ABSTRACT
The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Modification Methylases/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/pharmacology , Hematologic Neoplasms/drug therapy , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Crystallography, X-Ray , DNA Modification Methylases/chemistry , DNA Modification Methylases/genetics , DNA Modification Methylases/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Epigenesis, Genetic/drug effects , Female , Hematologic Neoplasms/genetics , Hematologic Neoplasms/immunology , Hematologic Neoplasms/mortality , Histocompatibility Antigens/chemistry , Histocompatibility Antigens/genetics , Histocompatibility Antigens/metabolism , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Interferons/immunology , Interferons/metabolism , Mice , Mice, Inbred BALB C , Microsomes, Liver , Molecular Docking Simulation , Survival Analysis , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.
Subject(s)
Arthritis, Experimental/genetics , Arthritis, Rheumatoid/genetics , Genetic Predisposition to Disease , Histocompatibility Antigens/genetics , Amino Acid Sequence , Amino Acids/genetics , Animals , Antibodies, Blocking/immunology , Antibodies, Blocking/pharmacology , Arthritis, Experimental/immunology , Arthritis, Rheumatoid/immunology , Binding Sites/genetics , Body Weight/drug effects , Body Weight/immunology , Disease Models, Animal , Genotype , Haplotypes/immunology , Histocompatibility Antigens/chemistry , Histocompatibility Antigens/immunology , Humans , Models, Molecular , Molecular Sequence Data , Polymorphism, Genetic/immunology , Protein Structure, Tertiary , Rats , Severity of Illness Index , Terpenes/immunologyABSTRACT
A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.
Subject(s)
Chromosomes, Human, Pair 5 , DNA, Complementary/chemistry , Histocompatibility Antigens/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , DNA, Complementary/isolation & purification , Histocompatibility Antigens/chemistry , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Tripartite Motif ProteinsABSTRACT
Two arginine-specific ADP-ribosyltransferase cDNAs (designated AT1 and AT2) were cloned from chicken bone marrow cells. Each cDNA encodes a different peptide of 312 amino acid residues. Homology of deduced amino acid sequences between AT1 and AT2 was 78.3%. We found all six combined peptide sequences of 222 amino acid residues derived from purified chicken heterophil ADP-ribosyltransferase (Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K., and Shimoyama, M. (1991) J. Biochem. (Tokyo) 110, 388-394) in the deduced amino acid sequence of AT1, with two amino acid mismatches. Arginine-specific ADP-ribosyltransferase activity was detected in culture medium of COS 7 cells transiently transfected with AT1 cDNA, while activity from the cells transfected with AT2 cDNA was found in both culture medium and cell lysate. AT1 transferase required 2-mercaptoethanol for the activity. The activity was inhibited in the presence of NaCl while AT2 enzyme was activated by either agent. On zymographic in situ gel analysis, estimated molecular masses of the AT1, AT2 and purified chicken heterophil transferases were 32, 34, and 27.5 kDa, respectively. Northern blot analysis with specific probes to AT1 or AT2 cDNAs revealed about a 1.5-kilobase message in chicken bone marrow cells but no signals were observed in heterophils, spleen, and liver of chicken or human HL-60 cells. Highly conserved regions were observed among the deduced amino acid sequences of AT1, AT2, rabbit skeletal muscle transferase, and rodent T-cell surface antigen RT6s.