DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation

Abstract

DNA methylation, histone modifications, and nucleosomal occupancy collaborate to cause silencing of tumor-related genes in cancer. The development of drugs that target these processes is therefore important for cancer therapy. Inhibitors of DNA methylation and histone deacetylation have been approved by the Food and Drug Administration for treatment of hematologic malignancies. However, drugs that target other mechanisms still need to be developed. Recently, 3-deazaneplanocin A (DZNep) was reported to selectively inhibit trimethylation of lysine 27 on histone H3 (H3K27me3) and lysine 20 on histone H4 (H4K20me3) as well as reactivate silenced genes in cancer cells. This finding opens the door to the pharmacologic inhibition of histone methylation. We therefore wanted to further study the mechanism of action of DZNep in cancer cells. Western blot analysis shows that DZNep globally inhibits histone methylation and is not selective. Two other drugs, sinefungin and adenosine dialdehyde, have similar effects as DZNep on H3K27me3. Intriguingly, chromatin immunoprecipitation of various histone modifications and microarray analysis show that DZNep acts through a different pathway than 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor. These observations give us interesting insight into how chromatin structure affects gene expression. We also determined the kinetics of gene activation to understand if the induced changes were somatically heritable. We found that upon removal of DZNep, gene expression is reduced to its original state. This suggests that there is a homeostatic mechanism that returns the histone modifications to their “ground state” after DZNep treatment. Our data show the strong need for further development of histone methylation inhibitors

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx)

“…Importantly, both treatments resulted in a marked decrease in global levels of H3K27me3 (Figure 6b), as previously reported, 25,40,41 whereas the levels of H3K9me3, another repressive mark, remained unchanged demonstrating that they act mainly through inhibition of EZH2-containing complexes at the used doses on the basis of previous reports for DZNep. 24,40,[42][43][44][45] Consistently with EZH2 silencingdependent effects, the appearance of apoptotic Annexin V-positive cells was seen in both DZNep-and MC1945-treated RH30 cells ( Figure 6c) associated with transcriptional upregulation of FBXO32 gene and decreased Myogenin levels after 72 h (Figure 6d). Collectively, these observations indicate that pharmacological approaches either favoring the degradation or blocking the catalytic functions of EZH2 affect the proliferative potential of PAX3-FOXO1-positive alveolar RMS cells and mirror the effect of siRNA-and short hairpin RNAmediated EZH2 depletion in derepressing FBXO32.…”

Epigenetic modification in chromatin machinery and its deregulation in pediatric brain tumors: Insight into epigenetic therapies

“…88,89 Interestingly, Kim and colleagues discovered that EZH2 enhances STAT3 activation by trimethylating lysine180 in STAT3, and it does so preferentially in glioma stem-like cells. 90 The use of the EZH2 inhibitor 3-deazaneplanocin A (DZNep) 91,92 and a highly selective EZH2 inhibitor GSK126 93 decreases STAT3 activation in glioma stem-like cells. This inhibition reverses the silencing of Polycomb target genes and diminishes STAT3 activity, suggesting therapeutic strategies.…”

Dynamic histone marks in the hippocampus and cortex facilitate memory consolidation

“…To examine whether the observed histone PTMs are causally linked to object memory formation, we blocked their activation in vivo using a cocktail of inhibitors containing staurosporine, a blocker of H3-specific kinases 24 , C646, an inhibitor of the histone acetyltransferase p300/CBP 25 , and 3-deazaneplanocin A, an inhibitor of histone methyltransferases 26 . These inhibitors effectively blocked histone phosphorylation, acetylation and methylation in the brain (Fig.…”

“…with a phosphatase/histone acetyl transferase/histone methyl transferase inhibitor cocktail consisting of 0.2 mg kg − 1 staurosporine (Cell Signaling), a kinase inhibitor that co-targets H3 kinases 24 , 10 nmol C646 (Tocris Bioscience), an inhibitor of the histone acetyl transferase p300/CBP 25 , and 2 mg kg − 1 3-deazaneplanocin A (Cayman), an inhibitor of histone methyltransferases 26 . The drugs were prepared in dimethylsulphoxide (Sigma).…”