HDAC10 in neuroblastoma chemoresistance

Abstract

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.