Histone Deacetylases 1 and 2 Regulate Microglia Function during Development, Homeostasis, and Neurodegeneration in a Context-Dependent Manner.

Microglia as tissue macrophages contribute to the defense and maintenance of central nervous system (CNS) homeostasis. Little is known about the epigenetic signals controlling microglia function in vivo. We employed constitutive and inducible mutagenesis in microglia to delete two class I histone deacetylases, Hdac1 and Hdac2. Prenatal ablation of Hdac1 and Hdac2 impaired microglial development. Mechanistically, the promoters of pro-apoptotic and cell cycle genes were hyperacetylated in absence of Hdac1 and Hdac2, leading to increased apoptosis and reduced survival. In contrast, Hdac1 and Hdac2 were not required for adult microglia survival during homeostasis. In a mouse model of Alzheimer's disease, deletion of Hdac1 and Hdac2 in microglia, but not in neuroectodermal cells, resulted in a decrease in amyloid load and improved cognitive impairment by enhancing microglial amyloid phagocytosis. Collectively, we report a role for epigenetic factors that differentially affect microglia development, homeostasis, and disease that could potentially be utilized therapeutically.

CBP/p300 and HDAC activities regulate H3K27 acetylation dynamics and zygotic genome activation in mouse preimplantation embryos.

Epigenome reprogramming after fertilization enables transcriptionally quiescent maternal and paternal chromatin to acquire a permissive state for subsequent zygotic genome activation (ZGA). H3K27 acetylation (H3K27ac) is a well-established chromatin marker of active enhancers and promoters. However, reprogramming dynamics of H3K27ac during maternal-to-zygotic transition (MZT) in mammalian embryos are not well-studied. By profiling the allelic landscape of H3K27ac during mouse MZT, we show that H3K27ac undergoes three waves of rapid global transitions between oocyte stage and 2-cell stage. Notably, germinal vesicle oocyte and zygote chromatin are globally hyperacetylated, with noncanonical, broad H3K27ac domains that correlate with broad H3K4 trimethylation (H3K4me3) and open chromatin. H3K27ac marks genomic regions primed for activation including ZGA genes, retrotransposons, and active alleles of imprinted genes. We show that CBP/p300 and HDAC activities play important roles in regulating H3K27ac dynamics and are essential for preimplantation development. Specifically, CBP/p300 acetyltransferase broadly deposits H3K27ac in zygotes to induce the opening of condensed chromatin at putative enhancers and ensure proper ZGA. On the contrary, HDACs revert broad H3K27ac domains to canonical domains and safeguard ZGA by preventing premature expression of developmental genes. In conclusion, coordinated activities of CBP/p300 and HDACs during mouse MZT are essential for ZGA and preimplantation development.

Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain.

Histone deacetylase 7 (HDAC7) is a member of the class IIa family of classical HDACs with important roles in cell development, differentiation, and activation, including in macrophages and other innate immune cells. HDAC7 and other class IIa HDACs act as transcriptional repressors in the nucleus but, in some cell types, they can also act in the cytoplasm to modify non-nuclear proteins and/or scaffold signalling complexes. In macrophages, HDAC7 is a cytoplasmic protein with both pro- and anti-inflammatory functions, with the latter activity involving activation of the pentose phosphate pathway (PPP) enzyme 6-phosphogluconate dehydrogenase (6PGD) and the generation of anti-inflammatory metabolite ribulose-5-phosphate. Here, we used ectopic expression systems and biochemical approaches to investigate the mechanism by which HDAC7 promotes 6PGD enzyme activity. We reveal that HDAC7 enzyme activity is not required for its activation of 6PGD and that the N-terminal protein-protein interaction domain of HDAC7 is sufficient to initiate this response. Mechanistically, the N-terminus of HDAC7 increases the affinity of 6PGD for NADP+, promotes the generation of a shorter form of 6PGD, and enhances the formation of higher order protein complexes, implicating its scaffolding function in engagement of the PPP. This contrasts with the pro-inflammatory function of HDAC7 in macrophages, in which it promotes deacetylation of the glycolytic enzyme pyruvate kinase M2 for inflammatory cytokine production.

Loss of adipose TET proteins enhances ß-adrenergic responses and protects against obesity by epigenetic regulation of ß3-AR expression.

ß-adrenergic receptor (ß-AR) signaling plays predominant roles in modulating energy expenditure by triggering lipolysis and thermogenesis in adipose tissue, thereby conferring obesity resistance. Obesity is associated with diminished ß3-adrenergic receptor (ß3-AR) expression and decreased ß-adrenergic responses, but the molecular mechanism coupling nutrient overload to catecholamine resistance remains poorly defined. Ten-eleven translocation (TET) proteins are dioxygenases that alter the methylation status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further oxidized derivatives. Here, we show that TET proteins are pivotal epigenetic suppressors of ß3-AR expression in adipocytes, thereby attenuating the responsiveness to ß-adrenergic stimulation. Deletion of all three Tet genes in adipocytes led to increased ß3-AR expression and thereby enhanced the downstream ß-adrenergic responses, including lipolysis, thermogenic gene induction, oxidative metabolism, and fat browning in vitro and in vivo. In mouse adipose tissues, Tet expression was elevated after mice ate a high-fat diet. Mice with adipose-specific ablation of all TET proteins maintained higher levels of ß3-AR in both white and brown adipose tissues and remained sensitive to ß-AR stimuli under high-fat diet challenge, leading to augmented energy expenditure and decreased fat accumulation. Consequently, they exhibited improved cold tolerance and were substantially protected from diet-induced obesity, inflammation, and metabolic complications, including insulin resistance and hyperlipidemia. Mechanistically, TET proteins directly repressed ß3-AR transcription, mainly in an enzymatic activity-independent manner, and involved the recruitment of histone deacetylases to increase deacetylation of its promoter. Thus, the TET-histone deacetylase-ß3-AR axis could be targeted to treat obesity and related metabolic diseases.

Probing class I histone deacetylases (HDAC) with proteolysis targeting chimera (PROTAC) for the development of highly potent and selective degraders.

Class I HDACs are considered promising targets for cancer due to their role in epigenetic modifications. The main challenges in developing a new, potent and non-toxic class I HDAC inhibitor are selectivity and appropriate pharmacokinetics. The PROTAC technique (Proteolysis Targeting Chimera) is a new method in drug development for the production of active substances that can degrade a protein of interest (POI) instead of inhibiting it. This technique will open the era to produce selective and potent drugs with a high margin of safety. Previously, we reported different inhibitors targeting class I HDACs functionalized with aminobenzamide or hydroxamate groups. In the current research work, we will employ PROTAC technique to develop class I HDAC degraders based on our previously reported inhibitors. We synthesized two series of aminobenzamide-based PROTACs and hydroxamate-based PROTACs and tested them in vitro against class I HDACs. To ensure their degradation, all of them were screened against HDAC2 as representative example of class I. The best candidates were evaluated at different concentrations at various HDAC subtypes. This resulted in the PROTAC (32a) (HI31.1) that degrades HDAC8 with a DC50 of 8.9 nM with a proper margin of selectivity against other isozymes. Moreover, PROTAC 32a is able to degrade HDAC6 with DC50 = 14.3 nM. Apoptotic study on leukemic cells (MV-4-11) displayed more than 50 % apoptosis took place at 100 nM. PROTAC 32a (HI31.1) showed a good margin of safety against normal cell line and proper chemical stability.

Synthesis and biological evaluation of ortho-phenyl phenylhydroxamic acids containing phenothiazine with improved selectivity for class IIa histone deacetylases.

Class IIa histone deacetylases (HDACs) have been linked to tumorigenesis in various cancers. Previously, we designed phenylhydroxamic acid LH4f as a potent class IIa HDAC inhibitor. However, it also unselectively inhibited class I and class IIb HDACs. To enhance the compound's selectivity towards class IIa HDACs, the ortho-phenyl group from the selective HDAC7 inhibitor 1 is incorporated into ortho position of the phenylhydroxamic acid in LH4f. Compared to LH4f, most resulting compounds displayed substantially improved selectivity towards the class IIa HDACs. Notably, compound 7 g exhibited the strongest HDAC9 inhibition with an IC50 value of 40 nM. Molecular modelling further identified the key interactions of compound 7 g bound to HDAC9. Compound 7 g significantly inhibited several human cancer cells, induced apoptosis, modulated caspase-related proteins as well as p38, and caused DNA damage. These findings suggest the potential of class IIa HDAC inhibitors as lead compounds for the development of cancer therapeutics.

Design and synthesis of bioreductive prodrugs of class I histone deacetylase inhibitors and their biological evaluation in virally transfected acute myeloid leukemia cells.

Although histone deacetylase (HDAC) inhibitors show promise in treating various types of hematologic malignancies, they have some limitations, including poor pharmacokinetics and off-target side effects. Prodrug design has shown promise as an approach to improve pharmacokinetic properties and to improve target tissue specificity. In this work, several bioreductive prodrugs for class I HDACs were designed based on known selective HDAC inhibitors. The zinc-binding group of the HDAC inhibitors was masked with various nitroarylmethyl residues to make them substrates of nitroreductase (NTR). The developed prodrugs showed weak HDAC inhibitory activity compared to their parent inhibitors. The prodrugs were tested against wild-type and NTR-transfected THP1 cells. Cellular assays showed that both 2-nitroimidazole-based prodrugs 5 and 6 were best activated by the NTR and exhibited potent activity against NTR-THP1 cells. Compound 6 showed the highest cellular activity (GI50 = 77 nM) and exhibited moderate selectivity. Moreover, activation of prodrug 6 by NTR was confirmed by liquid chromatography-mass spectrometry analysis, which showed the release of the parent inhibitor after incubation with Escherichia coli NTR. Thus, compound 6 can be considered a novel prodrug selective for class I HDACs, which could be used as a good starting point for increasing selectivity and for further optimization.

Neutrophil Elastase Degrades Histone Deacetylases and Sirtuin 1 in Primary Human Monocyte Derived Macrophages.

Neutrophil elastase (NE) is taken up by macrophages, retains intracellular protease activity, and induces a pro-inflammatory phenotype. However, the mechanism of NE-induced pro-inflammatory polarization of macrophages is not well understood. We hypothesized that intracellular NE degrades histone deacetylases (HDAC) and Sirtuins, disrupting the balance of lysine acetylation and deacetylation and resulting in nuclear to cytoplasmic translocation of a major alarmin, High Mobility Group Box 1 (HMGB1), a pro-inflammatory response in macrophages. Human blood monocytes were obtained from healthy donors or from subjects with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Monocytes were differentiated into blood monocyte derived macrophages (BMDMs) in vitro. Human BMDMs were exposed to NE or control vehicle, and the abundance of HDACs and Sirtuins was determined by Western blotting of total cell lysates or nuclear extracts or determined by ELISA. HDAC, Sirtuin, and Histone acetyltransferase (HAT) activities were measured. NE degraded most HDACs and Sirtuin (Sirt)1, resulting in decreased HDAC and sirtuin activities, with minimal change in HAT activity. We then evaluated whether the NE-induced loss of Sirt activity or loss of HDAC activities would alter the cellular localization of HMGB1. NE treatment or treatment with Trichostatin A (TSA), a global HDAC inhibitor, both increased HMGB1 translocation from the nucleus to the cytoplasm, consistent with HMGB1 activation. NE significantly degraded Class I and II HDAC family members and Sirt 1, which shifted BMDMs to a pro-inflammatory phenotype.

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development.

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.

Regulation of cholesterol metabolism: New players for an old physiological process.

Identified more than two centuries ago, cholesterol plays a pivotal role in human physiology. Since cholesterol metabolism is a physiologically significant process, it is not surprising that its alterations are associated with several pathologies. The discovery of new molecular targets or compounds able to modulate this sophisticated metabolism has been capturing the attention of research groups worldwide since many years. Endogenous and exogenous compounds are known to regulate cellular cholesterol synthesis and uptake, or reduce cholesterol absorption at the intestinal level, thereby regulating cholesterol homeostasis. However, there is a great need of new modulators and diverse new pathways have been uncovered. Here, after illustrating cholesterol metabolism and its well-known regulators, some new players of this important physiological process are also described.

Role of Histone Deacetylases in Monocyte Function in Health and Chronic Inflammatory Diseases.

Histone deacetylases (HDACs) are a family of 18 members that participate in the epigenetic regulation of gene expression. In addition to histones, some HDACs also deacetylate transcription factors and specific cytoplasmic proteins.Monocytes, as part of the innate immune system, maintain tissue homeostasis and help fight infections and cancer. In these cells, HDACs are involved in multiple processes including proliferation, migration, differentiation, inflammatory response, infections, and tumorigenesis. Here, a systematic description of the role that most HDACs play in these functions is reviewed. Specifically, some HDACs induce a pro-inflammatory response and play major roles in host defense. Conversely, other HDACs reprogram monocytes and macrophages towards an immunosuppressive phenotype. The right balance between both types helps monocytes to respond correctly to the different physiological/pathological stimuli. However, aberrant expressions or activities of specific HDACs are associated with autoimmune diseases along with other chronic inflammatory diseases, infections, or cancer.This paper critically reviews the interesting and extensive knowledge regarding the role of some HDACs in these pathologies. It also shows that as yet, very little progress has been made toward the goal of finding effective HDAC-targeted therapies. However, given their obvious potential, we conclude that it is worth the effort to develop monocyte-specific drugs that selectively target HDAC subtypes with the aim of finding effective treatments for diseases in which our innate immune system is involved.

DNA methylation and lipid metabolism are involved in GA-induced maize aleurone layers PCD as revealed by transcriptome analysis.

BACKGROUND: The aleurone layer is a part of many plant seeds, and during seed germination, aleurone cells undergo PCD, which is promoted by GA from the embryo. However, the numerous components of the GA signaling pathway that mediate PCD of the aleurone layers remain to be identified. Few genes and transcriptomes have been studied thus far in aleurone layers to improve our understanding of how PCD occurs and how the regulatory mechanism functions during PCD. Our previous studies have shown that histone deacetylases (HDACs) are required in GA-induced PCD of aleurone layer. To further explore the molecular mechanisms by which epigenetic modifications regulate aleurone PCD, we performed a global comparative transcriptome analysis of embryoless aleurones treated with GA or histone acetylase (HAT) inhibitors. RESULTS: In this study, a total of 7,919 differentially expressed genes (DEGs) were analyzed, 2,554 DEGs of which were found to be common under two treatments. These identified DEGs were involved in various biological processes, including DNA methylation, lipid metabolism and ROS signaling. Further investigations revealed that inhibition of DNA methyltransferases prevented aleurone PCD, suggesting that active DNA methylation plays a role in regulating aleurone PCD. GA or HAT inhibitor induced lipoxygenase gene expression, leading to lipid degradation, but this process was not affected by DNA methylation. However, DNA methylation inhibitor could regulate ROS-related gene expression and inhibit GA-induced production of hydrogen peroxide (H2O2). CONCLUSION: Overall, linking of lipoxygenase, DNA methylation, and H2O2 may indicate that GA-induced higher HDAC activity in aleurones causes breakdown of lipids via regulating lipoxygenase gene expression, and increased DNA methylation positively mediates H2O2 production; thus, DNA methylation and lipid metabolism pathways may represent an important and complex signaling network in maize aleurone PCD.

The role of histone deacetylases in embryonic development.

The basic units of chromatin are nucleosomes, that are made up of DNA wrapped around histone cores. Histone lysine residue is a common location for posttranslational modifications, with acetylation being the second most prevalent. Histone acetyltransferases (HATs/KATs) and histone deacetylases (HDACs/KDACs) regulate histone acetylation, which is important in gene expression control. HDACs/KDACs regulate gene expressions through the repression of the transcription machinery. HDAC/KDAC isoforms play a major role during various stages of embryo development and neurogenesis. In specific, class I and II HDACs/KDACs are involved in cardiac muscle differentiation and development. An insight into different pathways and genes associated with embryonic development, the effect of HDAC/KDAC activity during the embryonic stem cell differentiation, preimplantation, embryo development, gastrulation, and the role of different HDAC/KDAC inhibitors during the process of embryogenesis is summarized in the present review article.

Development of Lysine Crotonyl-Mimic Probe to Covalently Identify H3K27Cr Interacting Proteins.

Histone lysine crotonylation (Kcr) is one newly discovered acylation modification and regulates numerous pathophysiological processes. The binding affinity between Kcr and its interacting proteins is generally weak, which makes it difficult to effectively identify Kcr-interacting partners. Changing the amide of crotonyl to an ester increased reactivity with proximal cysteines and retained specificity for Kcr antibody. The probe "H3g27Cr" was designed by incorporating the ester functionality into a H3K27 peptide. Using this probe, multiple Kcr-interacting partners including STAT3 were successfully identified, and this has not been reported previously. Further experiments suggested that STAT3 possibly could form complexes with Histone deacetylase HDACs to downregulate the acetylation and crotonylation of Histone H3K27. Our unique design provided intriguing tools to further explore Kcr-interacting proteins and elucidate their working mechanisms.

Phenolic compounds as histone deacetylase inhibitors: binding propensity and interaction insights from molecular docking and dynamics simulations.

Histone deacetylases are well-established target enzymes involved in the pathology of different diseases including cancer and neurodegenerative disorders. The approved HDAC inhibitor drugs are associated with cellular toxicities. Different phenolic compounds have been shown to possess inhibitory activities against HDACs and are, therefore, considered safer alternatives to synthetic compounds. Here, we elucidated the binding mode and calculated the binding propensity of some of the top phenolic compounds against different isoforms representing different classes of Zn2+ ion-containing HDACs using the molecular docking approach. Our data reaffirmed the activity of the studied phenolic compounds against HDACs. Binding interaction analysis suggested that these compounds can block the activity of HDACs with or without binding to the active site zinc metal ion. Furthermore, molecular dynamics (MD) simulations were carried out on the selected crystal and docking complexes of each selected HDAC isoform. Analysis of root-mean-square displacement (RMSD) showed that the phenolic compounds demonstrated a stable binding mode over 50 ns in a way that is comparable to the cocrystal ligands. Together, these findings can aid future efforts in the search for natural inhibitors of HDACs.

Targeting histone deacetylases for heart diseases.

Histone deacetylases (HDACs) are responsible for the deacetylation of lysine residues in histone or non-histone substrates, leading to the regulation of many biological functions, such as gene transcription, translation and remodeling chromatin. Targeting HDACs for drug development is a promising way for human diseases, including cancers and heart diseases. In particular, numerous HDAC inhibitors have revealed potential clinical value for the treatment of cardiac diseases in recent years. In this review, we systematically summarize the therapeutic roles of HDAC inhibitors with different chemotypes on heart diseases. Additionally, we discuss the opportunities and challenges in developing HDAC inhibitors for the treatment of cardiac diseases.

Deacetylation of Histones and Non-histone Proteins in Inflammatory Diseases and Cancer Therapeutic Potential of Histone Deacetylase Inhibitors.

Epigenetic changes play an important role in the pathophysiology of autoimmune diseases such as allergic asthma, multiple sclerosis, lung diseases, diabetes, cystic fibrosis, atherosclerosis, rheumatoid arthritis, and COVID-19. There are three main classes of epigenetic alterations: post-translational modifications of histone proteins, control by non-coding RNA and DNA methylation. Since histone modifications can directly affect chromatin structure and accessibility, they can regulate gene expression levels. Abnormal expression and activity of histone deacetylases (HDACs) have been reported in immune mediated diseases. Increased acetylated levels of lysine residues have been suggested to be related to the overexpression of inflammatory genes. This review focuses on the effect of HDAC modifications on histone and non-histone proteins in autoimmune diseases. Furthermore, we discuss the potential therapeutic effect of HDAC inhibitors (HDACi) used in these diseases.

Reprogramming Carbohydrate Metabolism in Cancer and Its Role in Regulating the Tumor Microenvironment.

Altered metabolism has become an emerging feature of cancer cells impacting their proliferation and metastatic potential in myriad ways. Proliferating heterogeneous tumor cells are surrounded by other resident or infiltrating cells, along with extracellular matrix proteins, and other secretory factors constituting the tumor microenvironment. The diverse cell types of the tumor microenvironment exhibit different molecular signatures that are regulated at their genetic and epigenetic levels. The cancer cells elicit intricate crosstalks with these supporting cells, exchanging essential metabolites which support their anabolic processes and can promote their survival, proliferation, EMT, angiogenesis, metastasis and even therapeutic resistance. In this context, carbohydrate metabolism ensures constant energy supply being a central axis from which other metabolic and biosynthetic pathways including amino acid and lipid metabolism and pentose phosphate pathway are diverged. In contrast to normal cells, increased glycolytic flux is a distinguishing feature of the highly proliferative cancer cells, which supports them to adapt to a hypoxic environment and also protects them from oxidative stress. Such rewired metabolic properties are often a result of epigenetic alterations in the cancer cells, which are mediated by several factors including, DNA, histone and non-histone protein modifications and non-coding RNAs. Conversely, epigenetic landscapes of the cancer cells are also dictated by their diverse metabolomes. Altogether, this metabolic and epigenetic interplay has immense potential for the development of efficient anti-cancer therapeutic strategies. In this book chapter we emphasize upon the significance of reprogrammed carbohydrate metabolism in regulating the tumor microenvironment and cancer progression, with an aim to explore the different metabolic and epigenetic targets for better cancer treatment.

Targeting Epigenetic Regulators with HDAC and BET Inhibitors to Modulate Muscle Wasting.

Epigenetic changes contribute to the profound alteration in the transcriptional program associated with the onset and progression of muscle wasting in several pathological conditions. Although HDACs and their inhibitors have been extensively studied in the field of muscular dystrophies, the potential of epigenetic inhibitors has only been marginally explored in other disorders associated with muscle atrophy, such as in cancer cachexia and sarcopenia. BET inhibitors represent a novel class of recently developed epigenetic drugs that display beneficial effects in a variety of diseases beyond malignancies. Based on the preliminary in vitro and preclinical data, HDACs and BET proteins contribute to the pathogenesis of cancer cachexia and sarcopenia, modulating processes related to skeletal muscle mass maintenance and/or metabolism. Thus, epigenetic drugs targeting HDACs and BET proteins may emerge as promising strategies to reverse the catabolic phenotype associated with cachexia and sarcopenia. Further preclinical studies are warranted to delve deeper into the molecular mechanisms associated with the functions of HDACs and BET proteins in muscle atrophy and to establish whether their epigenetic inhibitors represent a prospective therapeutic avenue to alleviate muscle wasting.

Gut Microbiome Composition and Its Metabolites Are a Key Regulating Factor for Malignant Transformation, Metastasis and Antitumor Immunity.

The genetic and metabolomic abundance of the microbiome exemplifies that the microbiome comprises a more extensive set of genes than the entire human genome, which justifies the numerous metabolic and immunological interactions between the gut microbiota, macroorganisms and immune processes. These interactions have local and systemic impacts that can influence the pathological process of carcinogenesis. The latter can be promoted, enhanced or inhibited by the interactions between the microbiota and the host. This review aimed to present evidence that interactions between the host and the gut microbiota might be a significant exogenic factor for cancer predisposition. It is beyond doubt that the cross-talk between microbiota and the host cells in terms of epigenetic modifications can regulate gene expression patterns and influence cell fate in both beneficial and adverse directions for the host's health. Furthermore, bacterial metabolites could shift pro- and anti-tumor processes in one direction or another. However, the exact mechanisms behind these interactions are elusive and require large-scale omics studies to better understand and possibly discover new therapeutic approaches for cancer.