Targeting class I histone deacetylase 2 in MYC amplified group 3 medulloblastoma
- Jonas Ecker1,
- Ina Oehme1,
- Ralph Mazitschek2, 3,
- Andrey Korshunov4, 5,
- Marcel Kool6, 7,
- Thomas Hielscher8,
- Judit Kiss1, 9,
- Florian Selt1, 9,
- Carina Konrad1,
- Marco Lodrini1, 10,
- Hedwig E Deubzer1, 9, 10,
- Andreas von Deimling4, 5,
- Andreas E Kulozik9,
- Stefan M Pfister6, 7, 9,
- Olaf Witt1, 9 and
- Till Milde1, 9Email author
© Ecker et al.; licensee BioMed Central. 2015
Received: 12 March 2015
Accepted: 12 March 2015
Published: 3 April 2015
Medulloblastoma (MB) is the most frequent malignant brain tumor in children. Four subgroups with distinct genetic, epigenetic and clinical characteristics have been identified. Survival remains particularly poor in patients with Group 3 tumors harbouring a MYC amplification. We herein explore the molecular mechanisms and translational implications of class I histone deacetylase (HDAC) inhibition in MYC driven MBs.
Material and Methods
Expression of HDACs in primary MB subgroups was compared to normal brain tissue. A panel of MB cell lines, including Group 3 MYC amplified cell lines, were used as model systems. Cells were treated with HDAC inhibitors (HDACi) selectively targeting class I or IIa HDACs. Depletion of HDAC2 was performed. Intracellular HDAC activity, cellular viability, metabolic activity, caspase activity, cell cycle progression, RNA and protein expression were analyzed.
HDAC2 was found to be overexpressed in MB subgroups with poor prognosis (SHH, Group 3 and Group 4) compared to normal brain and the WNT subgroup. Inhibition of the enzymatic activity of the class I HDACs reduced metabolic activity, cell number, and viability in contrast to inhibition of class IIa HDACs. Increased sensitivity to HDACi was specifically observed in MYC amplified cells. Depletion of HDAC2 increased H4 acetylation and induced cell death. Simulation of clinical pharmacokinetics showed time-dependent on target activity that correlated with binding kinetics of HDACi compounds.
We conclude that HDAC2 is a valid drug target in patients with MYC amplified MB. HDACi should cover HDAC2 in their inhibitory profile and timing and dosing regimen in clinical trials should take binding kinetics of compounds into consideration.
Medulloblastomas (MB) are the most frequent malignant brain tumors in children, with approximately 60 and 300 newly diagnosed cases per year in Germany and the US, respectively . Brain tumors account for 38% of cancer-related deaths, while leukemias account for 24% only despite having a much higher incidence . Current treatment strategies for MB include aggressive surgery, cranio-spinal irradiation and adjuvant chemotherapy dependent on a risk stratification, which until very recently was solely based on clinical features such as histology and presence or absence of metastates at the time of primary diagnosis (e.g. HIT 2000 trial in Germany). However, it has recently been recognized that MB comprises four distinct molecular subgroups termed WNT, SHH, Group 3 and Group 4 , and newly opened clinical trials, such as the SIOP PNET5 trial (NCT02066220), include molecular markers such as beta-catenin in their risk stratification. Both WNT and SHH groups have been classified based on their characteristic activated oncogenic pathways, yet much less is known about the drivers of Group 3 and 4 MB [4,5]. Importantly, the majority of Group 3 tumors are characterized by high protein levels of cMYC, either induced by MYC amplification or by aberrant MYC expression [6,7], and MYC amplification is a marker for high-risk in Group 3 .
Each molecular subgroup can be divided further into different subtypes based on characteristic molecular aberrations, with different clinical courses in SHH, Group 3 and Group 4 , strongly suggesting additional biological heterogeneity in each subgroup. Indeed, the analysis of molecular biomarkers in individual subgroups reveals complex heterogeneity of MB subgroups down to the individual level, as has been shown for SHH  and Group 3  MB. While the WNT and the SHH subgroups are characterized by several recurrent focal mutations in their respective determining pathways, recurrent mutations are unexpectedly rare in Group 3 and Group 4 tumors [4,11,12]. However, several mechanisms of structural variation are recurrent in Group 3 and Group 4 tumors, including somatic copy number alterations, chromothripsis and tetraploidy [13,14], as well as a newly recognized mechanism termed enhancer hijacking that leads to aberrant oncogene expression .
More recently it has become evident that a driving element in Group 3 and Group 4 MBs are aberrations of genes associated with chromatin modification [5,15]. Most of these genes encode for histone mark reader proteins or members of chromatin modifying enzyme complexes, such as KDM6A , MLL2 , ZMYM3 and CHD7 . Somatic mutations, as well as aberrant expression and somatic copy number variations of chromatin modulators lead to altered H3K4 and H3K27 methylation profiles in Group 3 and Group 4 tumors . Finally, the novel MB candidate driver gene LIN28B was identified in Group 3 and 4 MB solely based on aberrant DNA methylation and overexpression of an alternative transcript .
While much insight has been gained into the relevance and function of histone methylation-dependent epigenetic events in Group 3 and Group 4 MB, much less is known about lysine acetylation- (or HDAC-) dependent epigenetic aberrations in MB at a chromatin-wide level. The zinc-dependent HDAC1 through HDAC11 comprise 11 members grouped into four classes (I, IIa, IIb, and IV) . In SHH MBs, SHH-induced HDAC activity is required for continued proliferation of cerebellar granule precursor cells . We and others have previously shown that HDACi treatment exerts anti-tumoral effects in MB in vitro and in vivo [20-24]. Our group has shown that distinct HDAC family members control specific oncogenic functions in pediatric neuronal cancer models including differentiation, cell cycle regulation, apoptosis, autophagy, chemotherapy resistance [25,26], and alterations in tumor suppressor pathways [27,28].
We have further demonstrated that specific HDAC isoforms are differentially expressed in MB [29,30], and found that expression of class IIa HDACs 5 and 9 correlates with cytogenetic aberrations and poor clinical outcome in the entire cohort of MB tumors, and high HDAC2 expression in group 3 MBs . With the recent advent of class-selective HDAC inhibitors (HDACis), such as the class IIa-selective HDACis MAZ1863 and MAZ1866  and selective substrates has opened the possibility of class-selective exploration of HDAC biology.
The aim of the presented study is to investigate the selective targeting of HDAC family members in a MB subgroup specific manner, and to elucidate the translational consequences.
Materials and methods
Patients and clinical samples
Material from patients of tissue microarray (TMA) set (paraffin embedded medulloblastoma samples) were randomly collected at the Department of Neuropathology, Burdenko Neurosurgical Institute (Moscow, Russia) between 1993 and 2011. Approval to link laboratory data to clinical data was obtained by the Institutional Review Board. Two neuropathologists confirmed the diagnoses according to the 2000 WHO classification. None of the patients had received irradiation or chemotherapy before collection of specimens. Metastatic state (M stage) was determined by magnetic resonance imaging and cerebro- spinal fluid cytopathology at diagnosis. Clinical and histopathologic data are summarized in Additional file 1: Table S1.
Cell lines, cell culture and siRNA-mediated knockdown
Cell lines and cell culture conditions have been described previously: MED8A, UW228-2, ONS76 and DAOY in , HD-MB03 in , D458 in . All cell lines had their identity confirmed and proven to be free of contamination by mycoplasma or viral contamination using the Multiplex cell Contamination Test (McCT) service . MYC status of all cell lines was confirmed by fluorescent in-situ hybridization (see below). siRNA transfection was performed as reported previously . siRNA reagents were purchased from Qiagen (Hilden, Germany) (see Additional file 2: Table S2).
RNA-isolation, cDNA synthesis, quantitative reverse transcription real-time PCR (qPCR) and gene expression analysis
RNA extraction, cDNA synthesis, quantitative real-time PCR, and software analysis was performed as reported previously . Primers were purchased from Qiagen (see Additional file 3: Table S3). Normal cerebellum RNA was purchased from Clontech (Mountain View, CA, USA).
The database analysis tool R2 (http://r2.amc.nl) was used to investigate HDAC1, 2, and 3 mRNA expression in brain tumors and normal brain tissues using publicly available datasets (Dataset: , Probesets: HDAC1 (201209_at), HDAC2 (201833_at), HDAC3 (216326_s_at).
Western blot (WB) and image processing
Protein concentrations of cell lysates were determined using the Thermo Scientific Pierce (Waltham, MA, USA) BCA Protein Assay Kit according to manufacturer’s instructions. The following antibodies were used: monoclonal mouse anti-human cMYC (1:200, catalog no. sc-40; Santa Cruz, Dallas, TX, USA), monoclonal mouse anti-human HDAC2 (1:1000; catalog no. sc-81599; Santa Cruz), polyclonal rabbit anti-human AcH4 (1:1000; catalog no. 06-866; Milipore, Billerica, MA, USA) and mouse monoclonal anti–β-actin (1:10000; catalog no. A5441; Sigma-Aldrich) and detected with Amersham ECL Prime Western Blotting Detection System (GE Healthcare, Little Chalfont, UK) on PVDF membrane with Chemi-Smart 5000 Technology (Vilber Lourmat, Eberhardzell, Germany). Uncropped images were contrast enhanced with Chemi-Capt 5000 (Vilber Lourmat) and subsequently cropped in Microsoft Office PowerPoint 2007 SP3 (Microsoft Corporation, Redmond, WA, USA).
TMA, IHC, and fluorescent in-situ hybridization (FISH)
DNA and RNA was extracted from the original tumors included in this TMA and analyzed by nanoString  and/or 450 k Array  as described previously to assign the molecular subgroups. For preparation of the TMA and IHC, see . For detection of HDAC2, the antibody No. ab32117 [Y461] (Abcam, Cambridge, UK) was used at 1:250 dilution. IHC was performed as reported previously . The scoring of the IHC was performed by two investigators (JE and TM), who were both blinded to the clinical information. Four staining intensity levels were defined and weightened with 0 for no staining, 1 for weak staining, 2 for intermediate staining and 3 for strong staining. The H-Score was calculated by summation of the percentages of area stained at each intensity level multiplied by the weightened intensity (i.e. 0, 1, 2, or 3) . The arithmetic mean of the scoring of both investigators was calculated.
MYC status of all cell lines was verified by FISH, as described previously . The probe used was Vysis LSI MYC (Cat. No.: 03 N87-020, Abbott, Abbott Park, IL, USA).
Bright-field images as well as IHC images were captured using an Olympus CX41 microscope with a Color View camera, and CellB 2.3 software (Olympus, Shinjuku, Tokyo, Japan).
HDAC inhibitors (HDACis)
Class IIa HDACis MAZ1863 (compound 6) and MAZ1866 (compound 13) have been described previously . MAZ1863 and MAZ1866, vorinostat (suberoylanilide hydroxamic acid, SAHA; Cat. No. S1047, Selleck Chemicals, Houston, TX, USA), and MS-275 (Cat. No. M4693-15A.25, Biomol GmbH, Hamburg, Germany) were dissolved in DMSO. HDACi and solvent controls were diluted in cell culture medium and added to the cell culture medium to achieve the indicated concentrations.
HDAC activity assay and washout experiments
0.5×10*5 cells in 100 μl medium were seeded in a 96-well plate and incubated for 24 hours at 37°C, 5% CO2. The artificial class I HDAC substrate Boc-Lys(Ac)-AMC (Cat. No. 1875, Bachem, Bubendorf, Switzerland), or class IIa HDAC substrate Boc-Lys(trifluoroacetyl)-AMC (Cat. No. 1985, Bachem) reconstituted in DMSO were diluted in cell culture medium for a final concentration of 50 μM. The developer solution was prepared with a working concentration of 2.5 mg/ml trypsin from porcine pancreas in DMEM, 2% of TritonX-100 and 10 μM of Trichostatin-A (TSA) and stored on ice until usage. Cell culture media was carefully removed and cells were treated with varying concentrations of HDACi (25 μl/well). After 5 min incubation at room temperature 25 μl of substrate dilution were added. After 30 min incubation at 37°C 50 μl of developer solution were added. After 30 min incubation at 37°C emitted fluorescence was measured in the plate reader FLUOstar OPTIMA (BMG Labtech, Ortenberg, Germany), and analyzed with OPTIMA Software, Version 2.00 R3 (Labtech).
For washout experiments cells were seeded as described above. After 24 hours incubation at 37°C the medium was carefully removed and the cells were treated with a dilution of an HDAC inhibitor and incubated at 37°C. After 90 minutes the inhibitor was carefully removed from the cells and cells were washed three times with 200 μl of fresh medium. At timepoints 0 h, 1 h, 3 h, 6 h, 12 h and 24 h after inhibitor removal HDAC activity was measured as described above.
Metabolic activity assay, cell counts and analysis of viability
The WST-1 assay (Roche, Basel, Switzerland) was used for the metabolic activity assays, these were performed as described . Cell counts and analysis of viability by trypan blue exclusion staining were performed using a ViCell XR counter (Beckman Coulter, Brea, CA, USA).
Measurement of the sub-G0 fraction and caspase-3–like activity
The sub-G0 fraction and caspase-3–like activity was measured as described . The positive control for the caspase-3-like activity consisted of untransfected cells treated with UV light (35 mJ/cm2) 12 h before caspase-3-like activity measurement.
Statistics analysis and graph editing
In vitro experiments were performed in a minimum of three biological replicates. Half-maximal effective concentrations (EC50) of HDACi were calculated using GraphPad Prism version 5.01 (GraphPad Software, La Jolla, CA, USA) for Windows. Results of treatments were compared using an unpaired t-test or One Way ANOVA test with Bonferroni’s multiple Comparison Test as indicated. p-values <0.05 were considered significant. Graphs were generated using GraphPad Prism version 5.01 and Microsoft Powerpoint for Mac 2011, Version 14.4.5.
MYC amplified medulloblastomas display differential expression of class I HDACs
We have previously shown that differential expression of HDAC family members occur in medulloblastoma. Whereas class IIa HDACs 5 and 9 correlate with specific cytogenetic aberrations and poor clinical outcome in the entire cohort of MB tumors , analysis of MB subgroup specific expression revealed a particular high level of HDAC2 in all three subgroups of MB associated with higher risk, i.e. SHH, group 3 and group 4 .
Inhibition of class I but not class IIa HDAC catalytic activity affects MYC amplified medulloblastoma cells
HDAC2 depletion induces cell death and attenuates cell growth
Simulation of clinical pharmacokinetics of HDACi in vitro uncovers challenges for translation
Successful treatment of MB remains a challenge in many patients, which suffer from therapy-related side effects, and the prognosis remains poor for many patients with Group 3 MB . Based on reports suggesting that epigenetic events seem to play an important role in this subgroup, we have investigated the selective targeting of HDAC family members in a subgroup specific manner.
We have previously found that the class IIa isoforms HDACs 5 and 9 are widely expressed on the protein level, and relatively overexpressed on the mRNA level in MB with poor prognosis (Chr 6q gain or 17q gain) ). Depletion of either HDAC5 or HDAC9 protein in MB cells resulted in a reduction of cell proliferation and increase in cell death . To investigate whether inhibition of the enzymatic deacetylase activity can phenocopy these effects, we tested novel selective class IIa HDAC inhibitors. Although these inhibitors strongly inhibited class IIa enzymatic activity in MB cells in dose-dependent fashion, we did not observe any obvious biological effect, such as reduction of metabolic activity in MB cells. These results suggest that the inhibition of class IIa enzymatic activity clearly did not confer the same biological effect as the reduction of HDAC5 or HDAC9 protein, suggesting that the enzymatic activity is not the main mechanism for the oncogenic effects of HDAC5 or 9 in MB cells. In contrast, inhibition of class I HDACs elicited a strong response, especially in MYC amplified cell lines. The enzymatic activity of class I and IIa HDACs therefore plays very divergent roles in MB biology. Indeed, a tyrosine residue in the catalytic site of class I HDACs potentiates the lysine deacetylation activity acting as a transition stabilizer . Due to a highly conserved mutation in the catalytic site of vertebrate class IIa HDACs, switching this tyrosine to a histidine residue, the deacetylating activity of class IIa HDACs on histone proteins is reduced more than a 1000-fold . The repressive effect of class IIa HDACs on gene expression therefore appears to be largely independent of their catalytic activity on histone proteins. This surprising finding is further emphasized by studies showing that a splice variant of HDAC9 lacking the catalytic HDAC domain represses the expression of MEF2 target genes just as effectively as wild-type HDAC9 protein . Finally it is being discussed that class IIa HDACs might play an important role in signal transduction independently of their enzymatic activity, either by bromodomain functioning as readers of epigenetic marks , or by shuttling between the nucleus and the cytoplasm  as has been shown for HDAC5  and HDAC7 , and this could well be true for HDAC5 and 9 in MB.
To verify target presence and elucidate the class I HDACs involved in MB biology, we carried out expression analyses and found class I HDAC2 to be the most strongly overexpressed in MB in general  and in the three MB subgroups SHH, group 3 and group 4 (associated with unfavourable or high risk) in particular. Our results indicate that MYC amplified cell lines have higher sensitivity to HDACi, that comprise class I HDACs 1, 2 and 3 in their inhibitory profile, than MYC single copy cell lines. A previous report studying the effect of the HDACi depsipetide (FK228), which most potently inhibits all class I HDACs, demonstrated that the tumor most sensitive to depsipeptide treatment tested (a CNS-PNET) had the highest expression of HDAC2 relative to HDAC1, and 3-7 . Furthermore, MYC amplification is a hallmark of Group 3 MB, and importantly the transcription factor cMYC has been described to govern the transcription of HDAC2 . We have previously shown that the epigenetic regulation of miR-183 in neuroblastoma involves MYCN and HDAC2 in the same complex . Based on these findings and consistent with our data, showing a significantly increased sensitivity for class I HDAC inhibiting agents in MYC amplified and HDAC2 overexpressing cell lines, the treatment of MYC amplified MB with HDAC inhibitors comprising class I HDACs in their inhibitory profile seems to be promising. Future studies should aim at the elucidation of the molecular interactions of cMYC and HDAC2, such as protein-protein interactions, feedback loops, and non-histone lysine deacetylation, governing the susceptibility of MYC amplified MB to HDACi.
Finally, whether HDACis will be efficacious for the treatment of solid tumors is still under debate. Many trials have failed to show meaningful response of solid tumors to HDACis  [45-48]. The root of failure to translate pre-clinical findings in general has been extensively discussed [59-61]. In general, insufficient pharmacological modeling of the clinical situation in terms of drugs concentrations and kinetics are the primarily criticized factors . Detailed recommendations to improve the predictive value of pre-clinical cancer studies have been developed , which include the use of appropriate models, and understanding of the clinical reality, i.e. knowledge of the limitations of pre-clinical experimental settings. Analysis of the models used in our studies confirmed the faithful recapitulation of the patients’ tumors by the Group 3 cell lines used in our study, as evidenced by MYC amplification, and MYC and HDAC2 expression analysis. To emulate in vitro the drug concentrations present in patients when treated with HDACis, we mimicked the clinical situation with appropriately low HDACi concentrations as well as washout experiments. Our results suggest that several reasons contribute to the failure of HDACis in the clinic despite their promising in vitro results. First, in the absence of a predictive biomarker, trial cohorts have been poorly pre-selected for patients responding with high sensitivity to HDACi treatment, suggesting the lack of efficacy. The most promising predictive biomarker identified in post-hoc analyses to date is IHC for HR23B, which has been shown to be a positive predictive marker for vorinostat in patients with cutaneous T-cell lymphoma  and for belinostat (PXD101) in patients with hepatocellular carcinoma . We here validate MYC amplification as a predictive and routinely applicable clinical biomarker for HDACi sensitivity of medulloblastoma patients. Second, HDACis have a very short inhibitory effect on HDACs when present only a fraction of a day. As this is the case in patients, but not in cell culture, both pre- and clinical studies need to recognize clinical as opposed to cell culture conditions, and subsequently of the limits of pre-clinical data. Pre-clinical studies need to investigate conditions present in patients, as performed in our washout experiments, and especially pay attention to the lifetime of the drug-target complex , as well use adequate and informative readouts . New dosing schedules could well be tested under these conditions, before being translated into the clinical setting. The difference of the half-life of the drug-target complex between vorinostat and MS-275 indicates that the lifetime of the drug-target complex is at least partially determined by the molecular set up of the inhibitory agent . The development of novel agents with increased half-life of the drug-target complex, such as panobinostat, could well be a promising strategy. On the clinical side, studies involving HDACis should demonstrate on target activity in tumor tissue in addition to simply monitoring drug levels and histone acetylation in PBMCs as a surrogate), and possibly develop novel dosing and/or application schedules, such as oral vs. intravenous application, extended release or infusion over several hours, multiple dosing with lower doses etc.
In summary, we conclude, that i) class I HDACs in general and HDAC2 in particular are a valid target in Group 3 MB, ii) MYC amplified MB are more sensitive to HDACi than MYC non-amplified MB and thus MYC amplification could serve as a positive predictive marker for HDACi treatment, and iii) that both inhibitory profiles and binding kinetics of compounds are of major importance when designing clinical trials using HDACis.
This work was supported by the MYCNet (BMBF), and the German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung). JE was supported by Talents in Medicine program of University Hospital Heidelberg and by The German National Academic Foundation (Studienstiftung des deutschen Volkes). IO was supported by the BMWi through SME to IO und DFG (OE 542/2-1) 2014 to IO. RM was supported by the National Institute of Health (P50CA086355).
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