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Histone methylation plays a key role in the regulation of chromatin structure, and its dynamics regulates important cellular processes. The investigation of the role of alterations in histone methylation in cancer has led to the identification of histone methyltransferases and demethylases as promising novel targets for therapy. Lysine-specific demethylase 1(LSD1, also known as KDM1A) is the first discovered histone lysine demethylase, with the ability to demethylase H3K4me1/2 and H3K9me1/2 at target loci in a context-dependent manner. LSD1 regulates the balance between self-renewal and differentiation of stem cells, and is highly expressed in various cancers, playing an important role in differentiation and self-renewal of tumor cells. In this review, we summarize recent studies about the LSD1, its role in normal and tumor cells, and the potential use of small molecule LSD1 inhibitors in therapy.

Key Points Histone deacetylases (HDACs) and histone acetylases (HATs) are enzymes that are responsible for deacetylating and acetylating, respectively, the amino-terminal tails of histones. These chromatin changes regulate transcription and many other nuclear events. Non-histone proteins (such as the oncosuppressor p53) and several cytoplasmic proteins are also regulated by HATs/HDACs. Studies on the molecular pathogenesis of acute myeloid leukaemias have shown that the aberrant recruitment of HDACs has an important role in leukaemogenesis. Leukaemia-associated fusion proteins (such as promyelocytic leukaemia (PML)–retinoic acid receptor (RAR) and acute myeloid leukaemia 1 (AML1)–ETO) recruit HDACs to repress the transcription of genes involved in differentiation (the fusion proteins therefore block differentiation) and impair the function of p53. Alterations in the expression and/or activity of HATs/HDACs have been also observed in solid tumours. Solid tumours show decreased levels of histone acetylation, which correlates with clinical outcome. HDAC inhibitors (HDACi) have been widely studied and belong to several chemical classes. HDACi exert cell-type-specific effects inducing apoptosis, cell-cycle arrest, and differentiation. In leukaemias, HDACi induce the expression of members of the tumour-necrosis factor-related apoptosis-inducing ligand (TRAIL) and FAS death receptor pathways. This induction is responsible for the pro-apoptotic effects of HDACi. Clinical trials for several HDACi have started, and HDACi-responsive tumours have been observed. How have the data from the initial clinical trials of histone deacetylase inhibitors (HDACi) improved our understanding of how these drugs work? And what key facts do we still need to understand about the biology of HDACi? Histone deacetylases (HDACs) are considered to be among the most promising targets in drug development for cancer therapy, and first-generation histone deacetylase inhibitors (HDACi) are currently being tested in phase I/II clinical trials. A wide-ranging knowledge of the role of HDACs in tumorigenesis, and of the action of HDACi, has been achieved. However, several basic aspects are not yet fully understood. Investigating these aspects in the context of what we now understand about HDACi action both in vitro and in vivo will further improve the design of optimized clinical protocols.

Understanding the cell cycle at the single-cell level is crucial for cellular biology and cancer research. While current methods using fluorescent markers have improved the study of adherent cells, non-adherent cells remain challenging. In this study, we addressed this gap by combining a specialized surface to enhance cell attachment, the FUCCI(CA)2 sensor, an automated image analysis pipeline, and a custom machine learning algorithm. This approach enabled precise measurement of cell cycle phase durations in non-adherent cells. This method was validated in acute myeloid leukemia cell lines NB4 and Kasumi-1, which have unique cell cycle characteristics, and we tested the impact of cell cycle-modulating drugs on NB4 cells. Our cell cycle analysis system, which is also compatible with adherent cells, is fully automated and freely available, providing detailed insights from hundreds of cells under various conditions. This report presents a valuable tool for advancing cancer research and drug development by enabling comprehensive, automated cell cycle analysis in both adherent and non-adherent cells.

Histone deacetylases (HDACs) are a key component of the epigenetic machinery regulating gene expression, and behave as oncogenes in several cancer types, spurring the development of HDAC inhibitors (HDACi) as anticancer drugs. This review discusses new results regarding the role of HDACs in cancer and the effect of HDACi on tumour cells, focusing on haematological malignancies, particularly acute myeloid leukaemia. Histone deacetylases may have opposite roles at different stages of tumour progression and in different tumour cell sub-populations (cancer stem cells), highlighting the importance of investigating these aspects for further improving the clinical use of HDACi in treating cancer.

Background Acute myeloid leukemia (AML) is a heterogeneous and aggressive blood cancer that results from diverse genetic aberrations in the hematopoietic stem or progenitor cells (HSPCs) leading to the expansion of blasts in the hematopoietic system. The heterogeneity and evolution of cancer blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. In this study, we investigated the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs at genetic and transcriptional levels, and unveiled the underlying pathways and genes contributing to recurrence. Methods Whole-exome sequencing was used to detect somatic mutations and large copy number variations (CNVs). Single cell RNA-seq was performed to investigate the clonal heterogeneity between Dx-Re pairs and amongst patients. Results scRNA-seq analysis revealed extensive expression differences between patients and Dx-Re pairs, even for those with the same -presumed- initiating events. Transcriptional differences between and within patients are associated with clonal composition and evolution, with the most striking differences in patients that gained large-scale copy number variations at relapse. These differences appear to have significant molecular implications, exemplified by a DNMT3A/FLT3-ITD patient where the leukemia switched from an AP-1 regulated clone at Dx to a mTOR signaling driven clone at Re. The two distinct AML1-ETO pairs share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells evolved from the Dx cells. Conclusions In summary, the single cell RNA data underpinned the tumor heterogeneity not only amongst patient blasts with similar initiating mutations but also between each Dx-Re pair. Our results suggest alternatively and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence.

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo . Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients. How breast cancer cells adapt to individual therapies targeting the oestrogen receptor alpha is poorly understood. Here the authors show resistance emerging through differential epigenetic reprogramming that activates the cholesterol biosynthesis pathway.

The COVID-19 pandemic has had a devastating impact worldwide and has been a great challenge for the scientific community. Vaccines against SARS-CoV-2 are now efficiently lessening COVID-19 mortality, although finding a cure for this infection is still a priority. An unbalanced immune response and the uncontrolled release of proinflammatory cytokines are features of COVID-19 pathophysiology and contribute to disease progression and worsening. Histone deacetylases (HDACs) have gained interest in immunology, as they regulate the innate and adaptative immune response at different levels. Inhibitors of these enzymes have already proven therapeutic potential in cancer and are currently being investigated for the treatment of autoimmune diseases. We thus tested the effects of different HDAC inhibitors, with a focus on a selective HDAC6 inhibitor, on immune and epithelial cells in in vitro models that mimic cells activation after viral infection. Our data indicate that HDAC inhibitors reduce cytokines release by airway epithelial cells, monocytes and macrophages. This anti-inflammatory effect occurs together with the reduction of monocytes activation and T cell exhaustion and with an increase of T cell differentiation towards a T central memory phenotype. Moreover, HDAC inhibitors hinder IFN-I expression and downstream effects in both airway epithelial cells and immune cells, thus potentially counteracting the negative effects promoted in critical COVID-19 patients by the late or persistent IFN-I pathway activation. All these data suggest that an epigenetic therapeutic approach based on HDAC inhibitors represents a promising pharmacological treatment for severe COVID-19 patients.

Background Acute myeloid leukemia (AML) is characterized by accumulation of aberrantly differentiated hematopoietic myeloid progenitor cells. The karyotyping-silent NUP98-NSD1 fusion is a molecular hallmark of pediatric AML and is associated with the activating FLT3-ITD mutation in > 70% of the cases. NUP98-NSD1 fusion protein promotes myeloid progenitor self-renewal in mice via unknown molecular mechanism requiring both the NUP98 and the NSD1 moieties. Methods We used affinity purification coupled to label-free mass spectrometry (AP-MS) to examine the effect of NUP98-NSD1 structural domain deletions on nuclear interactome binding. We determined their functional relevance in NUP98-NSD1 immortalized primary murine hematopoietic stem and progenitor cells (HSPC) by inducible knockdown, pharmacological targeting, methylcellulose assay, RT-qPCR analysis and/or proximity ligation assays (PLA). Fluorescence recovery after photobleaching and b-isoxazole assay were performed to examine the phase transition capacity of NUP98-NSD1 in vitro and in vivo. Results We show that NUP98-NSD1 core interactome binding is largely dependent on the NUP98 phenylalanine-glycine (FG) repeat domains which mediate formation of liquid-like phase-separated NUP98-NSD1 nuclear condensates. We identified condensate constituents including imitation switch (ISWI) family member SMARCA5 and BPTF (bromodomain PHD finger transcription factor), both members of the nucleosome remodeling factor complex (NURF). We validated the interaction with SMARCA5 in NUP98-NSD1 + patient cells and demonstrated its functional role in NUP98-NSD1/FLT3-ITD immortalized primary murine hematopoietic cells by genetic and pharmacological targeting. Notably, SMARCA5 inhibition did not affect NUP98-NSD1 condensates suggesting that functional activity rather than condensate formation per se is crucial to maintain the transformed phenotype. Conclusions NUP98-NSD1 interacts and colocalizes on the genome with SMARCA5 which is an essential mediator of the NUP98-NSD1 transformation in hematopoietic cells. Formation of NUP98-NSD1 phase-separated nuclear condensates is not sufficient for the maintenance of transformed phenotype, which suggests that selective targeting of condensate constituents might represent a new therapeutic strategy for NUP98-NSD1 driven AML.

Altered histone post-translational modifications are frequently associated with cancer. Here, we apply mass-spectrometry to study the epigenetic landscapes of breast cancer subtypes, with a particular focus on triple-negative breast cancers (TNBCs), a heterogeneous group lacking well-defined molecular targets and effective therapies. The analysis of over 200 tumors reveals epigenetic signatures that discriminate TNBCs from the other BC subtypes, and that distinguish TNBC patients with different prognoses. Employing a multi-OMICs approach integrating epigenomics, transcriptomics, and proteomics data, we investigate the mechanistic role of increased H3K4 methylation in TNBCs, demonstrating that H3K4me2 sustains the expression of genes associated with the TNBC phenotype. Through CRISPR-mediated editing, we establish a causal relationship between H3K4me2 and gene expression for several targets. Furthermore, treatment with H3K4 methyltransferase inhibitors reduce TNBC cell growth in vitro and in vivo. Collectively, our results unravel a novel epigenetic pathway implicated in TNBC pathogenesis and suggest new opportunities for targeted therapy. Using a histone focused multi-omics approach, here the authors show that increased H3K4 methylation drives triple-negative breast cancer phenotypes, revealing potential novel therapeutic avenues.

Different mechanisms have been implicated in the induction of senescence. Two of these mechanisms, the DNA damage response, which induces a replicative checkpoint, and the formation of heterochromatic foci, which leads to transcriptional repression, are found to act together in oncogene-expressing cells. Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescence-associated heterochromatic foci (SAHF). Here we show that in human fibroblasts resistant to premature p16 INK4a induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogene-induced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogene-expressing cells increase DDR signalling and lead to apoptosis. In vivo , a histone deacetylase inhibitor (HDACi) causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours.