The natural anti-tumor compound Celastrol targets a Myb-C/EBPß-p300 transcriptional module implicated in myeloid gene expression.

Myb is a key regulator of hematopoietic progenitor cell proliferation and differentiation and has emerged as a potential target for the treatment of acute leukemia. Using a myeloid cell line with a stably integrated Myb-inducible reporter gene as a screening tool we have previously identified Celastrol, a natural compound with anti-tumor activity, as a potent Myb inhibitor that disrupts the interaction of Myb with the co-activator p300. We showed that Celastrol inhibits the proliferation of acute myeloid leukemia (AML) cells and prolongs the survival of mice in an in vivo model of AML, demonstrating that targeting Myb with a small-molecule inhibitor is feasible and might have potential as a therapeutic approach against AML. Recently we became aware that the reporter system used for Myb inhibitor screening also responds to inhibition of C/EBPß, a transcription factor known to cooperate with Myb in myeloid cells. By re-investigating the inhibitory potential of Celastrol we have found that Celastrol also strongly inhibits the activity of C/EBPß by disrupting its interaction with the Taz2 domain of p300. Together with previous studies our work reveals that Celastrol independently targets Myb and C/EBPß by disrupting the interaction of both transcription factors with p300. Myb, C/EBPß and p300 cooperate in myeloid-specific gene expression and, as shown recently, are associated with so-called super-enhancers in AML cells that have been implicated in the maintenance of the leukemia. We hypothesize that the ability of Celastrol to disrupt the activity of a transcriptional Myb-C/EBPß-p300 module might explain its promising anti-leukemic activity.

Withaferin A, a natural compound with anti-tumor activity, is a potent inhibitor of transcription factor C/EBPß.

Recent work has shown that deregulation of the transcription factor Myb contributes to the development of leukemia and several other human cancers, making Myb and its cooperation partners attractive targets for drug development. By employing a myeloid Myb-reporter cell line we have identified Withaferin A (WFA), a natural compound that exhibits anti-tumor activities, as an inhibitor of Myb-dependent transcription. Analysis of the inhibitory mechanism of WFA showed that WFA is a significantly more potent inhibitor of C/EBPß, a transcription factor cooperating with Myb in myeloid cells, than of Myb itself. We show that WFA covalently modifies specific cysteine residues of C/EBPß, resulting in the disruption of the interaction of C/EBPß with the co-activator p300. Our work identifies C/EBPß as a novel direct target of WFA and highlights the role of p300 as a crucial co-activator of C/EBPß. The finding that WFA is a potent inhibitor of C/EBPß suggests that inhibition of C/EBPß might contribute to the biological activities of WFA.

Helenalin Acetate, a Natural Sesquiterpene Lactone with Anti-inflammatory and Anti-cancer Activity, Disrupts the Cooperation of CCAAT Box/Enhancer-binding Protein ß (C/EBPß) and Co-activator p300.

Recent work has demonstrated pro-oncogenic functions of the transcription factor CCAAT box/enhancer-binding protein ß (C/EBPß) in various tumors, implicating C/EBPß as an interesting target for the development of small-molecule inhibitors. We have previously discovered that the sesquiterpene lactone helenalin acetate, a natural compound known to inhibit NF-κB, is a potent C/EBPß inhibitor. We have now examined the inhibitory mechanism of helenalin acetate in more detail. We demonstrate that helenalin acetate is a significantly more potent inhibitor of C/EBPß than of NF-κB. Our work shows that helenalin acetate inhibits C/EBPß by binding to the N-terminal part of C/EBPß, thereby disrupting the cooperation of C/EBPß with the co-activator p300. C/EBPß is expressed in several isoforms from alternative translational start codons. We have previously demonstrated that helenalin acetate selectively inhibits only the full-length (liver-enriched activating protein* (LAP*)) isoform but not the slightly shorter (LAP) isoform. Consistent with this, helenalin acetate binds to the LAP* but not to the LAP isoform, explaining why its inhibitory activity is selective for LAP*. Although helenalin acetate contains reactive groups that are able to interact covalently with cysteine residues, as exemplified by its effect on NF-κB, the inhibition of C/EBPß by helenalin acetate is not due to irreversible reaction with cysteine residues of C/EBPß. In summary, helenalin acetate is the first highly active small-molecule C/EBPß inhibitor that inhibits C/EBPß by a direct binding mechanism. Its selectivity for the LAP* isoform also makes helenalin acetate an interesting tool to dissect the functions of the LAP* and LAP isoforms.

Targeting acute myeloid leukemia with a small molecule inhibitor of the Myb/p300 interaction.

The transcription factor Myb plays a key role in the hematopoietic system and has been implicated in the development of leukemia and other human cancers. Inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. However, because of a lack of suitable inhibitors, the feasibility of therapeutic approaches based on Myb inhibition has not been explored. We have identified the triterpenoid Celastrol as a potent low-molecular-weight inhibitor of the interaction of Myb with its cooperation partner p300. We demonstrate that Celastrol suppresses the proliferative potential of acute myeloid leukemia (AML) cells while not affecting normal hematopoietic progenitor cells. Furthermore, Celastrol prolongs the survival of mice in a model of an aggressive AML. Overall, our work demonstrates the therapeutic potential of a small molecule inhibitor of the Myb/p300 interaction for the treatment of AML and provides a starting point for the further development of Myb-inhibitory compounds for the treatment of leukemia and, possibly, other tumors driven by deregulated Myb.

Interaction and cooperation of the CCAAT-box enhancer-binding protein ß (C/EBPß) with the homeodomain-interacting protein kinase 2 (Hipk2).

CCAAT box/enhancer-binding protein ß (C/EBPß) is a bZip transcription factor that plays crucial roles in important cellular processes such as differentiation and proliferation of specific cell types. Previously, we showed that C/EBPß cooperates with the coactivator p300 through a novel mechanism that involves the C/EBPß-induced phosphorylation of multiple sites in the carboxyl-terminal domain of p300 by protein kinase Hipk2. We have now examined the interaction and cooperation of C/EBPß, p300, and Hipk2 in more detail. We show that Hipk2 and C/EBPß are direct physical binding partners whose interaction is mediated by sequences located in the amino-terminal and central domains of Hipk2 and the amino-terminal part of C/EBPß. In addition to phosphorylating p300 recruited to C/EBPß, Hipk2 also phosphorylates C/EBPß at sites that have previously been shown to plays key roles in the regulation of C/EBPß activity. Silencing of Hipk2 expression disrupts adipocyte differentiation of 3T3-L1 cells, a physiological C/EBPß-dependent differentiation process indicating that the cooperation of C/EBPß and Hipk2 is functionally relevant. Finally, we demonstrate that C/EBPα, a related C/EBP family member whose amino-terminal sequences differ significantly from that of C/EBPß, is unable to interact and cooperate with Hipk2. Instead, our data suggest that C/EBPα cooperates with the protein kinase Jnk to induce phosphorylation of p300. Overall, our data identify Hipk2 as a novel regulator of C/EBPß and implicate different protein kinases in the cooperation of p300 with C/EBPß and C/EBPα.