Abstract
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/EBPβ, 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/EBPβ by disrupting its interaction with the Taz2 domain of p300. Together with previous studies our work reveals that Celastrol independently targets Myb and C/EBPβ by disrupting the interaction of both transcription factors with p300. Myb, C/EBPβ 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/EBPβ-p300 module might explain its promising anti-leukemic activity.
Highlights
The transcription factor Myb plays a key role as a regulator of proliferation and differentiation of hematopoietic progenitor cells and has been implicated in the development of acute leukemia [1]
To investigate if Celastrol previously identified as a Myb inhibitor affects the activity of C/ EBPβ we performed luciferase assays with a reporter construct that is driven the promoter of the mim-1 gene, which contains high affinity C/EBP binding sites and is strongly activated by C/EBPβ [36]
To demonstrate under more physiological conditions that Celastrol inhibits C/EBPβ, we analyzed its effect on the transcriptional activation of an endogenous C/EBP target gene that is silent in fibroblasts but can be activated by ectopic expression of C/EBPβ or C/EBPα [15,33], thereby providing a read-out of C/EBP activity at a chromatin-embedded gene
Summary
The transcription factor Myb plays a key role as a regulator of proliferation and differentiation of hematopoietic progenitor cells and has been implicated in the development of acute leukemia [1]. In AML cells, super-enhancers are densely loaded with chromatin regulators such as p300 and BRD4 and hematopoietic transcription factors, such as Myb, C/EBPα, C/EBPβ, ERG, FLI1, and PU.1 [11] These studies have confirmed the important role of Myb and C/EBPs for the proliferation of AML cells, suggesting a mechanistic framework for the addiction of AML cells to high levels of Myb expression. We have previously established a myeloid reporter cell line as a screening tool to search for low molecular weight Myb inhibitors [23] These cells carry a stably integrated Myb-responsive reporter gene driven by the promoter and enhancer of the myeloid-specific chicken mim-1 gene, a Myb target gene whose expression is strongly induced by Myb in myeloid cells. Our data show that Celastrol is a highly potent inhibitor of C/EBPβ suggesting that the promising inhibitory effects of Celastrol on AML cells might be due to its ability to simultaneously target Myb and C/EBPβ
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