Transcription Factor MYB as Therapeutic Target: Current Developments.

The MYB protein is a pivotal player in the cellular transcriptional network, influencing major important processes such as cell proliferation, differentiation, and apoptosis. Because of its role in oncogenesis, MYB is now a compelling target for therapeutic interventions in cancer research. This review summarizes its molecular functions and current therapeutic approaches aiming to inhibit its oncogenic activity.

Natural Products with Antitumor Potential Targeting the MYB-C/EBPß-p300 Transcription Module.

The transcription factor MYB is expressed predominantly in hematopoietic progenitor cells, where it plays an essential role in the development of most lineages of the hematopoietic system. In the myeloid lineage, MYB is known to cooperate with members of the CCAAT box/enhancer binding protein (C/EBP) family of transcription factors. MYB and C/EBPs interact with the co-activator p300 or its paralog CREB-binding protein (CBP), to form a transcriptional module involved in myeloid-specific gene expression. Recent work has demonstrated that MYB is involved in the development of human leukemia, especially in acute T-cell leukemia (T-ALL) and acute myeloid leukemia (AML). Chemical entities that inhibit the transcriptional activity of the MYB-C/EBPß-p300 transcription module may therefore be of use as potential anti-tumour drugs. In searching for small molecule inhibitors, studies from our group over the last 10 years have identified natural products belonging to different structural classes, including various sesquiterpene lactones, a steroid lactone, quinone methide triterpenes and naphthoquinones that interfere with the activity of this transcriptional module in different ways. This review gives a comprehensive overview on the various classes of inhibitors and the inhibitory mechanisms by which they affect the MYB-C/EBPß-p300 transcriptional module as a potential anti-tumor target. We also focus on the current knowledge on structure-activity relationships underlying these biological effects and on the potential of these compounds for further development.

Activation of the oncogenic transcription factor B-Myb via multisite phosphorylation and prolyl cis/trans isomerization.

The oncogenic transcription factor B-Myb is an essential regulator of late cell cycle genes whose activation by phosphorylation is still poorly understood. We describe a stepwise phosphorylation mechanism of B-Myb, which involves sequential phosphorylations mediated by cyclin-dependent kinase (Cdk) and Polo-like kinase 1 (Plk1) and Pin1-facilitated peptidyl-prolyl cis/trans isomerization. Our data suggest a model in which initial Cdk-dependent phosphorylation of B-Myb enables subsequent Pin1 binding and Pin1-induced conformational changes of B-Myb. This, in turn, initiates further phosphorylation of Cdk-phosphosites, enabling Plk1 docking and subsequent Plk1-mediated phosphorylation of B-Myb to finally allow B-Myb to stimulate transcription of late cell cycle genes. Our observations reveal novel mechanistic hierarchies of B-Myb phosphorylation and activation and uncover regulatory principles that might also apply to other Myb family members. Strikingly, overexpression of B-Myb and of factors mediating its activation strongly correlates with adverse prognoses for tumor patients, emphasizing B-Myb's role in tumorigenesis.

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.

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.

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.

A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs.

An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4.

The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5'-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4.

PRMT4 is a novel coactivator of c-Myb-dependent transcription in haematopoietic cell lines.

Protein arginine methyltransferase 4 (PRMT4)-dependent methylation of arginine residues in histones and other chromatin-associated proteins plays an important role in the regulation of gene expression. However, the exact mechanism of how PRMT4 activates transcription remains elusive. Here, we identify the chromatin remodeller Mi2α as a novel interaction partner of PRMT4. PRMT4 binds Mi2α and its close relative Mi2ß, but not the other components of the repressive Mi2-containing NuRD complex. In the search for the biological role of this interaction, we find that PRMT4 and Mi2α/ß interact with the transcription factor c-Myb and cooperatively coactivate c-Myb target gene expression in haematopoietic cell lines. This coactivation requires the methyltransferase and ATPase activity of PRMT4 and Mi2, respectively. Chromatin immunoprecipitation analysis shows that c-Myb target genes are direct transcriptional targets of PRMT4 and Mi2. Knockdown of PRMT4 or Mi2α/ß in haematopoietic cells of the erythroid lineage results in diminished transcriptional induction of c-Myb target genes, attenuated cell growth and survival, and deregulated differentiation resembling the effects caused by c-Myb depletion. These findings reveal an important and so far unknown connection between PRMT4 and the chromatin remodeller Mi2 in c-Myb signalling.

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.

C/EBPß cooperates with MYB to maintain the oncogenic program of AML cells.

Studies on the role of transcription factor MYB in acute myeloid leukemia (AML) have identified MYB as a key regulator of a transcriptional program for self-renewal of AML cells. Recent work summarized here has now highlighted the CCAAT-box/enhancer binding protein beta (C/EBPß) as an essential factor and potential therapeutic target that cooperates with MYB and coactivator p300 in the maintenance of the leukemic cells.

Multimodal 4-arylchromene derivatives with microtubule-destabilizing, anti-angiogenic, and MYB-inhibitory activities.

Aim: Efficient and readily available anticancer drugs are sought as treatment options. For this reason, chromene derivatives were prepared using the one-pot reaction and tested for their anticancer and anti-angiogenic properties. Methods: 2-Amino-3-cyano-4-(aryl)-7-methoxy-4H-chromene compounds (2A-R) were repurposed or newly synthesized via a three-component reaction of 3-methoxyphenol, various aryl aldehydes, and malononitrile. We performed assays to study the inhibition of tumor cell growth [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromid (MTT) assay], effects on microtubules (immunofluorescence), cell cycle (flow-activated cell sorting analysis), angiogenesis (zebrafish model), and MYB activity (luciferase reporter assay). Fluorescence microscopy was applied for localization studies via copper-catalyzed azide-alkyne click reaction of an alkyne-tagged drug derivative. Results: Compounds 2A-C and 2F exhibited robust antiproliferative activities against several human cancer cell lines (50% inhibitory concentrations in the low nanomolar range) and showed potent MYB inhibition. The alkyne derivative 3 was localized in the cytoplasm after only 10 min of incubation. Substantial microtubule disruption and G2/M cell-cycle arrest were observed, where compound 2F stood out as a promising microtubule-disrupting agent. The study of anti-angiogenic properties showed that 2A was the only candidate with a high potential to inhibit blood vessel formation in vivo. Conclusion: The close interplay of various mechanisms, including cell-cycle arrest, MYB inhibition, and anti-angiogenic activity, led to identifying promising multimodal anticancer drug candidates.

Tumor suppressor protein Pdcd4 inhibits translation of p53 mRNA.

The tumor suppressor protein Pdcd4 is thought to suppress translation of mRNAs containing structured 5'-UTRs by interacting with translation initiation factor eIF4A and inhibiting its helicase activity. However, natural target mRNAs regulated by Pdcd4 so far are mostly unknown. Here, we identified p53 mRNA as a translational target of Pdcd4. We found that Pdcd4 is associated with p53 mRNA and suppresses its translation. The inhibitory effect of Pdcd4 on the translation of p53 mRNA depends on the ability of Pdcd4 to interact with eIF4A and is mediated by the 5'-UTR of p53 mRNA, which is able to form a stable stem-loop structure. We show that treatment of cells with DNA-damaging agents decreases the expression of Pdcd4. This suggests that translational suppression by Pdcd4 plays a role in maintaining a low level of p53 in unstressed cells and that this suppression is abrogated due to low levels of Pdcd4 after DNA damage. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA with a 5'-structured UTR and provides novel insight into the translational control of p53 expression.

Structure of the tandem MA-3 region of Pdcd4 protein and characterization of its interactions with eIF4A and eIF4G: molecular mechanisms of a tumor suppressor.

One of the key regulatory points of translation initiation is recruitment of the 43S preinitation complex to the 5' mRNA cap by the eIF4F complex (eIF4A, eIF4E, and eIF4G). The tumor suppressor protein Pdcd4 has been shown to inhibit cap-dependent translation by interacting tightly with the RNA helicase eIF4A via its tandem MA-3 domains. The NMR studies reported here reveal a fairly extensive and well defined interface between the two MA-3 domains in solution, which appears to be stabilized by a network of interdomain salt bridges and hydrogen bonds, and reveals a unique orientation of the two domains. Characterization of the stoichiometry of the Pdcd4-eIF4A complex suggests that under physiological conditions Pdcd4 binds to a single molecule of eIF4A, which involves contacts with both Pdcd4 MA-3 domains. We also show that contacts mediated by a conserved acidic patch on the middle MA-3 domain of Pdcd4 are essential for forming a tight complex with eIF4A in vivo, whereas the equivalent region of the C-terminal MA-3 domain appears to have no role in complex formation in vivo. The formation of a 1:1 eIF4A-Pdcd4 complex in solution is consistent with the reported presence in vivo of only one molecule of eIF4A in the eIF4F complex. Pdcd4 has also been reported to interact directly with the middle region of eIF4G, however, we were unable to obtain any evidence for even a weak, transient direct interaction.

C/EBPß sustains the oncogenic program of AML cells by cooperating with MYB and co-activator p300 in a transcriptional module.

Transcription factor MYB is a key regulator of gene expression in hematopoietic cells and has emerged as a novel drug target for acute myeloid leukemia (AML). Studies aiming to identify potential MYB inhibitors have found that the natural compound helenalin acetate (HA) inhibits viability and induces cell death and differentiation of AML cells by disrupting the MYB-induced gene expression program. Interestingly, CCAAT-box/enhancer binding protein ß (C/EBPß), a transcription factor known to cooperate with MYB and the co-activator p300 in myeloid cells, rather than MYB itself, was identified as the primary target of HA. This supports a model in which MYB, C/EBPß, and p300 form the core of a transcriptional module that is essential for maintenance of the proliferative potential of AML cells, highlighting a novel role for C/EBPß as a proleukemogenic factor.

Src-Family Protein Kinase Inhibitors Suppress MYB Activity in a p300-Dependent Manner.

Recent studies have disclosed transcription factor MYB as a potential drug target for malignancies that are dependent on deregulated MYB function, including acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often regarded as undruggable, successful targeting of MYB by low-molecular-weight compounds has recently been demonstrated. In an attempt to repurpose known drugs as novel MYB-inhibitory agents, we have screened libraries of approved drugs and drug-like compounds for molecules with MYB-inhibitory potential. Here, we present initial evidence for the MYB-inhibitory activity of the protein kinase inhibitors bosutinib, PD180970 and PD161570, that we identified in a recent screen. We show that these compounds interfere with the activity of the MYB transactivation domain, apparently by disturbing the ability of MYB to cooperate with the coactivator p300. We show that treatment of the AML cell line HL60 with these compounds triggers the up-regulation of the myeloid differentiation marker CD11b and induces cell death. Importantly, we show that these effects are significantly dampened by forced expression of an activated version of MYB, confirming that the ability to suppress MYB function is a relevant activity of these compounds. Overall, our work identifies several protein kinase inhibitors as novel MYB-inhibitory agents and suggests that the inhibition of MYB function may play a role in their pharmacological impact on leukemic cells.

Characterization of the MYB-inhibitory potential of the Pan-HDAC inhibitor LAQ824.

A large body of work has shown that MYB acts as a master transcription regulator in hematopoietic cells and has pinpointed MYB as a potential drug target for acute myeloid leukemia (AML). Here, we have examined the MYB-inhibitory potential of the HDAC inhibitor LAQ824, which was identified in a screen for novel MYB inhibitors. We show that nanomolar concentrations of LAQ824 and the related HDAC inhibitors vorinostat and panobinostat interfere with MYB function in two ways, by inducing its degradation and inhibiting its activity. Reporter assays show that the inhibition of MYB activity by LAQ824 involves the MYB transactivation domain and the cooperation of MYB with co-activator p300, a key MYB interaction partner and driver of MYB activity. In AML cells, LAQ824-induced degradation of MYB is accompanied by expression of myeloid differentiation markers and apoptotic and necrotic cell death. The ability of LAQ824 to inhibit MYB activity is supported by the observation that down-regulation of direct MYB target genes MYC and GFI1 occurs without apparent decrease of MYB expression already after 2 h of treatment with LAQ824. Furthermore, ectopic expression of an activated version of MYB In HL60 cells counteracts the induction of myeloid differentiation by LAQ824. Overall, our data identify LAQ824 and related HDAC inhibitors as potent MYB-inhibitory agents that exert dual effects on MYB expression and activity in AML cells.

A synthetic covalent ligand of the C/EBPß transactivation domain inhibits acute myeloid leukemia cells.

C/EBPß has recently emerged as a pro-leukemogenic transcription factor that cooperates with oncoprotein MYB to maintain proliferation and differentiation block of AML cells, making C/EBPß an interesting drug target for AML. Here we have studied the inhibitory potential and biological effects of a synthetic analog of the natural product helenalin, a known inhibitor of C/EBPß. The synthetic compound inhibits C/EBPß by covalent binding to cysteine residues in the transactivation domain, thereby causing up-regulation of differentiation-associated genes, cell death and reduced self-renewal potential of AML cells. Suppression of these effects by ectopic expression of C/EBPß or MYB and gene expression profiling validate C/EBPß as a relevant target of the helenalin-mimic and highlight its role as a pro-leukemogenic factor. Overall, our work demonstrates that the synthetic helenalin mimic acts as a covalent inhibitor of C/EBPß and identifies the cysteine residues in the transactivation domain of C/EBPß as ligandable sites. The helenalin mimic can be considered a potential "lead molecule" but needs further development towards more effective C/EBPß inhibitors before being used as a therapeutic agent.

A New Naphthopyran Derivative Combines c-Myb Inhibition, Microtubule-Targeting Effects, and Antiangiogenic Properties.

Based on the promising c-Myb inhibitor 1b, a series of 2-amino-4-aryl-4H-naphtho[1,2-b]pyran-3-carbonitriles (1a, 2a-q, 3a-g) were repurposed or newly synthesized via a three-component reaction of 1-naphthol, and various aryl aldehydes and malononitrile and screened for their c-Myb inhibitory activities. 1b also served as a lead compound for seven new naphthopyran derivatives (3a-f), which were cytotoxic with nanomolar IC50 values, to inhibit the polymerization of tubulin, and to destabilize microtubules in living cells. Especially, the alkyne 3f, originally made for intracellular localization studies using click chemistry, showed an overall high activity in all assays performed. A strong G2/M cell cycle arrest was detected, which resulted in a distinct increase in sub-G1 cells through the induction of effector caspases 3 and 7. Inhibition of angiogenesis was confirmed in vitro and in vivo. In summary, 3f was found to be a pleiotropic compound with high selectivity for cancer cells, combining c-Myb inhibitory, microtubule destabilizing, and antiangiogenic effects.