Venetoclax efficacy on acute myeloid leukemia is enhanced by the combination with butyrate.

Venetoclax has been approved recently for treatment of Acute myeloid leukemia (AML). Venetoclax is a BH3-mimetic and induces apoptosis via Bcl-2 inhibition. However, venetoclax's effect is still restrictive and a novel strategy is needed. In the present study, we demonstrate that sodium butyrate (NaB) facilitates the venetoclax's efficacy of cell death in AML cells. As a single agent, NaB or venetoclax exerted just a weak effect on cell death induction for AML cell line KG-1. The combination with NaB and venetoclax drastically induced cell death. NaB upregulated pro-apoptotic factors, Bax and Bak, indicating the synergistic effect by the collaboration with Bcl-2 inhibition by venetoclax. The combined treatment with NaB and venetoclax strongly cleaved a caspase substrate poly (ADP-ribose) polymerase (PARP) and a potent pan-caspase inhibitor Q-VD-OPh almost completely blocked the cell death induced by the combination, meaning that the combination mainly induced apoptosis. The combination with NaB and venetoclax also strongly induced cell death in another AML cell line SKNO-1 but did not affect chronic myeloid leukemia (CML) cell line K562, indicating that the effect was specific for AML cells. Our results provide a novel strategy to strengthen the effect of venetoclax for AML treatment.

Caspase inhibition improves viability and efficiency of liposomal transfection.

High transfection efficiency is the most important point for experiments of DNA and RNA introduction into cells. Decrease of cell viability during the transfection procedure is a crucial issue, resulting in transfection failure. However, the mechanism underlying cell growth inhibition has not been fully elucidated. Lipofection is frequently used for transfection experiments, whereases, depending on cell type, it causes a decrease in cell viability. The present study demonstrates here that a potent pan-caspase inhibitor Q-VD-OPh blocked cell death during the lipofection, indicating apoptosis was induced in lipofection. Moreover, Q-VD-OPh drastically increased transfected cells. This method provides easier and more effective transfection system of lipofection and may be useful for transfection of not only cell lines but also clinical uses such as gene therapy and nucleic acids vaccine.

Tumor necrosis factor­related apoptosis­inducing ligand is a novel transcriptional target of runt­related transcription factor 1.

Runt­related transcription factor 1 (RUNX1), which is also known as acute myeloid leukemia 1 (AML1), has been frequently found with genomic aberrations in human leukemia. RUNX1 encodes a transcription factor that can regulate the expression of hematopoietic genes. In addition, tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) performs an important function for malignant tumors in immune surveillance. However, the regulatory mechanism of TRAIL expression remain to be fully elucidated. In the present study, tetradecanoylphorbol 13­acetate­treated megakaryocytic differentiated K562 cells was used to examine the effect of RUNX1 on TRAIL expression. Luciferase assay series of TRAIL promoters for the cells co­transfected with RUNX1 and core­binding factor ß (CBFß) expression vectors were performed to evaluate the nature of TRAIL transcriptional regulation. Electrophoresis mobility shift assay of the RUNX1 consensus sequence of the TRAIL promoter with recombinant RUNX1 and CBFß proteins was also performed. BloodSpot database analysis for TRAIL expression in patients with acute myeloid leukemia were performed. The expression of TRAIL, its receptor Death receptor 4 and 5 and RUNX1 in K562 cells transfected with the RUNX1 expression vector and RUNX1 siRNA were evaluated by reverse transcription­quantitative PCR (RT­qPCR). TRAIL and RUNX1­ETO expression was also measured in Kasumi­1 cells transfected with RUNX1­ETO siRNA and in KG­1 cells transfected with RUNX1­ETO expression plasmid, both by RT­qPCR. Cell counting, lactate dehydrogenase assay and cell cycle analysis by flow cytometry were performed on Kasumi­1, KG­1, SKNO­1 and K562 cells treated with TRAIL and HDAC inhibitors sodium butyrate or valproic acid. The present study demonstrated that RUNX1 is a transcriptional regulator of TRAIL. It was initially found that the induction of TRAIL expression following the megakaryocytic differentiation of human leukemia cells was RUNX1­dependent. Subsequently, overexpression of RUNX1 was found to increase TRAIL mRNA expression by activating its promoter activity. Additional analyses revealed that RUNX1 regulated the expression of TRAIL in an indirect manner, because RUNX1 retained its ability to activate this promoter following the mutation of all possible RUNX1 consensus sites. Furthermore, TRAIL expression was reduced in leukemia cells carrying the t(8;21) translocation, where the RUNX1­ETO chimeric protein interfere with normal RUNX1 function. Exogenous treatment of recombinant TRAIL proteins was found to induce leukemia cell death. To conclude, the present study provided a novel mechanism, whereby TRAIL is a target gene of RUNX1 and TRAIL expression was inhibited by RUNX1­ETO. These results suggest that TRAIL is a promising agent for the clinical treatment of t(8;21) AML.

C11orf21, a novel RUNX1 target gene, is down-regulated by RUNX1-ETO.

The fusion protein RUNX1-ETO is an oncogenic transcription factor generated by t(8;21) chromosome translocation, which is found in FAB-M2-type acute myeloid leukemia (AML). RUNX1-ETO is known to dysregulate the normal RUNX1 transcriptional network, which should involve essential factors for the onset of AML with t(8;21). In this study, we screened for possible transcriptional targets of RUNX1 by reanalysis of public data in silico, and identified C11orf21 as a novel RUNX1 target gene because its expression was down-regulated in the presence of RUNX1-ETO. The expression level of C11orf21 was low in AML patient samples with t(8;21) and in Kasumi-1 cells, which carry RUNX1-ETO. Knockdown of RUNX1-ETO in Kasumi-1 cells restored C11orf21 expression, whereas overexpression of RUNX1 up-regulated C11orf21 expression. In addition, knockdown of RUNX1 in other human leukemia cells without RUNX-ETO, such as K562, led to a decrease in C11orf21 expression. Of note, the C11orf21 promoter sequence contains a consensus sequence for RUNX1 binding and it was activated by exogenously expressed RUNX1 based on our luciferase reporter assay. This luciferase signal was trans-dominantly suppressed by RUNX1-ETO and site-directed mutagenesis of the consensus site abrogated the reporter activity. This study demonstrated that C11orf21 is a novel transcriptional target of RUNX1 and RUNX1-ETO suppressed C11orf21 transcription in t(8;21) AML. Thus, through this in silico approach, we identified a novel transcriptional target of RUNX1, and the depletion of C11orf21, the target gene, may be associated with the onset of t(8;21) AML.

The orphan GPR50 receptor promotes constitutive TGFß receptor signaling and protects against cancer development.

Transforming growth factor-ß (TGFß) signaling is initiated by the type I, II TGFß receptor (TßRI/TßRII) complex. Here we report the formation of an alternative complex between TßRI and the orphan GPR50, belonging to the G protein-coupled receptor super-family. The interaction of GPR50 with TßRI induces spontaneous TßRI-dependent Smad and non-Smad signaling by stabilizing the active TßRI conformation and competing for the binding of the negative regulator FKBP12 to TßRI. GPR50 overexpression in MDA-MB-231 cells mimics the anti-proliferative effect of TßRI and decreases tumor growth in a xenograft mouse model. Inversely, targeted deletion of GPR50 in the MMTV/Neu spontaneous mammary cancer model shows decreased survival after tumor onset and increased tumor growth. Low GPR50 expression is associated with poor survival prognosis in human breast cancer irrespective of the breast cancer subtype. This describes a previously unappreciated spontaneous TGFß-independent activation mode of TßRI and identifies GPR50 as a TßRI co-receptor with potential impact on cancer development.

Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50.

BACKGROUND AND PURPOSE: Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT1 receptor and in the inactivation of the closely related orphan receptor GPR50. EXPERIMENTAL APPROACH: Chimeric MT1 -GPR50 receptors were generated and functionally analysed in terms of 2-[125 I]iodomelatonin binding, Gi /cAMP signalling and ß-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations. KEY RESULTS: MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of the MT1 receptor (ii) the most suitable features for melatonin binding in MT1 receptors and (iii) major predicted rearrangements upon MT1 receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 receptors and GPR50 respectively. CONCLUSIONS AND IMPLICATIONS: Our work demonstrated the crucial role of the E2 loop for MT1 receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Systematic protein-protein interaction mapping for clinically relevant human GPCRs.

G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.

Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1.

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.

Human Herpesvirus-6 U14 Induces Cell-Cycle Arrest in G2/M Phase by Associating with a Cellular Protein, EDD.

The human herpesvirus-6 (HHV-6) infection induces cell-cycle arrest. In this study, we found that the HHV-6-encoded U14 protein induced cell-cycle arrest at G2/M phase via an association with the cellular protein EDD, a mediator of DNA-damage signal transduction. In the early phase of HHV-6 infection, U14 colocalized with EDD dots in the nucleus, and similar colocalization was also observed in cells transfected with a U14 expression vector. When the carboxyl-terminal region of U14 was deleted, no association of U14 and EDD was observed, and the percentage of cells in G2/M decreased relative to that in cells expressing wild-type U14, indicating that the C-terminal region of U14 and the U14-EDD association are critical for the cell-cycle arrest induced by U14. These results indicate that U14 is a G2/M checkpoint regulator encoded by HHV-6.

[Oligomerization of human and viral 7TM proteins: a new viral strategy to manipulate host cells]. / Oligomérisation des protéines humaines et virales à sept domaines transmembranaires - Nouvelle stratégie virale pour manipuler la cellule hôte.

G protein-coupled receptors (GPCR), also called seven transmembrane domain (7TM) proteins, represent the largest family of membrane receptors with approximately 900 members in humans. Although a substantial number of 7TM proteins have been matched with endogenous ligands, for many of them no ligand has been identified raising questions about their function. Ligand-independent functions have been proposed for several of these so-called orphan 7TM proteins such as the modulation of the function of 7TM proteins with identified ligand through the formation of heteromeric complexes. Interestingly, viruses are using a similar strategy to hijack the host cell signaling machinery and to promote virus replication and dissemination. Indeed, to affect host cell function, several viruses encode orphan 7TM proteins that heteromerize either with other virally-encoded or with host-encoded 7TM proteins with identified ligands. This highlights the strategic importance of 7TM protein signaling and heteromerization for the regulation of cellular homeostasis.

Human cytomegalovirus-encoded UL33 and UL78 heteromerize with host CCR5 and CXCR4 impairing their HIV coreceptor activity.

Human cytomegalovirus (HCMV) encodes four 7-transmembrane-spanning (7TM) proteins, US28, US27, UL33, and UL78, which present important sequence homology with human chemokine receptors. Whereas US28 binds a large range of chemokines and disturbs host cell signaling at different levels, the others are orphans with largely unknown functions. Assembly of 2 different 7TM proteins into hetero-oligomeric complexes may profoundly change their respective functional properties. We show that HCMV-encoded UL33 and UL78 form heteromers with CCR5 and CXCR4 chemokine receptors in transfected human embryonic kidney 293T cells and monocytic THP-1 cells. Expression of UL33 and UL78 had pleiotropic, predominantly negative, effects on CCR5 and CXCR4 cell surface expression, ligand-induced internalization, signal transduction, and migration without modifying the chemokine binding properties of CCR5 and CXCR4. Importantly, the coreceptor activity of CCR5 and CXCR4 for HIV was largely impaired in the presence of UL33 and UL78 without affecting expression of the primary HIV entry receptor CD4 and its interaction with CCR5 and CXCR4. Collectively, we identified the first molecular function for the HCMV-encoded orphan UL33 and UL78 7TM proteins, namely the regulation of cellular chemokine receptors through receptor heteromerization.

Novel monoclonal antibodies for identification of multicentric Castleman's disease; Kaposi's sarcoma-associated herpesvirus-encoded vMIP-I and vMIP-II.

Recent studies have indicated that vMIP-I and vMIP-II play important roles in the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV)-related diseases due to the effects of these proteins on vascularization. We developed monoclonal antibodies against KSHV-encoded viral macrophage inflammatory protein-I (vMIP-I) and vMIP-II to study these expression profiles and reveal the pathogenesis of KSHV-related diseases. The MAbs against vMIP-I and vMIP-II reacted to KSHV-infected cell lines after lytic induction. Both vMIP-I and the vMIP-II gene products were detected 24 h post-induction with 12-O-tetradecanoylphorbol-13-acetate until 60 h in the cytoplasm of primary effusion lymphoma cell lines. In clinical specimens, both vMIP-I and vMIP-II gene products were detected in the tissues of patients with multicentric Castleman's disease. On the other hand, only vMIP-II was detected in a subset of Kaposi's sarcoma. We concluded that these antibodies might be powerful tools to elucidate the pathogenesis of KSHV-related diseases.

The 7-transmembrane protein homologue UL78 of the human cytomegalovirus forms oligomers and traffics between the plasma membrane and different intracellular compartments.

The human cytomegalovirus (HCMV) UL78 ORF is considered to encode an orphan 7-transmembrane receptor. However, until now, the UL78 protein (pUL78) has not been characterized. Here, we have investigated the expression of pUL78 and found it mainly associated with the endoplasmic reticulum. However, we provide evidence that pUL78 is also localized on the cell surface from where it is quickly endocytosed. Colocalization with adaptin and EEA-1 implies that at least a small amount of pUL78 is transported to the trans Golgi network and early endosomes. Using a bimolecular fluorescence complementation assay and co-immunoprecipitation experiments, we were able to find homomeric and heteromeric structure formations of pUL78 and the US28 protein, respectively. However, the absence of pUL78 had no effect on the accumulation of inositol phosphate triggered by the US28 protein. In summary, our results suggest that the UL78 protein of HCMV traffics between the cell surface and cytoplasm, from where it might be recycled via early endosomes.

History and biological significance of GPCR heteromerization in the neuroendocrine system.

G protein-coupled receptors (GPCRs) constitute a large family of seven transmembrane proteins that regulate major cellular functions. The important role of GPCRs in the neuroendocrine system is outlined by the great interest of pharmaceutical companies in developing new drugs targeting these receptors. GPCRs exist as monomers, but can also be organized in oligomeric structures composed of either homo- or heteromers. GPCR heteromerization may play an important role in modulating and fine-tuning GPCR function and signaling. The literature reports many examples of GPCR heteromers in vitro raising the question of the physiological relevance of these complexes in tissues. Considerable efforts are currently being directed towards conclusive evidence for the existence of GPCRs heteromers in vivo, a crucial step for the validation of the concept of GPCR heteromerization and future drug development. The present review will give a brief history of GPCR oligomerization and emphasize the importance and physiological relevance of GPCR heteromerization by discussing key examples of GPCR couples.

Heteromerization of human cytomegalovirus encoded chemokine receptors.

Human cytomegalovirus (HCMV) is a widespread pathogen that infects up to 80% of the human population and causes severe complications in immunocompromised patients. HCMV expresses four seven transmembrane (7TM) spanning/G protein-coupled receptors (GPCRs) - US28, US27, UL33 and UL78 - that show close homology to human chemokine receptors. While US28 was shown to bind several chemokines and to constitutively activate multiple signaling cascades, the function(s) of US27, UL33 and UL78 in the viral life cycle have not yet been identified. Here we investigated the possible interaction/heteromerization of US27, UL33 and UL78 with US28 and the functional consequences thereof. We provide evidence that these receptors not only co-localize, but also heteromerize with US28 in vitro. While the constitutive activation of the US28-mediated Gαq/phospholipase C pathway was not affected by receptor heteromerization, UL33 and UL78 were able to silence US28-mediated activation of the transcription factor NF-κB. Summarized, we provide evidence that these orphan viral receptors have an important regulatory capacity on the function of US28 and as a consequence, may ultimately impact on the viral life cycle of HCMV.

Reciprocal roles of cellular chemokine receptors and human herpesvirus 7-encoded chemokine receptors, U12 and U51.

Human herpesvirus 7 (HHV-7) is a member of the subfamily Betaherpesvirinae that exhibits a restricted cell tropism, preferentially infecting CD4+ T cells in vitro. HHV-7 encodes two functional chemokine receptors, U12 and U51. The human chemokines that act as ligands for these receptors have been identified as CCL22 (the natural ligand for CCR4) and CCL19 (the natural ligand for CCR7). It was found that murine L1.2 cells co-expressing CCR4 or CCR7 and U12 responded to both CCL22 and CCL19 in calcium-mobilization assays, but migrated in response only to the appropriate ligand for the expressed cellular receptor. Similar results were obtained with L1.2 cells co-expressing CCR4 or CCR7 with U51. These results suggest that the HHV-7 U12 and U51 receptors can function in concert with CCR4 and CCR7 in host-cell signalling pathways.

Human herpesvirus 7 open reading frames U12 and U51 encode functional beta-chemokine receptors.

Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily and infects mainly CD4+ T cells in vitro, infects children during infancy. HHV-7 contains two genes, U12 and U51, that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 and U51 genes, we cloned these genes and expressed the proteins in cells. U12 and U51 encoded functional calcium-mobilizing receptors for beta-chemokines, which include thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), EBI1-ligand chemokine (ELC), and secondary lymphoid-tissue chemokine (SLC), but not for other chemokines, suggesting that the chemokine selectivities of the U12 and U51 products were distinct from those of the known mammalian chemokine receptors. ELC and SLC induced migration in Jurkat cells stably expressing U12, but TARC and MDC did not. In contrast, none of these chemokines induced migration in Jurkat cells stably expressing U51. Together, these data indicate that the products of U12 and U51 may play important and different roles in the pathogenesis of HHV-7 through transmembrane signaling.

Human herpesvirus 7 open reading frame U12 encodes a functional beta-chemokine receptor.

Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily, infects mainly CD4+ T cells in vitro and infects children during infancy. After the primary infection, HHV-7 becomes latent. HHV-7 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 gene, we cloned the gene and expressed the U12 protein in cells. The U12 gene encoded a calcium-mobilizing receptor for the EBI1 ligand chemokine-macrophage inflammatory protein 3beta (ELC/MIP-3beta) but not for other chemokines, suggesting that the chemokine selectivity of the U12 gene product is distinct from that of the known mammalian chemokine receptors. These studies revealed that U12 activates distinct transmembrane signaling pathways that may mediate biological functions by binding with a beta-chemokine, ELC/MIP-3beta.