Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influence on MEF2-dependent transcription.

In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding.

Different class IIa HDACs repressive complexes regulate specific epigenetic responses related to cell survival in leiomyosarcoma cells.

Transcriptional networks supervising class IIa HDAC expression are poorly defined. Here we demonstrate that MEF2D is the key factor controlling HDAC9 transcription. This control, which is part of a negative feed-back loop during muscle differentiation, is hijacked in cancer. In leiomyosarcomas the MEF2D/HDAC9 vicious circuit sustains proliferation and cell survival, through the repression of the death receptor FAS. Comprehensive genome-wide studies demonstrate that HDAC4 and HDAC9 control different genetic programs and show both specific and common genomic binding sites. Although the number of MEF2-target genes commonly regulated is similar, only HDAC4 represses many additional genes that are not MEF2D targets. As expected, HDAC4-/- and HDAC9-/- cells increase H3K27ac levels around the TSS of the respective repressed genes. However, these genes rarely show binding of the HDACs at their promoters. Frequently HDAC4 and HDAC9 bind intergenic regions. We demonstrate that these regions, recognized by MEF2D/HDAC4/HDAC9 repressive complexes, show the features of active enhancers. In these regions HDAC4 and HDAC9 can differentially influence H3K27 acetylation. Our studies describe new layers of class IIa HDACs regulation, including a dominant positional effect, and can contribute to explain the pleiotropic actions of MEF2 TFs.

Changes in chromatin accessibility and transcriptional landscape induced by HDAC inhibitors in TP53 mutated patient-derived colon cancer organoids.

Here we present the generation and characterization of patient-derived organoids (PDOs) from colorectal cancer patients. PDOs derived from two patients with TP53 mutations were tested with two different HDAC inhibitors (SAHA and NKL54). Cell death induction, transcriptome, and chromatin accessibility changes were analyzed. HDACIs promote the upregulation of low expressed genes and the downregulation of highly expressed genes. A similar differential effect is observed at the level of chromatin accessibility. Only SAHA is a potent inducer of cell death, which is characterized by the upregulation of BH3-only genes BIK and BMF. Up-regulation of BIK is associated with increased accessibility in an intronic region that has enhancer properties. SAHA, but not NKL54, also causes downregulation of BCL2L1 and decreases chromatin accessibility in three distinct regions of the BCL2L1 locus. Both inhibitors upregulate the expression of innate immunity genes and members of the MHC family. In summary, our exploratory study indicates a mechanism of action for SAHA and demonstrate the low efficacy of NKL54 as a single agent for apoptosis induction, using two PDOs. These observations need to be validated in a larger cohort of PDOs.

Acetylation and Phosphorylation in the Regulation of Hypoxia-Inducible Factor Activities: Additional Options to Modulate Adaptations to Changes in Oxygen Levels.

O2 is essential for the life of eukaryotic cells. The ability to sense oxygen availability and initiate a response to adapt the cell to changes in O2 levels is a fundamental achievement of evolution. The key switch for adaptation consists of the transcription factors HIF1A, HIF2A and HIF3A. Their levels are tightly controlled by O2 through the involvement of the oxygen-dependent prolyl hydroxylase domain-containing enzymes (PHDs/EGNLs), the von Hippel-Lindau tumour suppressor protein (pVHL) and the ubiquitin-proteasome system. Furthermore, HIF1A and HIF2A are also under the control of additional post-translational modifications (PTMs) that positively or negatively regulate the activities of these transcription factors. This review focuses mainly on two PTMs of HIF1A and HIF2A: phosphorylation and acetylation.

HDACs and the epigenetic plasticity of cancer cells: Target the complexity.

Cancer cells must adapt to the hostile conditions of the microenvironment in terms of nutrition, space, and immune system attack. Mutations of DNA are the drivers of the tumorigenic process, but mutations must be able to hijack cellular functions to sustain the spread of mutant genomes. Transcriptional control is a key function in this context and is controlled by the rearrangement of the epigenome. Unlike genomic mutations, the epigenome of cancer cells can in principle be reversed. The discovery of the first epigenetic drugs triggered a contaminating enthusiasm. Unfortunately, the complexity of the epigenetic machinery has frustrated this enthusiasm. To develop efficient patient-oriented epigenetic therapies, we need to better understand the nature of this complexity. In this review, we will discuss recent advances in understanding the contribution of HDACs to the maintenance of the transformed state and the rational for their selective targeting.

Novel synthesis of 1,2-diaza-1,3-dienes with potential biological activity from cinnamic acids and diazonium salts of anilines.

Cinnamic acids are an important class of phenolic compounds, which have many beneficial effects on human health but are also interesting synthetic intermediates thanks to the presence of several reactive sites. While studying the reactivity of cinnamic acids with diazonium salts from aromatic amines, an unexpected reactivity has been discovered, leading to the formation of 1,2-diaza-1,3-dienes instead of traditional diazo-coupling products. The new compounds have been fully characterized by mono and bidimensional NMR spectroscopy and mass spectrometry. Preliminary studies on the biological activity of the compounds have been carried out testing both their antibacterial and antitumor activity, leading to promising results.

Proteotoxic stress-induced apoptosis in cancer cells: understanding the susceptibility and enhancing the potency.

Leiomyosarcoma (LMS) is aggressive cancer with few therapeutic options. LMS cells are more sensitive to proteotoxic stress compared to normal smooth muscle cells. We used small compound 2c to induce proteotoxic stress and compare the transcriptomic adaptations of immortalized human uterine smooth muscle cells (HUtSMC) and LMS cells SK-UT-1. We found that the expression of the heat shock proteins (HSPs) gene family is upregulated with higher efficiency in normal cells. In contrast, the upregulation of BH3-only proteins is higher in LMS cells. HSF1, the master regulator of HSP transcription, is sequestered into transcriptionally incompetent nuclear foci only in LMS cells, which explains the lower HSP upregulation. We also found that several compounds can enhance the cell death response to proteotoxic stress. Specifically, when low doses were used, an inhibitor of salt-inducible kinases (SIKs) and the inhibitor of IRE1α, a key element of the unfolded protein response (UPR), support proteotoxic-induced cell death with strength in LMS cells and without effects on the survival of normal cells. Overall, our data provide an explanation for the higher susceptibility of LMS cells to proteotoxic stress and suggest a potential option for co-treatment strategies.

A regulative epigenetic circuit supervised by HDAC7 represses IGFBP6 and IGFBP7 expression to sustain mammary stemness.

Background: In the breast, the pleiotropic epigenetic regulator HDAC7 can influence stemness. Materials & Methods: The authors used MCF10 cells knocked-out for HDAC7 to explore the contribution of HDAC7 to IGF1 signaling. Results: HDAC7 buffers H3K27ac levels at the IGFBP6 and IGFBP7 genomic loci and influences their expression. In this manner, HDAC7 can tune IGF1 signaling to sustain stemness. In HDAC7 knocked-out cells, RXRA promotes the upregulation of IGFBP6/7 mRNAs. By contrast, HDAC7 increases FABP5 expression, possibly through repression of miR-218. High levels of FABP5 can reduce the delivery of all-trans-retinoic acid to RXRA. Accordingly, the silencing of FABP5 increases IGFBP6 and IGFBP7 expression and reduces mammosphere generation. Conclusion: The authors propose that HDAC7 controls the uptake of all-trans-retinoic acid, thus influencing RXRA activity and IGF1 signaling.

HDAC4 degradation during senescence unleashes an epigenetic program driven by AP-1/p300 at selected enhancers and super-enhancers.

BACKGROUND: Cellular senescence is a permanent state of replicative arrest defined by a specific pattern of gene expression. The epigenome in senescent cells is sculptured in order to sustain the new transcriptional requirements, particularly at enhancers and super-enhancers. How these distal regulatory elements are dynamically modulated is not completely defined. RESULTS: Enhancer regions are defined by the presence of H3K27 acetylation marks, which can be modulated by class IIa HDACs, as part of multi-protein complexes. Here, we explore the regulation of class IIa HDACs in different models of senescence. We find that HDAC4 is polyubiquitylated and degraded during all types of senescence and it selectively binds and monitors H3K27ac levels at specific enhancers and super-enhancers that supervise the senescent transcriptome. Frequently, these HDAC4-modulated elements are also monitored by AP-1/p300. The deletion of HDAC4 in transformed cells which have bypassed oncogene-induced senescence is coupled to the re-appearance of senescence and the execution of the AP-1/p300 epigenetic program. CONCLUSIONS: Overall, our manuscript highlights a role of HDAC4 as an epigenetic reader and controller of enhancers and super-enhancers that supervise the senescence program. More generally, we unveil an epigenetic checkpoint that has important consequences in aging and cancer.

Enhancing Proteotoxic Stress in Leiomyosarcoma Cells Triggers Mitochondrial Dysfunctions, Cell Death, and Antitumor Activity in vivo.

Leiomyosarcomas are rare and aggressive tumors characterized by a complex karyotype. Surgical resection with or without radiotherapy and chemotherapy is the standard curative treatment. Unfortunately, a high percentage of leiomyosarcomas recurs and metastasizes. In these cases, doxorubicin and ifosfamide represent the standard treatment but with low response rates. Here, we evaluated the induction of proteotoxic stress as a possible strategy to kill leiomyosarcoma cells in a therapeutic perspective. We show that aggressive leiomyosarcomas coexist with high levels of proteotoxic stress. As a consequence, we hypothesized that leiomyosarcoma cells are vulnerable to further increases of proteotoxic stress. The small compound 2c is a strong inducer of proteotoxic stress. In leiomyosarcoma cells, it triggers cell death coupled to a profound reorganization of the mitochondrial network. By using stimulated emission depletion microscopy, we have unveiled the existence of DIABLO/SMAC clusters that are modulated by 2c. Finally, we have engineered a new version of 2c linked to polyethylene glycol though a short peptide, named 2cPP. This new prodrug is specifically activated by proteases present in the tumor microenvironment. 2cPP shows a strong antitumor activity in vivo against leiomyosarcomas and no toxicity against normal cells.

HDAC7-mediated control of tumour microenvironment maintains proliferative and stemness competence of human mammary epithelial cells.

HDAC7 is a pleiotropic transcriptional coregulator that controls different cellular fates. Here, we demonstrate that in human mammary epithelial cells, HDAC7 sustains cell proliferation and favours a population of stem-like cells, by maintaining a proficient microenvironment. In particular, HDAC7 represses a repertoire of cytokines and other environmental factors, including elements of the insulin-like growth factor signalling pathway, IGFBP6 and IGFBP7. This HDAC7-regulated secretome signature predicts negative prognosis for luminal A breast cancers. ChIP-seq experiments revealed that HDAC7 binds locally to the genome, more frequently distal from the transcription start site. HDAC7 can colocalize with H3K27-acetylated domains and its deletion further increases H3K27ac at transcriptionally active regions. HDAC7 levels are increased in RAS-transformed cells, in which this protein was required not only for proliferation and cancer stem-like cell growth, but also for invasive features. We show that an important direct target of HDAC7 is IL24, which is sufficient to suppress the growth of cancer stem-like cells.