Tumor cells activate autophagy in response to chemotherapy-induced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate that HDAC10 protects cancer cells from cytotoxic agents by mediating autophagy and identify this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. Moreover, these results propose a promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome. Significance Resistance to chemotherapy is one of the major challenges in oncology. Neuroblastoma is the most common extracranial solid tumor in childhood, and the successful response of high-risk patients to chemotherapy remains poor. Our work showed that the so far poorly studied histone deacetylase (HDAC)10 promotes autophagy-mediated cell survival and signals poor outcome in independent high-risk patient cohorts. Inhibition of HDAC10 sensitized tumor cells for cytotoxic drug treatment. These results offer HDAC10 as a potential biomarker for treatment response of high-risk tumors and open new avenues for developing selective treatment strategies to bypass drug resistance of these tumors.

For differentiation-defective malignancies, compounds that modulate transcription, such as retinoic acid and histone deacetylase (HDAC) inhibitors, are of particular interest. HDAC inhibitors are currently under investigation for the treatment of a broad spectrum of cancer diseases. However, one clinical drawback is class-specific toxicity of unselective inhibitors, limiting their full anticancer potential. Selective targeting of individual HDAC isozymes in defined tumor entities may therefore be an attractive alternative treatment approach. We have previously identified HDAC family member 8 (HDAC8) as a novel target in childhood neuroblastoma. Using small-molecule inhibitors, we now demonstrate that selective inhibition of HDAC8 exhibits antineuroblastoma activity without toxicity in two xenograft mouse models of MYCN oncogene-amplified neuroblastoma. In contrast, the unselective HDAC inhibitor vorinostat was more toxic in the same models. HDAC8-selective inhibition induced cell cycle arrest and differentiation in vitro and in vivo. Upon combination with retinoic acid, differentiation was significantly enhanced, as demonstrated by elongated neurofilament-positive neurites and upregulation of NTRK1. Additionally, MYCN oncogene expression was downregulated in vitro and tumor cell growth was markedly reduced in vivo. Mechanistic studies suggest that cAMP-response element-binding protein (CREB) links HDAC8- and retinoic acid-mediated gene transcription. In conclusion, HDAC-selective targeting can be effective in tumors exhibiting HDAC isozyme-dependent tumor growth in vivo and can be combined with differentiation-inducing agents.

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. 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.

Purpose: Pilocytic astrocytoma (PA) 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 PAs. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in PA remains unknown. Experimental Design: The patient-derived PA cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in PA. 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 PA tumors were analyzed by GEP and multiplex assay. Results: SASP factors were up-regulated in primary human and murine PA and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating PA cells. The SASP factors IL1B and IL6 were up-regulated in primary PA, 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 pan-BCL inhibitors. High IL1B and SASP expression in PA tumors was associated with favorable progression-free survival. Conclusions: We provide evidence for the SASP regulating OIS in pediatric PA, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in PA patients. Further investigation of the SASP driving the unpredictable growth of PAs, and its possible therapeutic application, is warranted.

The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75–78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs.

Histone deacetylase (HDAC) inhibitors are an emerging class of promising novel anticancer drugs. However, little is known which one of the 11 classical HDAC family members is the most relevant drug target for therapy. The first Phase I/II trials show that unselective inhibition of HDACs causes a variety of side effects. Therefore, identification and selective targeting of the most critical tumor entity-relevant HDAC family member may reduce unspecific effects and increase antitumor efficacy in the future. Here, we review the clinical relevance of a particular HDAC family member, HDAC8, in neuroblastoma biology, a highly malignant embryonal childhood cancer. HDAC8 expression correlates with poor outcome in neuroblastoma and selective HDAC8 inhibition induces differentiation. In contrast, the targeting of other HDAC family members results in a completely different phenotype. Because HDAC8-selective inhibitors are available, HDAC8 may be a potential drug target for neuroblastoma differentiation therapy using...