ABSTRACT
Lysine acetyltransferases (KATs) are a family of epigenetic enzymes involved in the regulation of gene expression; they represent a promising class of emerging drug targets. The frequent molecular dysregulation of these enzymes, as well as their mechanistic links to biological functions that are crucial to cancer, have led to exploration around the development of small-molecule inhibitors against KATs. Despite early challenges, recent advances have led to the development of potent and selective enzymatic and bromodomain (BRD) KAT inhibitors. In this review we discuss the discovery and development of new KAT inhibitors and their application as oncology therapeutics. Additionally, new chemically induced proximity approaches are presented, offering opportunities for unique target selectivity profiles and tissue-specific targeting of KATs. Emerging clinical data for CREB binding protein (CREBBP)/EP300 BRD inhibitors and KAT6 catalytic inhibitors indicate the promise of this target class in cancer therapeutics.
Subject(s)
Lysine Acetyltransferases , Neoplasms , Humans , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Lysine Acetyltransferases/chemistry , Lysine Acetyltransferases/genetics , Lysine Acetyltransferases/metabolism , Histones/metabolism , Lysine/metabolism , Neoplasms/drug therapyABSTRACT
MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient-derived xenograft (PDX) mouse models, antisense oligonucleotide-mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target.
Subject(s)
Exons , Melanoma/therapy , Nuclear Proteins/genetics , Oligonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins/genetics , Animals , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation , Humans , Melanoma/pathology , Mice , RNA-Binding Proteins/physiology , Serine-Arginine Splicing Factors , Tumor Suppressor Protein p53/physiologyABSTRACT
Recruitment of DNA repair factors and modulation of chromatin structure at sites of DNA double-strand breaks (DSBs) is a complex and highly orchestrated process. We developed a system that can induce DSBs rapidly at defined endogenous sites in mammalian genomes and enables direct assessment of repair and monitoring of protein recruitment, egress, and modification at DSBs. The tight regulation of the system also permits assessments of relative kinetics and dependencies of events associated with cellular responses to DNA breakage. Distinct advantages of this system over focus formation/disappearance assays for assessing DSB repair are demonstrated. Using ChIP, we found that nucleosomes are partially disassembled around DSBs during nonhomologous end-joining repair in G1-arrested mammalian cells, characterized by a transient loss of the H2A/H2B histone dimer. Nucleolin, a protein with histone chaperone activity, interacts with RAD50 via its arginine-glycine rich domain and is recruited to DSBs rapidly in an MRE11-NBS1-RAD50 complex-dependent manner. Down-regulation of nucleolin abrogates the nucleosome disruption, the recruitment of repair factors, and the repair of the DSB, demonstrating the functional importance of nucleosome disruption in DSB repair and identifying a chromatin-remodeling protein required for the process. Interestingly, the nucleosome disruption that occurs during DSB repair in cycling cells differs in that both H2A/H2B and H3/H4 histone dimers are removed. This complete nucleosome disruption is also dependent on nucleolin and is required for recruitment of replication protein A to DSBs, a marker of DSB processing that is a requisite for homologous recombination repair.
Subject(s)
DNA Breaks, Double-Stranded , G1 Phase Cell Cycle Checkpoints , Nucleosomes/metabolism , Phosphoproteins/metabolism , Protein Multimerization , RNA-Binding Proteins/metabolism , Recombinational DNA Repair , Acid Anhydride Hydrolases , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Histones/genetics , Histones/metabolism , Humans , MRE11 Homologue Protein , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleosomes/genetics , Phosphoproteins/genetics , RNA-Binding Proteins/genetics , Replication Protein A/genetics , Replication Protein A/metabolism , NucleolinABSTRACT
The ability of our cells to maintain genomic integrity is fundamental for protection from cancer development. Central to this process is the ability of cells to recognize and repair DNA damage and progress through the cell cycle in a regulated and orderly manner. In addition, protection of chromosome ends through the proper assembly of telomeres prevents loss of genetic information and aberrant chromosome fusions. Cells derived from patients with ataxia-telangiectasia (A-T) show defects in cell cycle regulation, abnormal responses to DNA breakage, and chromosomal end-to-end fusions. The identification and characterization of the ATM (ataxia-telangiectasia, mutated) gene product has provided an essential tool for researchers in elucidating cellular mechanisms involved in cell cycle control, DNA repair, and chromosomal stability.
Subject(s)
Ataxia Telangiectasia/genetics , Cell Cycle/genetics , DNA Damage/genetics , DNA Repair/genetics , Genetic Diseases, Inborn/genetics , Genomic Instability/genetics , Humans , Protein Serine-Threonine Kinases/genetics , Transcriptional Activation/geneticsABSTRACT
Psoralen plus UVA light (PUVA) is commonly used to treat psoriasis, a common skin disorder associated with rapid proliferation of cells. PUVA exerts its antiproliferative activity through formation of DNA monoadducts and interstrand cross-links (ICLs). However, this treatment may lead to skin malignancies as a direct result of inducing carcinogenic DNA damage. Inactivation of the p53 tumor suppressor gene is an important event in the development of skin cancer. p53 is rapidly phosphorylated and stabilized in response to DNA damage, and the induction of apoptosis by p53 is an important mechanism by which p53 exerts its tumor-suppressive activity. To better understand the mechanism by which PUVA treatment induces p53, we exposed human skin fibroblasts with PUVA under conditions that differentially produce monoadducts and ICLs and found that psoralen-induced ICLs induced phosphorylation of the Ser-15 site of p53 and apoptosis much more effectively than psoralen-induced monoadducts. The induction of p53 phosphorylation by psoralen ICLs did not require factors believed to be involved in the repair of psoralen ICLs [xeroderma pigmentosum (XP)-A, XP-C, XP-F, Cockayne's syndrome-B, Fanconi anemia] but did require the ataxia-telangiectasia and Rad3-related but not the ataxia-telangiectasia mutated kinase. Psoralen-induced ICLs blocked transcription and replication more efficiently than monoadducts, and induction of p53 and apoptosis correlated with doses causing interference with transcription rather than DNA replication. Our finding that cells underwent apoptosis preferentially during S-phase suggests that the combined blockade of transcription and DNA replication by psoralen ICLs during S-phase elicits a strong apoptotic response.
Subject(s)
Ataxia Telangiectasia , Cell Cycle Proteins/physiology , Cross-Linking Reagents/toxicity , DNA Damage/drug effects , Ficusin/toxicity , Protein Serine-Threonine Kinases/physiology , Tumor Suppressor Protein p53/biosynthesis , Ataxia Telangiectasia/chemically induced , Ataxia Telangiectasia/genetics , Ataxia Telangiectasia Mutated Proteins , Cell Line, Transformed , DNA Damage/radiation effects , Fibroblasts/drug effects , Fibroblasts/physiology , Humans , Tumor Suppressor Protein p53/genetics , Ultraviolet Rays/adverse effectsABSTRACT
The mechanisms by which DNA-damaging agents trigger the induction of the stress response protein p53 are poorly understood but may involve alterations of chromatin structure or blockage of either transcription or replication. Here we show that transcription-blocking agents can induce phosphorylation of the Ser-15 site of p53 in a replication-independent manner. Furthermore, microinjection of anti-RNA polymerase II antibodies into the nuclei of cells showed that blockage of transcription is sufficient for p53 accumulation even in the absence of DNA damage. This induction of p53 occurs by two independent mechanisms. First, accumulation of p53 is linked to diminished nuclear export of mRNA; and second, inhibition specifically of elongating RNA polymerase II complexes results in the phosphorylation of the Ser-15 site of p53 in a replication protein A (RPA)- and ATM and Rad3-related (ATR)-dependent manner. We propose that this transcription-based stress response involving RPA, ATR, and p53 has evolved as a DNA damage-sensing mechanism to safeguard cells against DNA damage-induced mutagenesis.
Subject(s)
Cell Cycle Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Replication Protein A/metabolism , Transcription, Genetic/genetics , Tumor Suppressor Protein p53/metabolism , Active Transport, Cell Nucleus , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Line , Cell Nucleus/metabolism , DNA Replication/genetics , Humans , Phosphorylation , Phosphoserine/metabolism , Protein Serine-Threonine Kinases/genetics , RNA Polymerase II/metabolism , RNA, Messenger/genetics , Replication Protein A/geneticsABSTRACT
BACKGROUND: The evolutionary conserved cyclin-dependent kinase phosphatase hCdc14A has been shown to play potential roles in the regulation of mitotic exit and in the centrosome duplication cycle. We have recently shown that hCdc14A also can interact with the tumor suppressor p53 both in vitro and in vivo and specifically dephosphorylates the ser315 site of p53 in vitro. In this study we developed antibodies against hCdc14A to investigate the expression and regulation of hCdc14A in human tissues and cancer cells. RESULTS: We show that hCdc14A is differentially expressed in human tissues and in 75 cancer cell lines examined. Treatments with the histone deacetylase inhibitor TSA, the demethylating agent 5-aza-2'-deoxycytodine or the proteasome inhibitor MG132 significantly induced expression of hCdc14A in cell lines expressing low or undetectable levels of hCdc14A. There was a strong bias for low expression of hCdc14A in cancer cell lines harboring wild-type p53, suggesting that high Cdc14A expression is not compatible with wild-type p53 expression. We present evidence for a role for hCdc14A in the dephosphorylation of the ser315 site of p53 in vivo and that hCdc14A forms a complex with Cdk1/cyclin B during interphase but not during mitosis. CONCLUSION: Our results that hCdc14A is differentially expressed in human cancer cells and that hCdc14A can interact with both p53 and the Cdk1/cyclin B complex may implicate that dysregulation of hCdc14A expression may play a role in carcinogenesis.
Subject(s)
CDC2 Protein Kinase/biosynthesis , Cyclin B/biosynthesis , Gene Expression Regulation, Neoplastic , Genes, p53 , Phosphoric Monoester Hydrolases/genetics , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cell Line , Cell Line, Tumor , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , Humans , Mitosis , Models, Biological , Phosphorylation , Proteasome Inhibitors , Protein Binding , Protein Tyrosine PhosphatasesABSTRACT
Interference with transcription triggers a stress response leading to the induction of the tumour suppressor p53. If transcription is not restored within a certain time frame cells may undergo apoptosis in a p53-dependent and independent manner. The mechanisms by which blockage of transcription induces apoptosis may involve diminished levels of anti-apoptotic factors, inappropriate accumulation of proteins in the nucleus, accumulation of p53 at mitochondria or complications during replication. Many chemotherapeutic agents currently used in the clinic interfere with transcription and this interference may contribute to their anti-cancer activities. Future efforts should be directed towards exploring whether interference of transcription could be used as an anti-cancer therapeutic strategy.
Subject(s)
Genetic Therapy/methods , Neoplasms/therapy , Transcription, Genetic/genetics , Apoptosis/genetics , Apoptosis/physiology , Humans , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/physiologyABSTRACT
Regulation of epidermal growth factor receptor (EGFR) signaling requires the concerted action of both positive and negative factors. While the existence of numerous molecules that stimulate EGFR activity has been well documented, direct biological inhibitors appear to be more limited in number and phylogenetic distribution. Kekkon1 (Kek1) represents one such inhibitor. Kek1 was initially identified in Drosophila melanogaster and appears to be absent from vertebrates and the invertebrate Caenorhabditis. To further investigate Kek1's function and evolution, we identified kek1 orthologs within dipterans. In D. melanogaster, kek1 is a transcriptional target of EGFR signaling during oogenesis, where it acts to attenuate receptor activity through an inhibitory feedback loop. The extracellular and transmembrane portion of Kek1 is sufficient for its inhibitory activity in D. melanogaster. Consistent with conservation of its role in EGFR signaling, interspecies comparisons indicate a high degree of identity throughout these regions. During formation of the dorsal-ventral axis Kek1 is expressed in dorsal follicle cells in a pattern that reflects the profile of receptor activation. D. virilis Kek1 (DvKek1) is also expressed dynamically in the dorsal follicle cells, supporting a conserved role in EGFR signaling. Confirming this, biochemical and transgenic assays indicate that DvKek1 is functionally interchangeable with DmKek1. Strikingly, we find that the cytoplasmic domain contains a region with the highest degree of conservation, which we have implicated in EGFR inhibition and dubbed the Kek tail (KT) box.