Histone deacetylases (HDACs) are important modulators of epigenetic gene regulation and additionally control the activity of non-histone protein substrates. While for HDACs 1-3 and 6 many potent selective inhibitors have been obtained, for other subtypes much less is known on selective inhibitors and the consequences of their inhibition. The present report describes the development of substituted benzhydroxamic acids as potent and selective HDAC8 inhibitors. Docking studies using available crystal structures have been used for structure-based optimization of this series of compounds. Within this study, we have investigated the role of HDAC8 in the proliferation of cancer cells and optimized hits for potency and selectivity, both in vitro and in cell culture. The combination of structure-based design, synthesis, and in vitro screening to cellular testing resulted in potent and selective HDAC8 inhibitors that showed anti-neuroblastoma activity in cellular testing.

Histone deacetylase 8 (HDAC8), a unique class I zinc-dependent HDAC, is an emerging target in cancer and other diseases. Its substrate repertoire extends beyond histones to many nonhistone proteins. Besides being a deacetylase, HDAC8 also mediates signaling via scaffolding functions. Aberrant expression or deregulated interactions with transcription factors are critical in HDAC8-dependent cancers. Many potent HDAC8-selective inhibitors with cellular activity and anticancer effects have been reported. We present HDAC8 as a druggable target and discuss inhibitors of different chemical scaffolds with cellular effects. Furthermore, we review HDAC8 activators that revert activity of mutant enzymes. Isotype-selective HDAC8 targeting in patients with HDAC8-relevant cancers is challenging, however, is promising to avoid adverse side effects as observed with pan-HDAC inhibitors.

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.

Background Postpancreatectomy morbidity remains significant even in high-volume centers and frequently results in delay or suspension of indicated adjuvant oncological treatment. This study investigated the short-term and long-term outcome after primary total pancreatectomy (PTP) and pylorus-preserving pancreaticoduodenectomy (PPPD) or Whipple procedure, with a special focus on administration of adjuvant therapy and oncological survival. Methods Patients who underwent PTP or PPPD/Whipple for periampullary cancer between January 2008 and December 2017 were retrospectively analyzed. Propensity score-matched analysis was performed to compare perioperative and oncological outcomes. Correspondingly, cases of rescue completion pancreatectomy (RCP) were analyzed. Results In total, 41 PTP and 343 PPPD/Whipple procedures were performed for periampullary cancer. After propensity score matching, morbidity (Clavien-Dindo classification (CDC) ≥ IIIa, 31.7% vs. 24.4%; p = 0.62) and mortality rates (7.3% vs. 2.4%, p = 0.36) were similar in PTP and PPPD/Whipple. Frequency of adjuvant treatment administration (76.5% vs. 78.4%; p = 0.87), overall survival (513 vs. 652 days; p = 0.47), and progression-free survival (456 vs. 454 days; p = 0.95) did not significantly differ. In turn, after RCP, morbidity (CDC ≥ IIIa, 85%) and mortality (40%) were high, and overall survival was poor (median 104 days). Indicated adjuvant therapy was not administered in 77%. Conclusions In periampullary cancers, PTP may provide surgical and oncological treatment outcomes comparable with pancreatic head resections and might save patients from RCP. Especially in selected cases with high-risk pancreatic anastomosis or preoperatively impaired glucose tolerance, PTP may provide a safe treatment alternative to pancreatic head resection.

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.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2234-8) contains supplementary material, which is available to authorized users.

Clinically relevant postoperative pancreatic fistula (CR-POPF) is a common severe surgical complication after pancreatic surgery. Current risk stratification systems mostly rely on intraoperatively assessed factors like manually determined gland texture or blood loss. We developed a preoperatively available image-based risk score predicting CR-POPF as a complication of pancreatic head resection. Frequency of CR-POPF and occurrence of salvage completion pancreatectomy during the hospital stay were associated with an intraoperative surgical (sFRS) and image-based preoperative CT-based (rFRS) fistula risk score, both considering pancreatic gland texture, pancreatic duct diameter and pathology, in 195 patients undergoing pancreatic head resection. Based on its association with fistula-related outcome, radiologically estimated pancreatic remnant volume was included in a preoperative (preFRS) score for POPF risk stratification. Intraoperatively assessed pancreatic duct diameter (p < 0.001), gland texture (p < 0.001) and high-risk pathology (p < 0.001) as well as radiographically determined pancreatic duct diameter (p < 0.001), gland texture (p < 0.001), high-risk pathology (p = 0.001), and estimated pancreatic remnant volume (p < 0.001) correlated with the risk of CR-POPF development. PreFRS predicted the risk of CR-POPF development (AUC = 0.83) and correlated with the risk of rescue completion pancreatectomy. In summary, preFRS facilitates preoperative POPF risk stratification in patients undergoing pancreatic head resection, enabling individualized therapeutic approaches and optimized perioperative management.

The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3–6 µM PCI-34051 or 10 µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05 µM (ALK-amplified) to 0.8 µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n = 88) and the German Neuroblastoma Trial cohort (n = 649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.

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