Bcr-TMP, a Novel Nanomolar-Active Compound That Exhibits Both MYB- and Microtubule-Inhibitory Activity

Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Here, we present the initial characterization of 2-amino-4-(3,4,5-trimethoxyphenyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile (Bcr-TMP), a nanomolar-active MYB-inhibitory compound identified in a screen for novel MYB inhibitors. Bcr-TMP affects MYB function in a dual manner by inducing its degradation and suppressing its transactivation potential by disrupting its cooperation with co-activator p300. Bcr-TMP also interferes with the p300-dependent stimulation of C/EBPβ, a transcription factor co-operating with MYB in myeloid cells, indicating that Bcr-TMP is a p300-inhibitor. Bcr-TMP reduces the viability of AML cell lines at nanomolar concentrations and induces cell-death and expression of myeloid differentiation markers. It also down-regulates the expression of MYB target genes and exerts stronger anti-proliferative effects on MYB-addicted primary murine AML cells and patient-derived ACC cells than on their non-oncogenic counterparts. Surprisingly, we observed that Bcr-TMP also has microtubule-disrupting activity, pointing to a possible link between MYB-activity and microtubule stability. Overall, Bcr-TMP is a highly potent multifunctional MYB-inhibitory agent that warrants further investigation of its therapeutic potential and mechanism(s) of action.

Evaluating the Influence of a G-Quadruplex Prone Sequence on the Transactivation Potential by Wild-Type and/or Mutant P53 Family Proteins through a Yeast-Based Functional Assay

P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at C-terminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic PUMA target gene can exert on the transactivation potential of full-length and N-terminal truncated P53 family α isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites.

Evolution and expansion of the RUNX2 QA repeat corresponds with the emergence of vertebrate complexity

Runt-related transcription factor 2 (RUNX2) is critical for the development of the vertebrate bony skeleton. Unlike other RUNX family members, RUNX2 possesses a variable poly-glutamine, poly-alanine (QA) repeat domain. Natural variation within this repeat is able to alter the transactivation potential of RUNX2, acting as an evolutionary ‘tuning knob’ suggested to influence mammalian skull shape. However, the broader role of the RUNX2 QA repeat throughout vertebrate evolution is unknown. In this perspective, we examine the role of the RUNX2 QA repeat during skeletal development and discuss how its emergence and expansion may have facilitated the evolution of morphological novelty in vertebrates.

The Influence of Quadruplex Structure in Proximity to P53 Target Sequences on the Transactivation Potential of P53 Alpha Isoforms

p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.

Induction of Hepatic Metabolic Functions by a Novel Variant of Hepatocyte Nuclear Factor 4γ

ABSTRACT Hepatocyte nuclear factor 4α (HNF4α) is a critical factor for hepatocyte differentiation. HNF4α expression is decreased in hepatocellular carcinoma (HCC), which suggests a role in repression of hepatocyte dedifferentiation. In the present study, hepatic expression of HNF4γ was increased in liver-specific Hnf4a-null mice. The HNF4γ whose expression was increased contained two variants, a known short variant, designated HNF4γ1, and a novel long variant, designated HNF4γ2. HNF4G2 mRNA was highly expressed in small intestine, and the transactivation potential of HNF4γ2 was the strongest among these variants, but the potential of HNF4γ1 was the lowest. Cotransfection experiments revealed that HNF4γ1 repressed HNF4α- and HNF4γ2-dependent transactivation, while HNF4γ2 promoted HNF4α-dependent transactivation. HNF4γ1 and HNF4γ2 were able to bind to the HNF4α binding sites with an affinity similar to that of HNF4α. Furthermore, HNF4γ2, but not HNF4γ1, robustly induced the expression of typical HNF4α target genes to a greater degree than HNF4α. Additionally, HNF4γ2 suppressed proliferation of hepatoma cells as well as HNF4α and HNF4γ1 did, and HNF4γ2 induced critical hepatic functions, such as glucose and urea production, and cytochrome P450 1A2 activity more strongly than HNF4α and HNF4γ1 did. These results indicate that HNF4γ2 has potential for redifferentiation of HCC and thus may be explored as a target for HCC therapy.

HIV-1 Tat potently stabilises Mdm2 and enhances viral replication

Murine double minute 2 (Mdm2) is known to enhance the transactivation potential of human immunodeficiency virus (HIV-1) Tat protein by causing its ubiquitination. However, the regulation of Mdm2 during HIV-1 infection and its implications for viral replication have not been well studied. Here, we show that the Mdm2 protein level increases during HIV-1 infection and this effect is mediated by HIV-1 Tat protein. Tat appears to stabilise Mdm2 at the post-translational level by inducing its phosphorylation at serine-166 position through AKT. Although p53 is one of the key players for Mdm2 induction, Tat-mediated stabilisation of Mdm2 appears to be independent of p53. Moreover, the non-phosphorylatable mutant of Mdm2 (S166A) fails to interact with Tat and shows decreased half-life in the presence of Tat compared with wild-type Mdm2. Furthermore, the non-phosphorylatable mutant of Mdm2 (S166A) is unable to support HIV-1 replication. Thus, HIV-1 Tat appears to stabilise Mdm2, which in turn enhances Tat-mediated viral replication. This study highlights the importance of post-translational modifications of host cellular factors in HIV-1 replication and pathogenesis.

Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50. RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. IMPORTANCE Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions.