HDAC10 in neuroblastoma chemoresistance

Neuroblastoma is the most common extracranial solid tumor in childhood, and characteristically displays a wide variety of clinical outcomes. While prognosis is generally favorable in low-risk and intermediate-risk tumors, outcome remains poor in high-risk neuroblastoma, and infaust in case of relapse. Multidrug resistance is frequent in high-risk neuroblastoma and remains to be one of the major factors limiting treatment success despite intensive multimodal therapy regimens, highlighting the need for novel treatment approaches capable of reducing neuroblastoma drug resistance. Histone deacetylases (HDACs) are involved in numerous cancer-relevant pathways and have become attractive anti-tumor targets due to their excellent druggability. Broadband inhibition of HDACs is, however, associated with dose-limiting side effects, which can be possibly circumvented by the inhibition of individual tumor-relevant isozymes. Previous work of our group has shown that high expression of class IIb histone deacetylase HDAC10 supports chemoresistance of neuroblastoma cells by promoting macroautophagy. Data suggested that HDAC10 was critical for lysosomal function, but the precise lysosomal role of HDAC10 and its cellular substrates remained unknown. The data presented in this study indicate that HDAC10 is crucial for lysosomal homeostasis in a number of highly drug-resistant neuroblastoma cell lines (SK-N-BE(2)-C, IMR-32, SK-N-AS) while being dispensable in others (Kelly, NB-1) and in non-transformed fibroblasts. In HDAC10-dependent cells, interference with HDAC10 function causes accumulation of lysosomes, a phenotype that is not observed in case of functional interference with the highly homologous class IIb member HDAC6. Depletion or inhibition of HDAC10 further interferes with downstream lysosomal processes such as lysosomal exocytosis, indicating that accumulating lysosomes are dysfunctional. Lysosomal accumulation and the inhibition of lysosomal exocytosis in turn promote intracellular accumulation of weakly basic chemotherapeutics such as doxorubicin, which does not remain sequestered in lysosomes but is also highly enriched in nuclei. Consequently, co-treatment with doxorubicin and HDAC10 inhibitors efficiently promotes cell death in treatment resistant neuroblastoma cell lines while sparing non-malignant cells. Lysosomal exocytosis is an important pro-survival mechanism under cytotoxic treatment. Inhibition of HDAC10, and thus lysosomal exocytosis, sensitizes cells not only by promoting doxorubicin accumulation, but also by inhibiting the process of lysosomal exocytosis itself. Moreover, interference with HDAC10 function promotes accumulation of DNA double-strand breaks (DSBs) both in absence and presence of doxorubicin, suggesting an additional role for HDAC10 in DSB repair. Preliminary data of mass spectrometric analyses of protein lysine acetylation after HDAC10 inhibition suggest that HDAC10 modulates acetylation of the V-ATPase subunit A and the Ku70/Ku80 complex member Ku80. It is thus conceivable that HDAC10 modulates lysosomal function at the level of lysosomal acidification, as well as DNA repair at the level of non-homologous end joining of DSBs. The recently published function of HDAC10 as N8-acetylspermidine deacetylase remains to be confirmed. Follow-up studies on the mechanistic role of HDAC10 could be greatly facilitated by a highly specific HDAC10 antibody. In this context, several promising HDAC10-reactive mouse hybridoma clones were generated, but recurring instability of the promising hybridoma clones delayed stable production of the antibody. In summary, in this thesis, a novel function of HDAC10 in regulation of lysosomal downstream mechanisms was identified and a previously published role of HDAC10 in DNA repair was confirmed. These mechanisms possess the translational potential to overcome drug resistance in combination with chemotherapies

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, i.e., 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

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 autophagyrelated 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

Shared genetic risk between eating disorder- and substance-use-related phenotypes:Evidence from genome-wide association studies

First published: 16 February 202

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 clinical drug targets. The isozyme HDAC10 contributes to chemotherapy resistance via inhibition of autophagic flux and has recently been described to be a polyamine deacetylase, but no studies directed toward selective HDAC10 inhibitors have been published. Herein, we disclose that the use of two complementary ligand-displacement assays has revealed unexpectedly potent HDAC10 binding of tubastatin A, which has been previously described as a highly selective HDAC6 inhibitor. We synthesized a targeted selection of tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10, but not HDAC6, binding. Only potent HDAC10 binders mimicked HDAC10 knockdown by causing dose-dependent accumulation of acidic vesicles in the BE(2)-C neuroblastoma cell line. Docking of inhibitors into human HDAC10 homology models indicated that a hydrogen-bond between a basic cap group nitrogen and the HDAC10 gatekeeper residue Glu272 was responsible for potent HDAC10 binding. Taken together, the presented assays and homology models provide an optimal platform for the development of HDAC10-selective inhibitors, as exemplified with the tubastatin A scaffold.<br /

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

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

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

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. Keywords: histone deacetylase