Comparing the Effect of Multiple Histone Deacetylase Inhibitors on SSTR2 Expression and [111In]In-DOTATATE Uptake in NET Cells

The aim of this study was to increase somatostatin type-2 receptor (SSTR2) expression on neuroendocrine tumor (NET) cells using histone deacetylase inhibitors (HDACis), potentially increasing the uptake of SSTR2-targeted radiopharmaceuticals and subsequently improving treatment efficacy of peptide receptor radionuclide therapy (PRRT). Human NET cell lines BON-1, NCI-H727, and GOT1 were treated with HDACis (i.e., CI-994, entinostat, LMK-235, mocetinostat, panobinostat, or valproic acid (VPA); entinostat and VPA were the HDACis tested in GOT1 cells) to examine SSTR2 mRNA expression levels and uptake of SSTR2-targeting radiotracer [111In]In-DOTATATE. Reversibility of the induced effects was examined after drug-withdrawal. Finally, the effect of VPA on radiosensitivity was investigated. A strong stimulatory effect in BON-1, NCI-H727, and GOT1 cells was observed after HDACi treatment, both on SSTR2 mRNA expression levels and [111In]In-DOTATATE uptake. The effects of the HDACis were largely reversible over a period of seven days, demonstrating largest reductions within the first day. The reversibility profile of the induced effects suggests that proper timing of HDACi treatment is most likely essential for a beneficial outcome. In addition to increasing SSTR2 expression levels, VPA enhanced the radiosensitivity of all cell lines. In conclusion, HDACi treatment increased SSTR2 expression, and radiosensitivity was also enhanced upon VPA treatment.

The HDAC inhibitor CI-994 acts as a molecular memory aid by facilitating synaptic and intra-cellular communication after learning

Long-term memory formation relies on synaptic plasticity, activity-dependent transcription and epigenetic modifications. Multiple studies have shown that HDAC inhibitor (HDACi) treatments can enhance individual aspects of these processes, and thereby act as putative cognitive enhancers. However, their mode of action is not fully understood. In particular, it is unclear how systemic application of HDACis, which are devoid of substrate specificity, can target pathways that promote memory formation. In this study, we explore the electrophysiological, transcriptional and epigenetic responses that are induced by CI-994, a class I HDAC inhibitor, combined with contextual fear conditioning (CFC) in mice. We show that CI-994-mediated improvement of memory formation is accompanied by enhanced long-term potentiation in the hippocampus, a brain region recruited by CFC, but not in the striatum, a brain region not primarily implicated in contextual memory formation. Furthermore, using a combination of bulk and single cell RNA sequencing, we find that synaptic plasticity-promoting gene expression cascades are more strongly engaged in the hippocampus than in the striatum, but only when HDACi treatment co-occurred with CFC, and not by either treatment alone. Lastly, using ChIP-sequencing, we show that the combined action of HDACi application and conditioning is required to elicit enhancer histone acetylation in pathways that may underlie improved memory performance. Together, our results indicate that systemic HDACi administration amplifies brain-region specific processes that are naturally induced by learning. These findings shed light onto the mode of action of HDACis as cognitive enhancers.Significance StatementMemory formation relies on a plethora of functions, including epigenetic modifications. Over the past years, multiple studies have indicated the potential of HDAC inhibitors (HDACi) to act as cognitive enhancers, but their mode of action is not fully understood. Here, we tested whether HDACi treatment improves memory formation via “cognitive epigenetic priming”, stipulating that HDACis – without inherent target specificity – specifically enhance plasticity-related processes. We found that combining HDACi with fear learning, but not either treatment alone, enhances synaptic plasticity as well as memory-promoting transcriptional signaling in the hippocampus, a brain area known to be recruited by fear learning, but not in others. These results lend experimental support to the theory of “cognitive epigenetic priming”.

Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines

Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.

Study of the antilymphoma activity of pracinostat reveals different sensitivities of DLBCL cells to HDAC inhibitors

Histone deacetylase inhibitors (HDACis) are antitumor agents with distinct efficacy in hematologic tumors. Pracinostat is a pan-HDACi with promising early clinical activity. However, similar to other HDACis, its activity as a single agent is limited. Diffuse large B-cell lymphoma (DLBCL) includes distinct molecular subsets or metabolically defined subtypes that rely in different ways on the B-cell receptor signaling pathway, oxidative phosphorylation, and glycolysis for their survival. The antitumor activity of pracinostat has not been determined in lymphomas. We performed preclinical in vitro activity screening of 60 lymphoma cell lines that included 25 DLBCLs. DLBCL cells belonging to distinct metabolic subtypes were treated with HDACis for 6 hours or 14 days followed by transcriptional profiling. DLBCL xenograft models enabled assessment of the in vivo antilymphoma activity of pracinostat. Combination treatments with pracinostat plus 10 other antilymphoma agents were performed. Western blot was used to assess acetylation levels of histone and nonhistone proteins after HDACi treatment. Robust antiproliferative activity was observed across all lymphoma histotypes represented. Focusing on DLBCL, we identified a low-sensitivity subset that almost exclusively consists of the oxidative phosphorylation (OxPhos)-DLBCL metabolic subtype. OxPhos-DLBCL cells also showed poorer sensitivity to other HDACis, including vorinostat. Transcriptomic analysis revealed fewer modulated transcripts but an enrichment of antioxidant pathway genes after HDACi treatment of OxPhos-DLBCLs compared with high-sensitivity B-cell receptor (BCR)–DLBCLs. Pharmacologic inhibition of antioxidant production rescued sensitivity of OxPhos-DLBCLs to pracinostat whereas BCR-DLBCLs were unaffected. Our study provides novel insights into the antilymphoma activity of pracinostat and identifies a differential response of DLBCL metabolic subtypes to HDACis.

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.

CXCL1 Clone Evolution Induced by the HDAC Inhibitor Belinostat Might Be a Favorable Prognostic Indicator in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer due to its lack of treatment options. Patients with TNBC frequently develop resistance to chemotherapy. As epigenetic-based antineoplastic drugs, histone deacetylase inhibitors (HDACis) have achieved particular efficacy in lymphoma but are less efficacious in solid tumors, and the resistance mechanism remains poorly understood. In this study, the GSE129944 microarray dataset from the Gene Expression Omnibus database was downloaded, and fold changes at the transcriptome level of a TNBC line (MDA-MB-231) after treatment with belinostat were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to identify the critical biological processes. Construction and analysis of the protein-protein interaction (PPI) network were performed to screen candidate genes related to cancer prognosis. A total of 465 DEGs were identified, including 240 downregulated and 225 upregulated genes. The cytokine-cytokine receptor pathway was identified as being significantly changed. Furthermore, the expression of CXCL1 was implicated as a favorable factor in the overall survival of breast cancer patients. With in vitro approaches, we also showed that belinostat could induce the expression of CXCL1 in another 2 TNBC cell lines (BT-549 and HCC-1937). We speculate that belinostat-induced CXCL1 expression could be one of the results of the stress clone evolution of cells after HDACi treatment. These findings provide new insights into clone evolution during HDACi treatment, which might guide us to a novel perspective that various mutation-targeted treatments should be implemented during the whole treatment cycle.

HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

Histone deacetylase inhibitors (HDACi) are epigenome modulating molecules that target histone and non-histone proteins and have been successfully used to target many types of cancer and immunological disorders. While HDACi’s effects on nuclear histone deacetylases are well characterized, their effect on non-nuclear, cytoplasmic molecules requires further investigation. In the current study we characterized the effects of class I/II HDACi, specifically, TSA, MS-275, and SAHA, on plasmacytoid dendritic cell (pDC) biology upon viral activation via the TLR7/9 pathway. TSA, MS-275, and SAHA, down-modulated the induction of IFN-α and TNF-α upon Influenza A virus (IAV; TLR7 signaling) and Herpes Simplex 1 (HSV-1; TLR9 signaling) stimulation in primary pDC. The HDACi inhibitory effect was more prominent for IAV-mediated responses than for HSV-1. While IFN-α induction was not associated with inhibition of IRF-7 upregulation in the presence of TSA or MS-275, IRF-7 upregulation was affected by SAHA, but only for IAV. Furthermore, TSA, but not MS-275, inhibited TLR7/9-induced expression of maturation markers, CD40, and CD86, but not CD40. In addition, HDACi treatment increased virally-induced shedding of CD62L. Mechanistically, TSA, MS-275, SAHA significantly decreased early IRF-7 and NF-κB nuclear translocation, which was preceded by a decline in phosphorylation of IRF-7 at Ser477/479 and NF-κB p-p65, except for MS-275. In summary, we propose that broad HDACi, but not class I HDACi, treatment can negatively impact early TLR7/9-mediated signaling, namely, the disruption of IRF-7 and NF-κB activation and translocation that lead to deleterious effects on pDC function.

Expression of Adenoviral E1A in Transformed Cells as an Additional Factor of HDACi-Dependent FoxO Regulation

The adenoviral early region 1A (E1A) protein has proapoptotic and angiogenic activity, along with its chemosensitizing effect, making it the focus of increased interest in the context of cancer therapy. It was previously shown that E1A-induced chemosensitization to different drugs, including histone deacetylases inhibitors (HDACi), appears to be mediated by Forkhead box O (FoxO) transcription factors. In this study, we explore the relationship between E1A expression and the modulation of FoxO activity with HDACi sodium butyrate (NaBut). We show here that the basal FoxO level is elevated in E1A-expressing cells. Prolonged NaBut treatment leads to the inhibition of the FoxO expression and activity in E1A-expressing cells. However, in E1A-negative cells, NaBut promotes the transactivation ability of FoxO over time. A more detailed investigation revealed that the NaBut-induced decrease of FoxO activity in E1A-expressing cells is due to the NaBut-dependent decrease in E1A expression. Therefore, NaBut-induced inhibition of FoxO in E1A-positive cells can be overcome under unregulated overexpression of E1A. Remarkably, the CBP/p300-binding domain of E1Aad5 is responsible for stabilization of the FoxO protein. Collectively, these data show that the expression of E1A increases the FoxO stability but makes the FoxO level more sensitive to HDACi treatment.

Dissociation of the DNMT3A-HDAC1 Repressor Complex Induces PD-L1 Expression

Mutations of DNMT3A are one the most frequently observed alterations in AML patients. The DNMT3A R882 mutation appears to confer a dominant-negative loss-of function effect and changes the DNA binding preference according to recent studies. DNMT3A R882 mutations are also found at increasing frequency with age in healthy elderly populations and are one of the earliest pre-malignant alterations in the clonal evolution progression to leukemia. Studies have shown that DNMT3A mutations decrease overall DNA methylation and through less clear mechanisms, also impact the epigenetic landscape by effecting changes in histone acetylation. Here we investigated potential mechanisms by which loss of DNMT3A activity changes histone acetylation. DNMT3A binds with many proteins that regulate chromatin biology and gene transcription. Among those interacting proteins, we focused on the DNMT3A-HDAC interaction and their regulation of target gene suppression. To investigate whether there are differences in binding of wild-type vs. mutant DNMT3A to HDACs, we performed immunoprecipitation and Western blotting assays using Myc- tagged wild-type and R882 mutated DNMT3A. We found that DNMT3A R882 mutants showed reduced interaction with HDAC1 and 2. In addition, upon treatment with HDAC inhibitors (HDACi), DNMT3A mutant protein was more easily dissociated from HDAC1/2 than was wild-type DNMT3A. Intriguingly, covalent modification of DNMT3A R882 by SUMO1 protein was significantly enhanced relative to wild type DNMT3A. Together, we suggest that the weak complex formation between mutant DNMT3A and HDACs results from augmented SUMOylation of the R882 mutant. Because the DNMT3A R882 mutation reduces its methyl transferase activity, we investigated which genes would be upregulated from the DNMT3A repressor complex. To do this, we established isogenic TF-1 cell lines that harbor haploid DNMT3A knockout (DNMT3A+/-) using the Cripsr-Cas9 system. We also treated cells with HDACi and 5-azacytidine (5-aza) which inhibit HDAC and DNMT, respectively. Interestingly, we discovered that PD-L1 expression is induced by HDACi and 5-aza treatment. Chemical inhibition by 5-aza or genetic inhibition by knockout reduces DNMT3A activity and synergized with HDACi to increase PD-L1 expression. Flow cytometry analysis also demonstrated increased membrane PD-L1 expression in response to HDACi. We also found out that DNMT3A+/- resulted in higher Histone H3K27 acetylation, which is known as a gene activation mark. Higher H3K27 acetylation in DNMT3A+/- cell confirms the findings by other groups but the mechanisms by which this occurs are unknown. We suggest that haploinsufficiency of DNMT3A results in a reduced DNMT3A-HDAC interaction leading to higher H3K27 acetylation and increased PD-L1 expression. Our results also revealed that HDACi treatment induced cell cycle arrest, DNA damage and apoptosis at increasing levels in DNMT3A+/- cell. Even though the DNMT3A+/- TF-1 showed increased sensitivity to HDACi treatment, we observed a correlation of higher phosphor- ERK1/2 and PD-L1 levels in the surviving cells. The enhanced expression of PD-L1 and activation of ERK1/2 may explain in part how mutated DNMT3A contributes to drug resistance and immune checkpoint avoidance. Many oncology clinical trials are underway utilizing HDACi. However, the questions of which mutational backgrounds might be most sensitive to these agents and how to best combine them with other agents remain to be answered. To test whether reduced DNMT3A activity increases PD-L1 expression in vivo, we crossed floxed DNMT3A mice with Mx1-Cre mice. After 4 weeks of induction of Cre recombinase by injecting pIpC in the progeny carrying both genetically engineered changes, lineage depleted mouse BM cells were analyzed for PD-L1 expression using quantitative RT-PCR. BM cells derived from DNMT3A knockout mice showed increased expression of PD-L1 compared to wild-type mice. Treatment of these BM cells with an HDACi and/or 5-aza resulted in a synergistic induction of PD-L1 expression for the combination. Taken together, we suggest that mutant DNMT3A induces higher H3K27 acetylation along with PD-L1 expression due to a looser complex between HDAC1 and mutant DNMT3A. Therefore, we suggest that combined treatment with an HDACi and an immune checkpoint inhibitor targeting the PD-L1/PD-1 axis might be a promising strategy for treating DNMT3A mutant AML patients. Disclosures Small: InSilico Medicine: Membership on an entity's Board of Directors or advisory committees; Pharos I, B & T: Consultancy, Research Funding.