chaetocin and Leukemia--Myeloid--Acute

Epigenetic modifying enzymes have a crucial role in the pathogenesis of acute myeloid leukemia (AML). Methylation of lysine 9 on histone H3 by the methyltransferase G9a and SUV39H1 is associated with inhibition of tumor suppressor genes. We studied the effect of G9a and SUV39H1 inhibitors on viability and differentiation of AML cells and tested the cytotoxicity induced by combination of G9a and SUV39H1 inhibitors and various epigenetic drugs. The SUV39H1 inhibitor (chaetocin) and the G9a inhibitor (UNC0638) caused cell death in AML cells at high concentrations. However, only chaetocin-induced CD11b expression and differentiation of AML cells at non-cytotoxic concentration. HL-60 and KG-1a cells were more sensitive to chaetocin than U937 cells. Long-term incubation of chaetocin led to downregulation of SUV39H1 and reduction of H3K9 tri-methylation in HL-60 and KG-1a cells. Combination of chaetocin with suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor) or JQ (a BET (bromodomain extra terminal) bromodomain inhibitor) showed synergistic cytotoxicity. Conversely, no synergism was found by combining chaetocin and UNC0638. More importantly, chaetocin-induced differentiation and combined cytotoxicity were also found in the primary cells of AML patients. Collectively, the SUV39H1 inhibitor chaetocin alone or in combination with other epigenetic drugs may be effective for the treatment of AML.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Cells, Cultured; DNA Methylation; Drug Synergism; Epigenesis, Genetic; Humans; Immunoblotting; Leukemia, Myeloid, Acute; Methyltransferases; Piperazines; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction

Silencing of SOCS1, a TSG, has been detected in various malignancies, including AML. However, the underlying mechanism of SOCS1 inactivation remains elusive. In this study, we explored the role of histone methylation in SOCS1 expression in AML cells. By ChIP assay, we demonstrated that G9a and SUV39H1, two enzymes catalyzing H3K9 methylation, were physically associated with the SOCS1 promoter, and treatment with chaetocin, a histone methyltransferase inhibitor, suppressed H3K9 methylation on the SOCS1 promoter and enhanced SOCS1 expression. Furthermore, knockdown of G9a and SUV39H1 by siRNA could also induce SOCS1 expression. On the other hand, SOCS1 knockdown by shRNA eliminated chaetocin-induced cell apoptosis. To investigate further whether any transcription factor was involved in H3K9 methylation-related SOCS1 repression, we scanned the sequences of the SOCS1 gene promoter and found two binding sites for Gfi-1, a transcription repressor. By DNA pull-down and ChIP assays, we showed that Gfi-1 directly bound the SOCS1 promoter, and ectopic Gfi-1 expression suppressed STAT5-induced SOCS1 promoter activation. In contrast, Gfi-1 knockdown by shRNA enhanced SOCS1 expression and inhibited STAT5 expression. Moreover, the knockdown of G9a completely rescued the repressive effect of Gfi-1 on STAT5A-induced SOCS1 promoter activation. Collectively, our study indicates that the expression of Gfi-1 contributes to SOCS1 silencing in AML cells through epigenetic modification, and suppression of histone methyltransferase can provide new insight in AML therapy.

Topics: Apoptosis; Cell Line, Tumor; DNA-Binding Proteins; Gene Expression Regulation, Leukemic; Gene Knockdown Techniques; Histones; Humans; Leukemia, Myeloid, Acute; Methylation; Piperazines; Promoter Regions, Genetic; Protein Binding; Repressor Proteins; RNA Interference; STAT5 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Transcription Factors; Transcription, Genetic; Transcriptional Activation

SUV39H1 is a histone 3 lysine 9 (H3K9)-specific methyltransferase that is important for heterochromatin formation and the regulation of gene expression. Chaetocin specifically inhibits SUV39H1, resulted in H3K9 methylation reduction as well as reactivation of silenced genes in cancer cells. Histone deacetylase (HDAC) inhibitors inhibit deacetylases and accumulate high levels of acetylation lead to cell cycle arrest and apoptosis. In this study, we demonstrated that treatment with chaetocin enhanced apoptosis in human leukemia HL60, KG1, Kasumi, K562, and THP1 cells. In addition, chaetocin induced the expression of cyclin-dependent kinase inhibitor 2B (p15), E-cadherin (CDH1) and frizzled family receptor 9 (FZD9) through depletion of SUV39H1 and reduced H3K9 methylation in their promoters. Co-treatment with chaetocin and HDAC inhibitor trichostatin A (TSA) dramatically increased apoptosis and produced greater activation of genes. Furthermore, this combined treatment significantly increased loss of SUV39H1 and reduced histone H3K9 trimethylation responses accompanied by increased acetylation. Importantly, co-treatment with chaetocin and TSA produced potent antileukemic effects in leukemia cells derived from patients. These in vitro findings suggest that combination therapy with SUV39H1 and HDAC inhibitors may be of potential value in the treatment of leukemia.

Topics: Acetylation; Adolescent; Adult; Aged; Apoptosis; Cadherins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p15; DNA Methylation; Enzyme Inhibitors; Frizzled Receptors; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Histones; HL-60 Cells; Humans; Hydroxamic Acids; K562 Cells; Leukemia; Leukemia, Myeloid, Acute; Male; Middle Aged; Piperazines; Promoter Regions, Genetic; Young Adult

Epigenetic deregulation is involved in acute myeloid leukemia (AML) pathogenesis and epigenetic targeting drugs are in clinical trial. Since the first results with histone-deacetylase inhibitors in AML are controversial, novel single and combined treatments need to be explored. It is tempting to combine chromatin-targeting drugs. SUV39H1, the main methyl-transferase for lysine 9 tri-methylation on histone H3, interacts with oncogenes involved in AML and acts as a transcriptional repressor for hematopoietic differentiation and immortalization. We report here that pharmacological inhibition of SUV39H1 by chaetocin induces apoptosis in leukemia cell lines in vitro and primary AML cells ex vivo, and that it interferes with leukemia growth in vivo. Chaetocin treatment upregulates reactive oxygen species (ROS) production as well as the transcription of death-receptor-related genes, in a ROS-dependent manner, leading to death receptor-dependent apoptosis. In addition to its direct inhibition by chaetocin, SUV39H1 is indirectly modulated by chaetocin-induced ROS. Accordingly, chaetocin potentiates other anti-AML drugs, in a ROS-dependent manner. The decryption of a dual mechanism of action against AML involving both direct and indirect SUV39H1 modulation represents an innovative read-out for the anticancer activity of chaetocin and for its synergy with other anti-AML drugs, suggesting new therapeutic combination strategies in AML.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspases; Humans; Leukemia, Myeloid, Acute; Methyltransferases; Mice; Piperazines; Reactive Oxygen Species; Receptors, Death Domain; Repressor Proteins; U937 Cells; Xenograft Model Antitumor Assays