Class I HDAC inhibition reduces DNA damage repair capacity of MYC-amplified medulloblastoma cells.

PURPOSE: MYC-driven Group 3 medulloblastoma (MB) (subtype II) is a highly aggressive childhood brain tumor. Sensitivity of MYC-driven MB to class I histone deacetylase inhibitors (HDACi) has been previously demonstrated in vitro and in vivo. In this study we characterize the transcriptional effects of class I HDACi in MYC-driven MB and explore beneficial drug combinations. METHODS: MYC-amplified Group 3 MB cells (HD-MB03) were treated with class I HDACi entinostat. Changes in the gene expression profile were quantified on a microarray. Bioinformatic assessment led to the identification of pathways affected by entinostat treatment. Five drugs interfering with these pathways (olaparib, idasanutlin, ribociclib, selinexor, vinblastine) were tested for synergy with entinostat in WST-8 metabolic activity assays in a 5 × 5 combination matrix design. Synergy was validated in cell count and flow cytometry experiments. The effect of entinostat and olaparib on DNA damage was evaluated by γH2A.X quantification in immunoblotting, fluorescence microscopy and flow cytometry. RESULTS: Entinostat treatment changed the expression of genes involved in 22 pathways, including downregulation of DNA damage response. The PARP1 inhibitors olaparib and pamiparib showed synergy with entinostat selectively in MYC-amplified MB cells, leading to increased cell death, decreased viability and increased formation of double strand breaks, as well as increased sensitivity to additional induction of DNA damage by doxorubicin. Non-MYC-amplified MB cells and normal human fibroblasts were not susceptible to this triple treatment. CONCLUSION: Our study identifies the combination of entinostat with olaparib as a new potential therapeutic approach for MYC-driven Group 3 MB.

Class I HDAC inhibitor entinostat synergizes with PLK1 inhibitors in MYC-amplified medulloblastoma cells.

PURPOSE: We and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy. METHODS: We used a gene expression dataset to identify PLK1 as a second target in MB cells and validated the relevance of PLK1 in MB. We measured cell metabolic activity, viability, and cycle progression in MB cells after treatment with PLK1-specific inhibitors (PLK1i). Chou-Talalay synergy calculations were used to determine the nature of class I HDACi entinostat and PLK1i interaction which was validated. Finally, the clinical potential of the combination was assessed in the in vivo experiment. RESULTS: MYC-amplified tumor cells are highly sensitive towards treatment with ATP-competitive PLK1i as a monotherapy. Entinostat and PLK1i in combination act synergistically in MYC-driven MB cells, exerting cytotoxic effects at clinically relevant concentrations. The downstream effect is exerted via MYC-related pathways, pointing out the potential of MYC amplification as a clinically feasible predictive biomarker for patient selection. While entinostat significantly extended survival of mice implanted with orthotopic MYC-amplified MB PDX, there was no evidence of the improvement of survival when treating the animals with the combination. CONCLUSION: The combination of entinostat and PLK1i showed synergistic interaction in vitro, but not in vivo. Therefore, further screening of blood-brain barrier penetrating PLK1i is warranted to determine the true potential of the combination as no on-target activity was observed after PLK1i volasertib treatment in vivo.

ERBB and P-glycoprotein inhibitors break resistance in relapsed neuroblastoma models through P-glycoprotein.

Chemotherapy resistance is a persistent clinical problem in relapsed high-risk neuroblastomas. We tested a panel of 15 drugs for sensitization of neuroblastoma cells to the conventional chemotherapeutic vincristine, identifying tariquidar, an inhibitor of the transmembrane pump P-glycoprotein (P-gp/ABCB1), and the ERBB family inhibitor afatinib as the top resistance breakers. Both compounds were efficient in sensitizing neuroblastoma cells to vincristine in trypan blue exclusion assays and in inducing apoptotic cell death. The evaluation of ERBB signaling revealed no functional inhibition, that is, dephosphorylation of the downstream pathways upon afatinib treatment but direct off-target interference with P-gp function. Depletion of ABCB1, but not ERRB4, sensitized cells to vincristine treatment. P-gp inhibition substantially broke vincristine resistance in vitro and in vivo (zebrafish embryo xenograft). The analysis of gene expression datasets of more than 50 different neuroblastoma cell lines (primary and relapsed) and more than 160 neuroblastoma patient samples from the pediatric precision medicine platform INFORM (Individualized Therapy For Relapsed Malignancies in Childhood) confirmed a pivotal role of P-gp specifically in neuroblastoma resistance at relapse, while the ERBB family appears to play a minor part.

Evaluation of Antitumor and On-Target Activity of HDAC Inhibitors with the Zebrafish Embryo Xenograft Model.

Reliable preclinical drug testing models for cancer research are urgently needed with zebrafish embryo models emerging as a powerful vertebrate model for xenotransplantation studies. Here, we describe the evaluation of toxicity, efficacy, and on-target activity of histone deacetylase (HDAC) inhibitors in a zebrafish embryo yolk sac xenotransplantation model of medulloblastoma and neuroblastoma cells. For this, we performed toxicity assays with our zebrafish drug library consisting of 28 clinically relevant targeted as well as chemotherapeutic drugs with zebrafish embryos. We further engrafted zebrafish embryos with fluorescently labeled pediatric tumor cells (SK-N-BE(2)-C, HD-MB03, or MED8A) and monitored the progression after HDAC inhibitor treatment of xenotransplanted tumors through tumor volume measurements with high-content confocal microscopy in a multi-well format. The on-target activity of HDAC inhibitors was verified through immunohistochemistry staining on paraffin-embedded early larvae. Overall, the zebrafish embryo xenotransplantation model allows for fast and cost-efficient in vivo evaluation of targeted drug toxicity, efficacy, and on-target activity in the field of precision oncology.

Synthesis, Biochemical, and Cellular Evaluation of HDAC6 Targeting Proteolysis Targeting Chimeras.

Histone deacetylases are considered promising epigenetic targets for chemical protein degradation due to their diverse roles in physiological cellular functions and in the diseased state. Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that hijack the cell's ubiquitin-proteasome system (UPS). One of the promising targets for this approach is histone deacetylase 6 (HDAC6), which is highly expressed in several types of cancers and is linked to the aggressiveness of tumors. In the present work, we describe the synthesis of HDAC6 targeting PROTACs based on previously synthesized benzohydroxamates selectively inhibiting HDAC6 and how to assess their activities in different biochemical in vitro assays and in cellular assays. HDAC inhibition was determined using fluorometric assays, while the degradation ability of the PROTACs was assessed using western blot analysis.

Aza-SAHA Derivatives Are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation and Phenocopy HDAC10 Knockout.

We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group ("aza-scan") into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one-atom replacement (C→N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ-748, with potency and selectivity demonstrated by cellular and biochemical target engagement, as well as thermal shift assays. Cocrystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling confirmed exquisite cellular HDAC10-selectivity of DKFZ-748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, validated for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limiting in vitro tumor model, DKFZ-748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ-748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings.

Design, Synthesis and Biological Characterization of Histone Deacetylase 8 (HDAC8) Proteolysis Targeting Chimeras (PROTACs) with Anti-Neuroblastoma Activity.

In addition to involvement in epigenetic gene regulation, histone deacetylases (HDACs) regulate multiple cellular processes through mediating the activity of non-histone protein substrates. The knockdown of HDAC8 isozyme is associated with the inhibition of cell proliferation and apoptosis enhancement in several cancer cell lines. As shown in several studies, HDAC8 can be considered a potential target in the treatment of cancer forms such as childhood neuroblastoma. The present work describes the development of proteolysis targeting chimeras (PROTACs) of HDAC8 based on substituted benzhydroxamic acids previously reported as potent and selective HDAC8 inhibitors. Within this study, we investigated the HDAC8-degrading profiles of the synthesized PROTACs and their effect on the proliferation of neuroblastoma cells. The combination of in vitro screening and cellular testing demonstrated selective HDAC8 PROTACs that show anti-neuroblastoma activity in cells.

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement.

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.

Identification of histone deacetylase 10 (HDAC10) inhibitors that modulate autophagy in transformed cells.

Histone deacetylases (HDACs) are a family of 18 epigenetic modifiers that fall into 4 classes. Histone deacetylase inhibitors (HDACi) are valid tools to assess HDAC functions. HDAC6 and HDAC10 belong to the class IIb subgroup of the HDAC family. The targets and biological functions of HDAC10 are ill-defined. This lack of knowledge is due to a lack of specific and potent HDAC10 inhibitors with cellular activity. Here, we have synthesized and characterized piperidine-4-acrylhydroxamates as potent and highly selective inhibitors of HDAC10. This was achieved by targeting the acidic gatekeeper residue Glu274 of HDAC10 with a basic piperidine moiety that mimics the interaction of the polyamine substrate of HDAC10. We have confirmed the binding modes of selected inhibitors using X-ray crystallography. Promising candidates were selected based on their specificity by in vitro profiling using recombinant HDACs. The most promising HDAC10 inhibitors 10c and 13b were tested for specificity in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene. By immunoblot experiments we assessed the hyperacetylation of histones and tubulin-α, which are class I and HDAC6 substrates, respectively. As validated test for HDAC10 inhibition we used flow cytometry assessing autolysosome formation in neuroblastoma and AML cells. We demonstrate that 10c and 13b inhibit HDAC10 with high specificity over HDAC6 and with no significant impact on class I HDACs. The accumulation of autolysosomes is not a consequence of apoptosis and 10c and 13b are not toxic for normal human kidney cells. These data show that 10c and 13b are nanomolar inhibitors of HDAC10 with high specificity. Thus, our new HDAC10 inhibitors are tools to identify the downstream targets and functions of HDAC10 in cells.

Functional Therapeutic Target Validation Using Pediatric Zebrafish Xenograft Models.

The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing. In such an approach, patient-derived tumor cells are exposed to a library of approved oncological drugs in a physiological setting, e.g., in the form of animal avatars injected with patient tumor cells. We used molecularly fully characterized tumor samples from the INFORM study to compare drug screen results of individual patient-derived cell models in functional assays: (i) patient-derived spheroid cultures within a few days after tumor dissociation; (ii) tumor cells reisolated from the corresponding mouse PDX; (iii) corresponding long-term organoid-like cultures and (iv) drug evaluation with the corresponding zebrafish PDX (zPDX) model. Each model had its advantage and complemented the others for drug hit and drug combination selection. Our results provide evidence that in vivo zPDX drug screening is a promising add-on to current functional drug screening in precision medicine platforms.

Combining APR-246 and HDAC-Inhibitors: A Novel Targeted Treatment Option for Neuroblastoma.

APR-246 (Eprenetapopt/PRIMA-1Met) is a very potent anti-cancer drug in clinical trials and was initially developed as a p53 refolding agent. As an alternative mode of action, the elevation of reactive oxygen species (ROS) has been proposed. Through an in silico analysis, we investigated the responses of approximately 800 cancer cell lines (50 entities; Cancer Therapeutics Response Portal, CTRP) to APR-246 treatment. In particular, neuroblastoma, lymphoma and acute lymphocytic leukemia cells were highly responsive. With gene expression data from the Cancer Cell Line Encyclopedia (CCLE; n = 883) and patient samples (n = 1643) from the INFORM registry study, we confirmed that these entities express low levels of SLC7A11, a previously described predictive biomarker for APR-246 responsiveness. Combining the CTRP drug response data with the respective CCLE gene expression profiles, we defined a novel gene signature, predicting the effectiveness of APR-246 treatment with a sensitivity of 90% and a specificity of 94%. We confirmed the predicted APR-246 sensitivity in 8/10 cell lines and in ex vivo cultures of patient samples. Moreover, the combination of ROS detoxification-impeding APR-246 with approved HDAC-inhibitors, known to elevate ROS, substantially increased APR-246 sensitivity in cell cultures and in vivo in two zebrafish neuroblastoma xenograft models. These data provide evidence that APR-246, in combination with HDAC-inhibitors, displays a novel potent targeted treatment option for neuroblastoma patients.

Broad-Spectrum HDAC Inhibitors Promote Autophagy through FOXO Transcription Factors in Neuroblastoma.

Depending on context and tumor stage, deregulation of autophagy can either suppress tumorigenesis or promote chemoresistance and tumor survival. Histone deacetylases (HDACs) can modulate autophagy; however, the exact mechanisms are not fully understood. Here, we analyze the effects of the broad-spectrum HDAC inhibitors (HDACi) panobinostat and vorinostat on the transcriptional regulation of autophagy with respect to autophagy transcription factor activity (Transcription factor EB-TFEB, forkhead boxO-FOXO) and autophagic flux in neuroblastoma cells. In combination with the late-stage autophagic flux inhibitor bafilomycin A1, HDACis increase the number of autophagic vesicles, indicating an increase in autophagic flux. Both HDACi induce nuclear translocation of the transcription factors FOXO1 and FOXO3a, but not TFEB and promote the expression of pro-autophagic FOXO1/3a target genes. Moreover, FOXO1/3a knockdown experiments impaired HDACi treatment mediated expression of autophagy related genes. Combination of panobinostat with the lysosomal inhibitor chloroquine, which blocks autophagic flux, enhances neuroblastoma cell death in culture and hampers tumor growth in vivo in a neuroblastoma zebrafish xenograft model. In conclusion, our results indicate that pan-HDACi treatment induces autophagy in neuroblastoma at a transcriptional level. Combining HDACis with autophagy modulating drugs suppresses tumor growth of high-risk neuroblastoma cells. These experimental data provide novel insights for optimization of treatment strategies in neuroblastoma.

Reduced chromatin binding of MYC is a key effect of HDAC inhibition in MYC amplified medulloblastoma.

BACKGROUND: The sensitivity of myelocytomatosis oncogene (MYC) amplified medulloblastoma to class I histone deacetylase (HDAC) inhibition has been shown previously; however, understanding the underlying molecular mechanism is crucial for selection of effective HDAC inhibitors for clinical use. The aim of this study was to investigate the direct molecular interaction of MYC and class I HDAC2, and the impact of class I HDAC inhibition on MYC function. METHODS: Co-immunoprecipitation and mass spectrometry were used to determine the co-localization of MYC and HDAC2. Chromatin immunoprecipitation (ChIP) sequencing and gene expression profiling were used to analyze the co-localization of MYC and HDAC2 on DNA and the impact on transcriptional activity in primary tumors and a MYC amplified cell line treated with the class I HDAC inhibitor entinostat. The effect on MYC was investigated by quantitative real-time PCR, western blot, and immunofluorescence. RESULTS: HDAC2 is a cofactor of MYC in MYC amplified medulloblastoma. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein, inducing a downregulation of MYC activated genes (MAGs) and upregulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and by distinct enhancer-box distribution. CONCLUSIONS: Our data elucidate the molecular interaction of MYC and HDAC2 and support a model in which inhibition of class I HDACs directly targets MYC's transactivating and transrepressing functions.

Rapid In Vivo Validation of HDAC Inhibitor-Based Treatments in Neuroblastoma Zebrafish Xenografts.

The survival rate among children with relapsed neuroblastomas continues to be poor, and thus new therapeutic approaches identified by reliable preclinical drug testing models are urgently needed. Zebrafish are a powerful vertebrate model in preclinical cancer research. Here, we describe a zebrafish neuroblastoma yolk sac model to evaluate efficacy and toxicity of histone deacetylase (HDAC) inhibitor treatments. Larvae were engrafted with fluorescently labeled, genetically diverse, established cell lines and short-term cultures of patient-derived primary cells. Engrafted tumors progressed locally and disseminated remotely in an intact environment. Combination treatments involving the standard chemotherapy doxorubicin and HDAC inhibitors substantially reduced tumor volume, induced tumor cell death, and inhibited tumor cell dissemination to the tail region. Hence, this model allows for fast, cost-efficient, and reliable in vivo evaluation of toxicity and response of the primary and metastatic tumor sites to drug combinations.

Design and Synthesis of Dihydroxamic Acids as HDAC6/8/10 Inhibitors.

We report the synthesis and evaluation of a class of selective multitarget agents for the inhibition of HDAC6, HDAC8, and HDAC10. The concept for this study grew out of a structural analysis of the two selective inhibitors Tubastatin A (HDAC6/10) and PCI-34051 (HDAC8), which we recognized share the same N-benzylindole core. Hybridization of the two inhibitor structures resulted in dihydroxamic acids with benzyl-indole and -indazole core motifs. These substances exhibit potent activity against HDAC6, HDAC8, and HDAC10, while retaining selectivity over HDAC1, HDAC2, and HDAC3. The best substance inhibited the viability of the SK-N-BE(2)C neuroblastoma cell line with an IC50 value similar to a combination treatment with Tubastatin A and PCI-34051. This compound class establishes a proof of concept for such hybrid molecules and could serve as a starting point for the further development of enhanced HDAC6/8/10 inhibitors.

Selective Inhibition of Histone Deacetylase 10: Hydrogen Bonding to the Gatekeeper Residue is Implicated.

The discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understanding the biological functions of individual HDACs and for validating HDACs as drug targets. The isozyme HDAC10 contributes to chemotherapy resistance and has recently been described to be a polyamine deacetylase, but no studies toward selective HDAC10 inhibitors have been published. Using two complementary assays, we found Tubastatin A, an HDAC6 inhibitor, to potently bind HDAC10. We synthesized Tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10 binding. HDAC10 inhibitors mimicked knockdown by causing dose-dependent accumulation of acidic vesicles in a neuroblastoma cell line. Furthermore, docking into human HDAC10 homology models indicated that a hydrogen bond between a cap group nitrogen and the gatekeeper residue Glu272 was responsible for potent HDAC10 binding. Taken together, our data provide an optimal platform for the development of HDAC10-selective inhibitors, as exemplified with the Tubastatin A scaffold.

The Senescence-associated Secretory Phenotype Mediates Oncogene-induced Senescence in Pediatric Pilocytic Astrocytoma.

PURPOSE: Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown.Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay. RESULTS: SASP factors were upregulated in primary human and murine pilocytic astrocytoma and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating pilocytic astrocytoma cells. The SASP factors IL1B and IL6 were upregulated in primary pilocytic astrocytoma, and both pathways were regulated during OIS in DKFZ-BT66. Stimulation with rIL1B but not rIL6 reduced growth of DKFZ-BT66 cells and induced the SASP. Anti-inflammatory treatment with dexamethasone induced regrowth of senescent cells and inhibited the SASP. Senescent DKFZ-BT66 cells responded to senolytic BCL2 inhibitors. High IL1B and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. CONCLUSIONS: We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in patients with pilocytic astrocytoma. Further investigation of the SASP driving the unpredictable growth of pilocytic astrocytomas, and its possible therapeutic application, is warranted.

Dual role of HDAC10 in lysosomal exocytosis and DNA repair promotes neuroblastoma chemoresistance.

Drug resistance is a leading cause for treatment failure in many cancers, including neuroblastoma, the most common solid extracranial childhood malignancy. Previous studies from our lab indicate that histone deacetylase 10 (HDAC10) is important for the homeostasis of lysosomes, i.e. acidic vesicular organelles involved in the degradation of various biomolecules. Here, we show that depleting or inhibiting HDAC10 results in accumulation of lysosomes in chemotherapy-resistant neuroblastoma cell lines, as well as in the intracellular accumulation of the weakly basic chemotherapeutic doxorubicin within lysosomes. Interference with HDAC10 does not block doxorubicin efflux from cells via P-glycoprotein inhibition, but rather via inhibition of lysosomal exocytosis. In particular, intracellular doxorubicin does not remain trapped in lysosomes but also accumulates in the nucleus, where it promotes neuroblastoma cell death. Our data suggest that lysosomal exocytosis under doxorubicin treatment is important for cell survival and that inhibition of HDAC10 further induces DNA double-strand breaks (DSBs), providing additional mechanisms that sensitize neuroblastoma cells to doxorubicin. Taken together, we demonstrate that HDAC10 inhibition in combination with doxorubicin kills neuroblastoma, but not non-malignant cells, both by impeding drug efflux and enhancing DNA damage, providing a novel opportunity to target chemotherapy resistance.

The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines.

High histone deacetylase (HDAC) 8 and HDAC10 expression levels have been identified as predictors of exceptionally poor outcomes in neuroblastoma, the most common extracranial solid tumor in childhood. HDAC8 inhibition synergizes with retinoic acid treatment to induce neuroblast maturation in vitro and to inhibit neuroblastoma xenograft growth in vivo. HDAC10 inhibition increases intracellular accumulation of chemotherapeutics through interference with lysosomal homeostasis, ultimately leading to cell death in cultured neuroblastoma cells. So far, no HDAC inhibitor covering HDAC8 and HDAC10 at micromolar concentrations without inhibiting HDACs 1, 2 and 3 has been described. Here, we introduce TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid), a novel HDAC6/8/10 inhibitor for neuroblastoma therapy. TH34 is well-tolerated by non-transformed human skin fibroblasts at concentrations up to 25 µM and modestly impairs colony growth in medulloblastoma cell lines, but specifically induces caspase-dependent programmed cell death in a concentration-dependent manner in several human neuroblastoma cell lines. In addition to the induction of DNA double-strand breaks, HDAC6/8/10 inhibition also leads to mitotic aberrations and cell-cycle arrest. Neuroblastoma cells display elevated levels of neuronal differentiation markers, mirrored by formation of neurite-like outgrowths under maintained TH34 treatment. Eventually, after long-term treatment, all neuroblastoma cells undergo cell death. The combination of TH34 with plasma-achievable concentrations of retinoic acid, a drug applied in neuroblastoma therapy, synergistically inhibits colony growth (combination index (CI) < 0.1 for 10 µM of each). In summary, our study supports using selective HDAC inhibitors as targeted antineoplastic agents and underlines the therapeutic potential of selective HDAC6/8/10 inhibition in high-grade neuroblastoma.

Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance.

Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g., via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here we show that both static 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D-grown cells are substantially different from those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models more closely mimic tumor (patho-)physiology, including the upregulation of tumor relevant pathways, such as autophagy.