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Purpose: Histone deacetylase inhibitors (HDACi) are promising anticancer drugs. Although some HDACi have entered the clinic, the mechanism(s) underlying their tumor selectivity are poorly understood.Experimental Design and Results: Using gene expression analysis, we define a core set of six genes commonly regulated in acute myeloid leukemia (AML) blasts and cell lines. MYC, the most prominently modulated, is preferentially altered in leukemia. Upon HDACi treatment, c-Myc is acetylated at lysine 323 and its expression decreases, leading to TRAIL activation and apoptosis. c-Myc binds to the TRAIL promoter on the proximal GC box through SP1 or MIZ1, impairing TRAIL activation. HDACi exposure triggers TRAIL expression, altering c-Myc-TRAIL binding. These events do not occur in normal cells. Excitingly, this inverse correlation between TRAIL and c-Myc is supported by HDACi treatment ex vivo of AML blasts and primary human breast cancer cells. The predictive value of c-Myc to HDACi responsiveness is confirmed in vivo in AML patients undergoing HDACi-based clinical trials.Conclusions: Collectively, our findings identify a key role for c-Myc in TRAIL deregulation and as a biomarker of the anticancer action of HDACi in AML. The potential improved patient stratification could pave the way toward personalized therapies. Clin Cancer Res; 23(10); 2542–55. ©2016 AACR.

supplementary chemistry

SAHA effects in different cell lines.

Supplementary Tables

Schematic representation of the TRAIL promoter, its analyzed regions and the occupancy of epi-marks analyzed under indicated conditions.

Epigenetic status of the TRAIL promoter region after HDACi exposure.

On line Materials and Methods

c-Myc down-regulation modulates DR5 and p21 genes.

SAHA effects in normal cells.

Suppl. Methods

Purpose: Alteration in cell death is a hallmark of cancer. A functional role regulating survival, apoptosis, and necroptosis has been attributed to RIP1/3 complexes.Experimental Design: We have investigated the role of RIP1 and the effects of MC2494 in cell death induction, using different methods as flow cytometry, transcriptome analysis, immunoprecipitation, enzymatic assays, transfections, mutagenesis, and in vivo studies with different mice models.Results: Here, we show that RIP1 is highly expressed in cancer, and we define a novel RIP1/3–SIRT1/2–HAT1/4 complex. Mass spectrometry identified five acetylations in the kinase and death domain of RIP1. The novel characterized pan-SIRT inhibitor, MC2494, increases RIP1 acetylation at two additional sites in the death domain. Mutagenesis of the acetylated lysine decreases RIP1-dependent cell death, suggesting a role for acetylation of the RIP1 complex in cell death modulation. Accordingly, MC2494 displays tumor-selective potential in vitro, in leukemic blasts ex vivo, and in vivo in both xenograft and allograft cancer models. Mechanistically, MC2494 induces bona fide tumor-restricted acetylated RIP1/caspase-8–mediated apoptosis. Excitingly, MC2494 displays tumor-preventive activity by blocking 7,12-dimethylbenz(α)anthracene–induced mammary gland hyperproliferation in vivo.Conclusions: These preventive features might prove useful in patients who may benefit from a recurrence-preventive approach with low toxicity during follow-up phases and in cases of established cancer predisposition. Thus, targeting the newly identified RIP1 complex may represent an attractive novel paradigm in cancer treatment and prevention. Clin Cancer Res; 24(12); 2886–900. ©2018 AACR.

HDACI-specific effects on c-Myc acetylation.

Effects of c-Myc overexpression, p21shRNA and TRAILshRNA (A).

Primer sequence, location and product of amplification size.

Supplementary Figures

HDACi effects in different cell lines.

PDF file - 147KB, Differences in gene expression between ATRA and MC2392.

PDF file - 187KB, MC2392 specificity and selectivity for PML-RARalpha binding sites.

PDF file - 160KB, Supplemental Materials and Methods. Supplemental Table 1. Primers. Supplemental Table 2. Analysis of the transcription level of the 148 genes associated with apoptosis revealed main differences between ATRA and MC2392. Supplemental Table 3. Primers used. Supplemental Table 4. Primers used in qPCR ChIP.

HDAC inhibitors (HDACi) are widely used in the clinic to sensitize tumorigenic cells for treatment with other anticancer compounds. The major drawback of HDACi is the broad inhibition of the plethora of HDAC-containing complexes. In acute promyelocytic leukemia (APL), repression by the PML-RARα oncofusion protein is mediated by an HDAC-containing complex that can be dissociated by pharmacologic doses of all trans retinoic acid (ATRA) inducing differentiation and cell death at the expense of side effects and recurrence. We hypothesized that the context-specific close physical proximity of a retinoid and HDACi-binding protein in the repressive PML-RARα-HDAC complex may permit selective targeting by a hybrid molecule of ATRA with a 2-aminoanilide tail of the HDAC inhibitor MS-275, yielding MC2392. We show that MC2392 elicits weak ATRA and essentially no HDACi activity in vitro or in vivo. Genome-wide epigenetic analyses revealed that in NB4 cells expressing PML-RARα, MC2392 induces changes in H3 acetylation at a small subset of PML-RARα–binding sites. RNA-seq reveals that MC2392 alters expression of a number of stress-responsive and apoptotic genes. Concordantly, MC2392 induced rapid and massive, caspase-8–dependent cell death accompanied by RIP1 induction and ROS production. Solid and leukemic tumors are not affected by MC2392, but expression of PML-RARα conveys efficient MC2392-induced cell death. Our data suggest a model in which MC2392 binds to the RARα moiety and selectively inhibits the HDACs resident in the repressive complex responsible for the transcriptional impairment in APLs. Our findings provide proof-of-principle of the concept of a context-dependent targeted therapy. Cancer Res; 74(8); 2328–39. ©2014 AACR.

PDF file - 37KB, ChIP analysis for MC2392 specific target genes.

PDF file - 91KB, Death pathways modulation by MC2392.

How do organisms regulate the correct balance between the production of “new” cells and the elimination of the “old” ones, remains an important biology issue under investigation. Cell(s) death represents a fundamental process involved in organism development and cell homeostasis, whose alteration is considered one hallmark of cancer and lead to drug resistance and consequently treatment failure. The recent re-classification of cell death has identified new molecular programs in which several proteins have a pivotal role. Several studies have highlighted a direct link between epigenetic modifications and cell death mechanisms. Different epi-modifications have been described, capable of regulating diverse key players implicated in cell death, leading to uncontrolled proliferation of cancer cells. Scientific efforts are focused on the understanding the epigenetic regulation of cell death mechanisms by developing tools and/or new epi-molecules able to overcome cell death resistance. The development of new epi-molecular tools can overcome cell death deregulation thus potentially improving the sensitivity to the anti-tumor therapies. This chapter focuses on the main epigenetic deregulations in cell death mechanisms in cancer.

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/-) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/- mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/–) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/– mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

Introduction: Acetylation of histone and non-histone proteins is a post-translational modification mostly associated with activation of gene transcription. The first histone acetyltransferase (HAT) identified as modifying newly synthesized histone H4 at Lys5 and Lys12 was a type B HAT named HAT1. Although it was the first HAT to be discovered, it remains one of the most poorly studied enzymes in its class. In addition to its well-established role in the cytoplasm, recent findings have revealed new and intriguing aspects about the function of HAT1 in the nucleus. Purpose: Uncovering the non-canonical role of HAT1 first in DNA damage repair, then in chromatin assembly and chromatin maturation prompted in-depth investigations into the functional role of this protein in several biological processes. The involvement of HAT1 in regulating different pathways associated with a wide range of diseases, including cancer, and its contradictory role in carcinogenesis lead us to elucidate its role. Specifically, recent studies have shown that lower expression of HAT1 is associated with the pathogenesis of lung cancer 1, while in hepatocellular carcinoma, nasopharyngeal cancer, and pancreatic cancer 2–4 is highly expressed, acting as an oncogene, and linked to poor prognosis 4. Results and Conclusions: Immunoprecipitation experiment of overexpressed HAT1 combined with MS/MS analysis will be important to assess in which protein complexes HAT1 resides in breast cancer cell line. Moreover, genomics will be used for seeking possible molecular markers that can contribute at activating oncogenes or inactivating tumor suppressor genes. The main goal of this project is to understand HAT1 molecular mechanisms in cancer in order to develop novel strategy for cancer therapy. While efforts to identify molecules able to block its enzymatic activity are ongoing, the development of new compounds targeting one of its interactors could provide another entry point for HAT1 inhibition. REFERENCES

PURPOSE: Programmed cell death (PCD) is an important cellular event responsible for the maintenance of cellular homeostasis. More studies have suggested that the imbalance between cell survival and cell death leads to tumorigenesis, a process in which cells escape from PCDs. INTRODUCTION: Among the well-known cell death pathways, necroptosis has been defined to be a regulated necrotic cell death, which mediators belong both to the apoptotic and necrotic pathways [1]. The process can be triggered by a variety of stimuli and requires the activation of receptor-interacting protein (RIP) kinases RIPK1 and RIPK3, as well as mixed lineage kinase domain-like protein (MLKL) [2]. RESULTS AND CONCLUSIONS: A recent study has correlated the acetylation of RIP to the tumorigenesis and cell death process. The molecular complex mediated by RIPK1 shows the co-presence of proteins with different enzymatic activities such as acetyltransferase, kinase and deacetylase elucidating cancer not only as a genetic but also an epigenetic disease [3]. Since RIPK1 is overexpressed in several cancer cell types and it is involved in either survival or apoptosis and necroptosis, one of the main goals of the project is to determine how to modulate the RIP1-complex, in order to lead malignant cells to cell death. To reach this aim, the interactome study in leukaemia cell line will be performed to clarify the involvement of RIPK1. While the roles of RIPK3 and MLKL in leukemogenesis are already known, the novelty is to understand “how” RIPK1 triggers different cellular pathways that leads cells to make a critical decision either to survive or die. Starting from the characterization of Necrostatins, the project includes the identification of new modulators of RIPK1 which could regulate the activated molecular complex in different cellular processes and eventually to make necroptosis a pharmacologically controllable event.

A specific aptameric sequence has been immobilized on short polyethyleneglycol (PEG) interface on gold nano-film deposited on a D-shaped plastic optical fiber (POFs) probe, and the protein binding has been monitored exploiting the very sensitive surface plasmon resonance (SPR) phenomenon. The receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein has been specifically used to develop an aptasensor. Surface analysis techniques coupled to fluorescence microscopy and plasmonic analysis have been utilized to characterize the biointerface. Spanning a wide protein range (25 ÷ 1000 nM), the SARS-Cov-2 spike protein was detected with a Limit of Detection (LoD) of about 37 nM. Different interferents (BSA, AH1N1 hemagglutinin protein and MERS spike protein) have been tested confirming the specificity of our aptasensor. Finally, a preliminary test in diluted human serum encouraged its application in a point-of-care device, since POF-based aptasensor represent a potentially low-cost compact biosensor, characterized by a rapid response, a small size and could be an ideal laboratory portable diagnostic tool., Graphical abstract Image 1

Simple Summary A new form of programmed necrosis called necroptosis has emerged. This new and well-documented type of programmed cell death is involved in several human diseases, including cancer. RIPK1, the main mediator of necroptosis, in response to different stimuli, activates several molecular pathways leading to inflammation, cell survival, or cell death. Targeting necroptosis could be a new strategy for advanced therapies. In this review, we focus on the biological relevance of this type of programmed cell death and its main executor RIPK1 in pathogenesis to find novel potential clinical intervention strategies. Abstract Regulated cell death mechanisms are essential for the maintenance of cellular homeostasis. Evasion of cell death is one of the most important hallmarks of cancer. Necroptosis is a caspase independent form of regulated cell death, investigated as a novel therapeutic strategy to eradicate apoptosis resistant cancer cells. The process can be triggered by a variety of stimuli and is controlled by the activation of RIP kinases family as well as MLKL. The well-studied executor, RIPK1, is able to modulate key cellular events through the interaction with several proteins, acting as strategic crossroads of several molecular pathways. Little evidence is reported about its involvement in tumorigenesis. In this review, we summarize current studies on the biological relevance of necroptosis, its contradictory role in cancer and its function in cell fate control. Targeting necroptosis might be a novel therapeutic intervention strategy in anticancer therapies as a pharmacologically controllable event.

Simple Summary Cancer therapy relies on the employment of different strategies aimed at inducing cancer cell death through different mechanisms, including DNA damage and apoptosis induction. One of the key regulators of these pathways is the epigenetic enzyme SIRT6, which has been shown to have a dichotomous function in cell fate determination and, consequently, cancer initiation and progression. In this review, we aim to summarize the current knowledge on the role of SIRT6 in cancer. We show that it can act as both tumor suppressor and promoter, even in the same cancer type, depending on the biological context. We then describe the most promising modulators of SIRT6 which, through enzyme activation or inhibition, may impair tumor growth. These molecules can also be used for the elucidation of SIRT6 function, thereby advancing the current knowledge on this crucial protein. Abstract Sirtuin 6 (SIRT6) is a NAD+-dependent nuclear deacylase and mono-ADP-ribosylase with a wide spectrum of substrates. Through its pleiotropic activities, SIRT6 modulates either directly or indirectly key processes linked to cell fate determination and oncogenesis such as DNA damage repair, metabolic homeostasis, and apoptosis. SIRT6 regulates the expression and activity of both pro-apoptotic (e.g., Bax) and anti-apoptotic factors (e.g., Bcl-2, survivin) in a context-depending manner. Mounting evidence points towards a double-faced involvement of SIRT6 in tumor onset and progression since the block or induction of apoptosis lead to opposite outcomes in cancer. Here, we discuss the features and roles of SIRT6 in the regulation of cell death and cancer, also focusing on recently discovered small molecule modulators that can be used as chemical probes to shed further light on SIRT6 cancer biology and proposed as potential new generation anticancer therapeutics.

The discovery and development of isoform-selective histone deacetylase (HDAC) inhibitor is a challenging task because of the sequence homology among HDAC enzymes. In the present work, novel tetrahydro benzo[b]thiophene-3-carbonitrile based benzamides were designed, synthesized, and evaluated as HDAC inhibitors. Pharmacophore modeling was our main design strategy, and two novel series of tetrahydro benzo[b]thiophene-3-carbonitrile derivatives with piperidine linker (series 1) and piperazine linker (series 2) were identified as HDAC inhibitors. Among all the synthesised compounds, 9h with 4-(aminomethyl) piperidine linker and 14n with piperazine linker demonstrated good activity against human HDAC1 and HDAC6, respectively. Both the compounds also exhibited good antiproliferative activity against several human cancer cell lines. Both these compounds (9h and 14n) also induced cell cycle arrest and apoptosis in U937 and MDA-MB-231 cancer cells. Overall, for the first time, this research discovered potent isoform-selective HDAC inhibitors using cyclic linker instead of the aliphatic chain and aromatic ring system, which were reported in known HDAC inhibitors.

Positive Regulatory Domain (PRDM) gene family members commonly express two main molecular variants, the PR-plus isoform usually acting as tumor suppressor and the PR-minus one functioning as oncogene. Accordingly, PRDM2/RIZ encodes for RIZ1 (PR-plus) and RIZ2 (PR-minus). In human cancers, genetic or epigenetic modifications induce RIZ1 silencing with an expression level imbalance in favor of RIZ2 that could be relevant for tumorigenesis. Additionally, in estrogen target cells and tissues, estradiol increases RIZ2 expression level with concurrent increase of cell proliferation and survival. Several attempts to study RIZ2 function in HeLa or MCF-7 cells by its over-expression were unsuccessful. Thus, we over-expressed RIZ2 in HEK-293 cells, which are both RIZ1 and RIZ2 positive but unresponsive to estrogens. The forced RIZ2 expression increased cell viability and growth, prompted the G2-to-M phase transition and organoids formation. Accordingly, microarray analysis revealed that RIZ2 regulates several genes involved in mitosis. Consistently, RIZ silencing in both estrogen-responsive MCF-7 and -unresponsive MDA-MB-231 cells induced a reduction of cell proliferation and an increase of apoptosis rate. Our findings add novel insights on the putative RIZ2 tumor-promoting functions, although additional attempts are warranted to depict the underlying molecular mechanism.

A novel technique was demonstrated that overcome important drawbacks to crosslink cells by irradiation with ultrashort UV laser pulses (L-crosslinking). To use this technique coupled to Chromatin ImmunoPrecipitation (ChIP) in a high throughput context, a pre-screening fast method needs to be implemented to set up suitable irradiation conditions of the cell sample for efficient L-crosslinking with no final and long ChIP analysis. Here a fast method is reported where living human cells have been first transfected with a vector coding for Estrogen Receptor {\alpha} (ER{\alpha}), linked to Green Florescent protein (ER{\alpha}-GFP), so that the well-known interaction between the Estrogen Receptor Elements (ERE) region of the cell DNA and the ER{\alpha} protein can be detected by studying the fluorometric response of the irradiated cells. The damage induced to cells by UV irradiation is characterized by looking at DNA integrity, proteins stability and cellular viability. A second novel approach is presented to analyze or re-visit DNA and RNA sequences and their molecular configurations. This approach is based on methods derived from Chern-Simons super-gravity adapted to describe mutations in DNA/RNA strings, as well as interactions between nucleic acids. As a preliminary case we analyze the KRAS human gene sequence and some of its mutations. Interestingly, our model shows how the Chern-Simons current are capable to characterize the mutations within a sequence, in particular giving a quantitative indication of the mutation likelihood., Comment: 33 pages, 5 figures

Some species of clover are reported to have beneficial effects in human diseases. However, little is known about the activity of the forage plant Trifolium repens, or white clover, which has been recently found to exert a hepatoprotective action. Scientific interest is increasingly focused on identifying new drugs, especially natural products and their derivatives, to treat human diseases including cancer. We analyzed the anticancer effects of T. repens in several cancer cell lines. The phytochemical components of T. repens were first extracted in a methanol solution and then separated into four fractions by ultra-high-performance liquid chromatography. The effects of the total extract and each fraction on cancer cell proliferation were analyzed by MTT assay and Western blotting. T. repens and, more robustly, its isoflavonoid-rich fraction showed high cytotoxic effects in chronic myelogenous leukemia (CML) K562 cells, with IC50 values of 1.67 and 0.092 mg/mL, respectively. The block of cell growth was associated with a total inhibition of BCR-ABL/STAT5 and activation of the p38 signaling pathways. In contrast, these strongly cytotoxic effects did not occur in normal cells. Our findings suggest that the development of novel compounds derived from phytochemical molecules contained in Trifolium might lead to the identification of new therapeutic agents active against CML.