Therapeutic targeting of ACLY in T-ALL in vivo.

T-cell Acute Lymphoblastic Leukemia (T-ALL) is a hematological malignancy in need of novel therapeutic approaches. Here, we identify the ATP-citrate lyase ACLY as a novel therapeutic target in T-ALL. Our results show that ACLY is overexpressed in T-ALL, and its expression correlates with NOTCH1 activity. To test the effects of ACLY in leukemia progression and the response to NOTCH1 inhibition, we developed an isogenic model of NOTCH1-induced Acly conditional knockout leukemia. Importantly, we observed intrinsic antileukemic effects upon loss of ACLY, which further synergized with NOTCH1 inhibition in vivo . Gene expression profiling analyses showed that the transcriptional signature of ACLY loss very significantly correlates with the signature of NOTCH1 inhibition in vivo , with significantly downregulated pathways related to oxidative phosphorylation, electron transport chain, ribosomal biogenesis and nucleosome biology. Consistently, metabolomic profiling upon ACLY loss revealed a metabolic crisis with accumulation of nucleotide intermediates and reduced levels of several amino acids. Overall, our results identify a link between NOTCH1 and ACLY and unveil ACLY as a novel promising target for T-ALL treatment.

Lineage diversification of pigeon paramyxovirus effect re-emergence potential in chickens.

Genotype VI-paramyxovirus (GVI-PMV1) is a major cause of epidemic Newcastle-like disease in Columbiformes. This genotype of avian paramyxovirus type 1 has diversified rapidly since its introduction into the US in 1982 resulting in two extant lineages, which have different population growth properties. Although some GVI-PMV1s replicate poorly in chickens, it is possible that variants with different replicative or pathogenic potential in chickens exist among the genetically-diverse GVI-PMV1s strains. To determine if variants of Columbiform GVI-PMV1 with different phylogenetic affiliations have distinct phenotypic properties in chickens, we investigated the replicative properties of 10 naturally circulating pigeon-derived isolates representing four subgroups of GVI-PMV1 in primary chicken lung epithelial cells and in chicken embryos. Our data demonstrate that GVI-PMV1 variants have different infection phenotypes in their chicken source host and that properties reflect subgroup affiliation. These subgroup replicative properties are consistent with observed dynamics of viral population growth.

Successful establishment and global dispersal of genotype VI avian paramyxovirus serotype 1 after cross species transmission.

The evolutionary history of avian paramyxovirus serotype 1 (PMV1), which includes the agents of Newcastle disease (ND), is characterized by a series of strain emergence events since viruses in this family were first recognized in the 1920s. Despite the importance of ND to the poultry industry, little is known about PMV1 strain emergence events and the subsequent dispersal and evolution of new strains. The genotype VI-PMV1 was first identified in the 1980s and has been named pigeon paramyxovirus-1 (PPMV1) because of unusual host specificity with Columbiformes (Collins et al., 1996); it has been responsible for panzootics in both chickens and pigeons during that time. Here, we used evolutionary analyses to characterize the emergence of this contemporary PMV1 lineage. We demonstrate that GVI-PMV1 arose through cross-species transmission events from Galliformes (i.e. chicken) to Columbiformes, and quickly established in pigeon populations. Our studies revealed a close association between the time of viral emergence and panzootic events of this virus. The virus appeared first in Southeastern Europe and quickly spread across the European continent, which became the epicenter for global virus dissemination. With new viral gene sequences, we show that GVI-PMV1 viruses currently circulating in North America resulted from multiple invasion events from Europe, one associated with an exotic European Columbiformes species, and that extant lineages have diversified locally. This study extends our understanding of successful viral emergence subsequent to cross-species transmission and dispersal patterns of newly emerged avian viruses, which may improve surveillance awareness and disease control of this and other important avian pathogens.

Polymorphic integrations of an endogenous gammaretrovirus in the mule deer genome.

Endogenous retroviruses constitute a significant genomic fraction in all mammalian species. Typically they are evolutionarily old and fixed in the host species population. Here we report on a novel endogenous gammaretrovirus (CrERVγ; for cervid endogenous gammaretrovirus) in the mule deer (Odocoileus hemionus) that is insertionally polymorphic among individuals from the same geographical location, suggesting that it has a more recent evolutionary origin. Using PCR-based methods, we identified seven CrERVγ proviruses and demonstrated that they show various levels of insertional polymorphism in mule deer individuals. One CrERVγ provirus was detected in all mule deer sampled but was absent from white-tailed deer, indicating that this virus originally integrated after the split of the two species, which occurred approximately one million years ago. There are, on average, 100 CrERVγ copies in the mule deer genome based on quantitative PCR analysis. A CrERVγ provirus was sequenced and contained intact open reading frames (ORFs) for three virus genes. Transcripts were identified covering the entire provirus. CrERVγ forms a distinct branch of the gammaretrovirus phylogeny, with the closest relatives of CrERVγ being endogenous gammaretroviruses from sheep and pig. We demonstrated that white-tailed deer (Odocoileus virginianus) and elk (Cervus canadensis) DNA contain proviruses that are closely related to mule deer CrERVγ in a conserved region of pol; more distantly related sequences can be identified in the genome of another member of the Cervidae, the muntjac (Muntiacus muntjak). The discovery of a novel transcriptionally active and insertionally polymorphic retrovirus in mammals could provide a useful model system to study the dynamic interaction between the host genome and an invading retrovirus.

Modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus.

The immune surveillance system protects host cells from viral infection, and viruses have evolved to escape this system for efficient proliferation in the host. Host cells produce cytokines and chemokines in response to viral infection, and among such effector molecules, type I interferons are the principal antiviral cytokines and therefore effective targets for viruses to disarm host surveillance. Porcine reproductive and respiratory syndrome virus (PRRSV) expresses proteins that circumvent the IFN response and other cellular processes, and to compensate the small coding capacity of PRRSV, these proteins are multifunctional. To date, at least four viral proteins have been identified and studied as viral antagonists of host defenses: N as a structural protein and three non-structural proteins, Nsp1 (Nsp1α and Nsp1ß), Nsp2, and Nsp11. Among these, N and Nsp1 are nuclear-cytoplasmic proteins distributed in both the nucleus and cytoplasm of cells. Nsp1 and Nsp2 are viral proteases while Nsp11 is an endoribonuclease. This review describes the current understanding of the role of these proteins in modulating the host innate immune responses. Blocking against virus-mediated inhibition of the innate response may lead to the future development of effective vaccines. The understanding of viral mechanisms modulating the normal cellular processes will be a key to the design of an effective control strategy for PRRS.

Compensatory upregulation of tyrosine kinase Etk/BMX in response to androgen deprivation promotes castration-resistant growth of prostate cancer cells.

We previously showed that targeted expression of non-receptor tyrosine kinase Etk/BMX in mouse prostate induces prostate intraepithelial neoplasia, implying a possible causal role of Etk in prostate cancer development and progression. Here, we report that Etk is upregulated in both human and mouse prostates in response to androgen ablation. Etk expression seems to be differentially regulated by androgen and interleukin 6 (IL-6), which is possibly mediated by the androgen receptor (AR) in prostate cancer cells. Our immunohistochemical analysis of tissue microarrays containing 112 human prostate tumor samples revealed that Etk expression is elevated in hormone-resistant prostate cancer and positively correlated with tyrosine phosphorylation of AR (Pearson correlation coefficient rho = 0.71, P < 0.0001). AR tyrosine phosphorylation is increased in Etk-overexpressing cells, suggesting that Etk may be another tyrosine kinase, in addition to Src and Ack-1, which can phosphorylate AR. We also showed that Etk can directly interact with AR through its Src homology 2 domain, and such interaction may prevent the association of AR with Mdm2, leading to stabilization of AR under androgen-depleted conditions. Overexpression of Etk in androgen-sensitive LNCaP cells promotes tumor growth while knocking down Etk expression in hormone-insensitive prostate cancer cells by a specific shRNA that inhibits tumor growth under androgen-depleted conditions. Taken together, our data suggest that Etk may be a component of the adaptive compensatory mechanism activated by androgen ablation in prostate and may play a role in hormone resistance, at least in part, through direct modulation of the AR signaling pathway.

Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells.

Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

Exploratory Spatial Analysis of in vitro Respiratory Syncytial Virus Co-infections.

The cell response to virus infection and virus perturbation of that response is dynamic and is reflected by changes in cell susceptibility to infection. In this study, we evaluated the response of human epithelial cells to sequential infections with human respiratory syncytial virus strains A2 and B to determine if a primary infection with one strain will impact the ability of cells to be infected with the second as a function of virus strain and time elapsed between the two exposures. Infected cells were visualized with fluorescent markers, and location of all cells in the tissue culture well were identified using imaging software. We employed tools from spatial statistics to investigate the likelihood of a cell being infected given its proximity to a cell infected with either the homologous or heterologous virus. We used point processes, K-functions, and simulation procedures designed to account for specific features of our data when assessing spatial associations. Our results suggest that intrinsic cell properties increase susceptibility of cells to infection, more so for RSV-B than for RSV-A. Further, we provide evidence that the primary infection can decrease susceptibility of cells to the heterologous challenge virus but only at the 16 h time point evaluated in this study. Our research effort highlights the merits of integrating empirical and statistical approaches to gain greater insight on in vitro dynamics of virus-host interactions.

Regulation of androgen receptor activity by tyrosine phosphorylation.

The androgen receptor (AR) is essential for the growth of prostate cancer cells. Here, we report that tyrosine phosphorylation of AR is induced by growth factors and elevated in hormone-refractory prostate tumors. Mutation of the major tyrosine phosphorylation site in AR significantly inhibits the growth of prostate cancer cells under androgen-depleted conditions. The Src tyrosine kinase appears to be responsible for phosphorylating AR, and there is a positive correlation of AR tyrosine phosphorylation with Src tyrosine kinase activity in human prostate tumors. Our data collectively suggest that growth factors and their downstream tyrosine kinases, which are elevated during hormone-ablation therapy, can induce tyrosine phosphorylation of AR and such modification may be important for prostate tumor growth under androgen-depleted conditions.

Tyrosine kinase Etk/BMX is up-regulated in human prostate cancer and its overexpression induces prostate intraepithelial neoplasia in mouse.

The nonreceptor tyrosine kinase Etk/BMX was originally identified from the human prostate xenograft CWR22. Here, we report that Etk is up-regulated in human prostate tumor specimens surveyed. Knocking down Etk expression by a specific small interfering RNA (siRNA) in prostate cancer cells attenuates cell proliferation, suggesting an essential role of Etk for prostate cancer cell survival and growth. Targeted expression of Etk in mouse prostate epithelium results in pathologic changes resembling human prostatic intraepithelial neoplasia, indicating that up-regulation of Etk may contribute to prostate cancer development. A marked increase of luminal epithelial cell proliferation was observed in the Etk transgenic prostate, which may be attributed in part to the elevated activity of Akt and signal transducers and activators of transcription 3 (STAT3). More interestingly, the expression level of acetyltransferase cyclic AMP-responsive element binding protein-binding protein (CBP) is also increased in the Etk transgenic prostate as well as in a prostate cancer cell line overexpressing Etk, concomitant with elevated histone 3 acetylation at lysine 18 (H3K18Ac). Down-modulation of Etk expression by a specific siRNA leads to a decrease of H3 acetylation in prostate cancer cell lines. Our data suggest that Etk may also modulate chromatin remodeling by regulating the activity of acetyltransferases, such as CBP. Given that Etk may exert its effects in prostate through modulation of multiple signaling pathways altered in human prostate cancer, the Etk transgenic mouse model may be a useful tool for studying the functions of Etk and identification of new molecular markers and drug targets relevant to human diseases.

Pituitary adenylate cyclase-activating polypeptide 27 is a functional ligand for formyl peptide receptor-like 1.

Although the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the regulation of several immune responses, its target receptors and signaling mechanisms have yet to be fully elucidated in immune cells. In this study, we found that PACAP27, but not PACAP38, specifically stimulated intracellular calcium mobilization and ERK phosphorylation in human neutrophils. Moreover, formyl peptide receptor-like 1 (FPRL1) was identified as a PACAP27 receptor, and PACAP27 was found to selectively stimulate intracellular calcium increase in FPRL1-transfected rat basophil leukocytes-2H3 cell lines. In addition, PACAP27-induced calcium increase and ERK phosphorylation were specifically inhibited by an FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp-Trp (WRW4), thus supporting the notion that PACAP27 acts on FPRL1. In terms of the functional role of PACAP27, we found that the peptide stimulated CD11b surface up-regulation and neutrophil chemotactic migration, and that these responses were completely inhibited by WRW4. The interaction between PACAP27 and FPRL1 was analyzed further using truncated PACAPs and chimeric PACAPs using vasoactive intestinal peptide, and the C-terminal region of PACAP27 was found to perform a vital function in the activation of FPRL1. Taken together, our study suggests that PACAP27 activates phagocytes via FPRL1 activation, and that this results in proinflammatory behavior, involving chemotaxis and the up-regulation of CD11b.

Synergism of cytoplasmic kinases in IL6-induced ligand-independent activation of androgen receptor in prostate cancer cells.

IL6 is a pleiotropic cytokine which has been implicated in ligand-independent activation of androgen receptor in prostate cancer cells. Here, we present the evidence that two cytoplasmic kinases Pim1 and Etk are involved in this process. We showed that Pim1 is expressed in all prostate cancer cell lines examined. Both the expression level and the kinase activity of Pim1 are regulated by IL6 in these cells. Furthermore, we showed that IL6 downstream tyrosine kinase Etk can induce tyrosine phosphorylation of Pim1 which is correlated with its kinase activity. Mutation of the conserved Tyrosine 218 in the activation loop results in reduced kinase activity of Pim1. Interestingly, Etk can also be activated by Pim1 when they are coexpressed in prostate cancer cells, suggesting a possible positive feedback loop between Etk and Pim1. It appears that both Pim1 and Etk are required for IL6-induced activation of androgen receptor-mediated transcription in prostate cancer cells because overexpression of the kinase-deficient form of either Pim1 or Etk dramatically blocks the IL6 effect. Coexpression of the two kinases together but neither one alone is sufficient to activate ARE-containing promoter. Taken together, our data suggest a synergism of Ser/Thr kinase Pim1 and tyrosine kinase Etk in IL6 signaling and provide new insights into ligand-independent activation of androgen receptor in prostate cancer cells.

Selective activation of small GTPase RhoA by tyrosine kinase Etk through its pleckstrin homology domain.

Etk/Bmx is a member of the Btk family tyrosine kinase, which contains an N-terminal pleckstrin homology domain. Etk has been shown to play a pivotal role in the regulation of various cellular processes including differentiation, apoptosis, and cell motility. Here we present evidence that Etk is a modulator of the small GTPase RhoA. Etk and RhoA both are translocated to the plasma membrane and can form a complex upon serum stimulation in C2C12 cells. Etk interacts with RhoA but not other closely related small GTPases such as Cdc42 and Rac1, suggesting a specific modulation of RhoA by Etk. Our results demonstrate that Etk activates RhoA and enhances Rho-mediated stress fiber formation and transcription activity in a pleckstrin homology domain-dependent manner. Furthermore, Etk disrupts the interaction between RhoA and Rho-GDI (guanine nucleotide dissociation inhibitor) and promotes the membrane translocation of RhoA. Our data suggest that Etk plays an important role in regulation of RhoA-mediated signaling.

Interaction between tyrosine kinase Etk and a RUN domain- and FYVE domain-containing protein RUFY1. A possible role of ETK in regulation of vesicle trafficking.

Etk/BMX tyrosine kinase is involved in regulation of various cellular processes including proliferation, differentiation, motility, and apoptosis. Through a yeast two-hybrid screening for the effectors of Etk, a new gene family designated as RUFY was identified. The RUFY gene family (RUFY1 and RUFY2) contains an N-terminal RUN domain and a C-terminal FYVE domain with two coiled-coil domains in-between. They appear to be homologues of a recently identified mouse Rabip4 (Cormant, M., Mari, M., Galmiche, A., Hofman, P., and Le Marchand-Brustel, Y. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 1637-1642). RUFY proteins are localized predominantly to endosomes as evidenced by their co-localization with early endosome antigen marker (EEA1). Etk interacts with RUFY1 through its SH3 and SH2 domains. RUFY1 is tyrosine-phosphorylated and appears to be a substrate of Etk. The RUFY1 mutant lacking the phosphorylation sites failed to go to the endosomes. Furthermore, overexpression of Etk in COS-1 and B82L cells resulted in increased plasma membrane localization of the epidermal growth factor receptor and delayed its induced endocytosis in COS-1 cells. The effects of Etk were blocked by the FYVE domain of RUFY1. Interestingly, the FYVE domain of RUFY1 is targeted to the plasma membrane through an interaction between its proline-rich motif and the SH3 domain of Etk or possibly some other membrane-associated SH3 domain-containing protein(s), whereas the lipid binding activity of the FYVE domain is not required. Our data suggest that Etk may be involved in regulation of endocytosis through its interaction with an endosomal protein RUFY1.

Suppressive Effects of Edible Thai Plants on Superoxide and Nitric Oxide Generation.

We screened ethanol extracts from a total of 134 species of edible Thai plants for their suppressive effects on superoxide (O2(-)) generation using a xanthine (XA)-xanthine oxidase (XOD) assay system. When the extracts were tested at a concentration of 500 &mgr;/g/ml, 28.4% significantly suppressed O2(-) generation. Of these active extracts, it was found that in 17.9% of cases the action was due to XOD inhibition, in 1.5% due to O2(-) scavenging activity, and in 9% due to both XOD inhibition and O2(-) scavenging. In addition, some plant extracts (25 species) which had been known to possibly possess anti-tumor promoting activity were tested for O2(-) and NO generation in cellular systems. In this test, 13 species exhibited strong inhibitory activity toward both O2(-) and NO generation. From the fruit pods of Oroxylum indicum (Bignoniaceae), a traditional vegetable in Thailand, two flavones, oroxylin A and chrysin, and a triterpene carboxylic acid, ursolic acid (UA), were identified as inhibitors of O2(-) generation in XA/XOD system. These compounds also showed marked inhibitory effects on the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced O2(-) generation in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. Our results suggest that, as we have reported earlier, edible Thai plants are promising sources of antioxidants with chemopreventive potential.