. 2014;15:305–16.View ArticlePubMedGoogle Scholar
- Yu J, Qiu S, Ge Q, Wang Y, Wei H, Guo D, Chen S, Liu S, Li S, Xing H, Rao Q, Wang J, Wang M. A novel SAHA-bendamustine hybrid induces apoptosis of leukemia cells. Oncotarget. 2015;6:20121–31.PubMedPubMed CentralGoogle Scholar
- Teng DC, Sun J, An YQ, Hu ZH, Liu P, Ma YC, Han B, Shi Y. Role of PHLPP1 in inflammation response: its loss contributes to gliomas development and progression. Int Immunopharmacol. 2016;34:229–34.View ArticlePubMedGoogle Scholar