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Primary effusion lymphoma (PEL) constitutes a subset of non-Hodgkin lymphoma whose incidence is highly increased in the context of HIV infection. Kaposi sarcomaassociated herpesvirus is the causative agent of PEL. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in cell proliferation and survival, and this pathway is dysregulated in many different cancers, including PEL, which display activated PI3K, Akt, and mammalian target of rapamycin (mTOR) kinases. PELs rely heavily on PI3K/Akt/mTOR signaling, are dependent on autocrine and paracrine growth factors, and also have a poor prognosis with reported median survival times of less than 6 months. We compared different compounds that inhibit the PI3K/Akt/mTOR pathway in PEL. Although compounds that modulated activity of only a single pathway member inhibited PEL proliferation, the use of a novel compound, NVP-BEZ235, that dually inhibits both PI3K and mTOR kinases was significantly more efficacious in culture and in a PEL xenograft tumor model. NVP-BEZ235 was effective at low nanomolar concentrations and has oral bioavailability. We also report a novel mechanism for NVP-BEZ235 involving the suppression of multiple autocrine and paracrine growth factors required for lymphoma survival. IntroductionThe phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in cell proliferation and cell survival. PI3K activation stimulates the production of phosphatidylinositol 3,4,5-triphosphate, which results in activation of the kinases PDK1 and Akt. The lipid phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein is a negative regulator of this pathway. Akt kinase promotes cell survival by phosphorylating, and thereby inactivating, proapoptotic factors, such as the FOXO transcription factor family, GSK-3␤, caspase-9, and Bad. [1][2][3][4] Phosphorylation of Bad and the FOXO transactivators prevent apoptosis. Akt also phosphorylates p27, a negative regulator of the cell cycle, thereby preventing cell cycle arrest. In addition, Akt activation leads to phosphorylation and activation of the mammalian target of rapamycin (mTOR), a kinase that stimulates protein synthesis and cell proliferation.Activated mTOR protein can associate with raptor and mLST8/G␤L to form the mTORC1 complex. The mTORC1 complex induces phosphorylation of p70 S6 kinase (S6K), leading to phosphorylation and activation of the ribosomal protein S6. mTORC1 also inhibits 4E-BP1, a repressor of eukaryotic initiation factor eIF4E. This arm of the mTOR pathway is rapamycin-sensitive. In contrast, the mTORC2 complex, which consists of mTOR, mLST8/G␤L, mSin1, and Rictor, is insensitive to the effects of rapamycin. mTORC2 functions in a feedback loop that phosphorylates and activates Akt by phosphorylation at Ser473. 5 Hence, inhibitors of PI3K/Akt probably have broader effects than mTOR inhibitors.The nutrient sensor, AMP activated kinase (AMPK), is a negative regulator of mTORC1. 6 AMPK controls cellular homeostasis by regulating energy production withi...

Epstein-Barr virus (EBV) establishes a latent form of infection in memory B cells, while antibody-secreting plasma cells often harbor the lytic form of infection. The switch between latent and lytic EBV infection is mediated by the two viral immediate-early proteins BZLF1 (Z) and BRLF1 (R), which are not expressed in latently infected B cells. Here we demonstrate that a cellular transcription factor that plays an essential role in plasma cell differentiation, X-box-binding protein 1 (XBP-1), also activates the transcription of the two EBV immediate-early gene promoters. In reporter gene assays, XBP-1 alone was sufficient to activate the R promoter, whereas the combination of XBP-1 and protein kinase D (PKD) was required for efficient activation of the Z promoter. Most importantly, the expression of XBP-1 and activated PKD was sufficient to induce lytic viral gene expression in EBV-positive nasopharyngeal carcinoma cells and lymphoblastoid cells, while an XBP-1 small interfering RNA inhibited constitutive lytic EBV gene expression in lymphoblastoid cells. These results suggest that the plasma cell differentiation factor XBP-1, in combination with activated PKD, can mediate the reactivation of EBV, thereby allowing the viral life cycle to be intimately linked to plasma cell differentiation. Epstein-Barr virus (EBV)is the causative agent of infectious mononucleosis and is associated with B-cell lymphomas, nasopharyngeal carcinomas, gastric carcinomas, and other malignancies (26,45). EBV causes lytic infection in normal oral epithelial cells (32, 51) while usually establishing one of the latent forms of infection in circulating memory B cells. In contrast, tonsillar B cells that express antigens specific for plasma markers commonly harbor the lytic form of EBV infection, which results in the production of infectious viral particles (10,29,30).The switch from latent to lytic EBV infection is mediated by the immediate-early (IE) protein BZLF1 (Z) and the immediate-early/early protein BRLF1 (R) (1,16,57). Z and R are transcription factors that activate each other's transcription and together are sufficient to activate the entire lytic viral gene expression cascade (17,49). In latently infected cells, the promoters driving Z and R expression (Zp and Rp) are inactive. Therefore, the activation of Zp and Rp by cellular transcription factors is the crucial initial step required for lytic viral gene expression. B-cell receptor engagement activates lytic EBV gene expression in some B-cell lines in vitro and activates both EBV IE promoters in reporter gene assays (23). Although several different individual cellular transcription factors can activate one or both of the two EBV IE promoters in reporter gene assays (23), to date these factors have not been shown to be sufficient for the efficient reactivation of lytic viral gene expression from the endogenous viral genome in latently infected cells.While there is a strong correlation between plasma cell differentiation and lytic EBV gene expression in human tonsils, it is not pr...

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with B-cell lymphomas, nasopharyngeal carcinoma, gastric carcinomas, and other malignancies (44, 56). EBV primarily infects two cell types, epithelial cells and B cells (44,50). In normal oral epithelial cells, the virus exists in a lytic state (34, 37, 44, 51), whereas EBV infection of B cells usually results in a latent type of infection (34,44). However, in a small percentage of B cells, the virus periodically reactivates and replicates in the lytic manner. This reactivation is initiated by expression of the two immediateearly genes, BZLF1 and BRLF1 (8,13,15,33,46,47,51,55), which encode the transcriptional activators Z and R, respectively.Z is a bZip protein homologous to the cellular proteins c-Fos and c-Jun and binds directly to AP-1 and AP-1-like motifs (known as Z-responsive elements [ZREs]) present in many lytic viral promoters (12,19,21). Z expression results in activation of lytic EBV gene expression in essentially all EBVpositive cell lines. Z initially activates expression of the BRLF1 immediate-early promoter (Rp) by binding directly to three somewhat atypical ZRE sites in Rp, referred to as ZRE-1, ZRE-2, and the recently identified ZRE-3 (6, 41). Z and R together then activate the expression of the entire complement of lytic EBV genes, thus inducing lytic EBV infection.The EBV genome in latently infected cells is extensively methylated (18,39). Methylation likely suppresses the expression of lytic viral genes, thus helping to maintain latent infection. In addition, methylation of viral promoters driving LMP-1 and EBNA-2 expression is associated with the most stringent form of viral latency (type I) (2,18,28,30,38,52), in which LMP-1 and EBNA-2 are not expressed. It has been proposed that DNA methylation causes transcriptional repression by multiple different mechanisms, including modification of the histone acetylation state and prevention of transcription factor binding to DNA (5,

RhaS activates transcription of the Escherichia coli rhaBAD and rhaT operons in response to L-rhamnose and is a member of the AraC/XylS family of transcription activators. We wished to determine whether 70 might be an activation target for RhaS. We found that 70 K593 and R599 appear to be important for RhaS activation at both rhaBAD and rhaT, but only at truncated promoters lacking the binding site for the second activator, CRP. To determine whether these positively charged 70 residues might contact RhaS, we constructed alanine substitutions at negatively charged residues in the C-terminal domain of RhaS. Substitutions at four RhaS residues, E181A, D182A, D186A, and D241A, were defective at both truncated promoters. Finally, we assayed combinations of the RhaS and 70 substitutions and found that RhaS D241 and 70 R599 met the criteria for interacting residues at both promoters. Molecular modeling suggests that 70 R599 is located in very close proximity to RhaS D241; hence, this work provides the first evidence for a specific residue within an AraC/XylS family protein that may contact 70 . More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with 70 may be conserved.

In conclusion, the striking antileukemic activity of singleagent dasatinib in this case of CBF leukemia provides a rationale for its combination with standard chemotherapy, and recently the German-Austrian AMLSG Cooperative Group has initiated a phase II clinical study in patients with newly diagnosed CBF AML. 16 For patients with relapsed RUNX1/RUNX1T1-positive leukemia and comorbidities or a reduced performance status precluding aggressive therapy, a trial with single-agent dasatinib may be warranted. Conflict of interestThe authors declare no conflict of interest. Acknowledgements

Kaposi sarcoma-associated herpesvirus (KSHV) is associated with 3 different human malignancies: Kaposi sarcoma (KS), primary effusion lymphoma, and multicentric Castleman disease. The KS lesion is driven by KSHV-infected endothelial cells and is highly dependent on autocrine and paracrine factors for survival and growth. We report that latent KSHV infection increases the vascular permeability of endothelial cells. Endothelial cells with latent KSHV infection display increased Rac1 activation and activation of its downstream modulator, p21-activated kinase 1 (PAK1). The KSHV-infected cells also exhibit increases in tyrosine phosphorylation of vascular endothelial (VE)-cadherin and ␤-catenin, whereas total levels of these proteins remained unchanged, suggesting that latent infection disrupted endothelial cell junctions. Consistent with these findings, we found that KSHVinfected endothelial cells displayed increased permeability compared with uninfected endothelial cells. Knockdown of Rac1 and inhibition of reactive oxygen species (ROS) resulted in decreased permeability in the KSHV-infected endothelial cells. We further demonstrate that the KSHV K1 protein can activate Rac1. Rac1 was also highly activated in KSHVinfected endothelial cells and KS tumors. In conclusion, KSHV latent infection increases Rac1 and PAK1 activity in endothelial cells, resulting in the phosphorylation of VE-cadherin and ␤-catenin and leading to the disassembly of cell junctions and to increased vascular permeability of the infected endothelial cells. (Blood. 2011;118(19):5344-5354) IntroductionThe endothelial cell barrier function is regulated by vascular endothelial (VE)-cadherin-containing adherens junctions in addition to tight junctions. 1 VE-cadherin is involved in maintaining the integrity of endothelial cell junctions by preventing the disassembly of the endothelial barrier and regulating the movement of macromolecules through the endothelium. 1-3 However, upon VEGF stimulation, these normal endothelial cell junctions are reorganized to allow the extravasation of cellular factors. 4 This involves the disruption of VE-cadherin at the adherens junction 2,4,5 and internalization of VE-cadherin from the cell surface. 6 VEGF stimulation leads to the induction of Rac1 activity 7,8 and its downstream effector, p21-activated kinase 1 (PAK1). 8 In addition, Rac1 has also been shown to regulate VE-cadherin phosphorylation through the generation of reactive oxygen species (ROS). 9,10 Kaposi sarcoma (KS) is a multifocal vascular tumor of mixed cellular composition. KS lesions are composed of a mixed population of cells, including spindle-shaped endothelial cells and infiltrating leukocytes. 11,12 KS is the most common neoplasm in patients with AIDS. Areas that have the highest HIV burden, such as sub-Saharan Africa, also have the highest rate of KS. KSassociated herpesvirus (KSHV) is the etiological agent found in all epidemiologic forms of KS, 13 and viral genomic DNA is present in AIDS-associated KS, as well as in HIV-negative classic and tra...

RhaS, an AraC family protein, activates rhaBADtranscription by binding to rhaI, a site consisting of two 17-bp inverted repeat half-sites. In this work, amino acids in RhaS that make base-specific contacts with rhaI were identified. Sequence similarity with AraC suggested that the first contacting motif of RhaS was a helix-turn-helix. Assays of rhaB-lacZactivation by alanine mutants within this potential motif indicated that residues 201, 202, 205, and 206 might contact rhaI. The second motif was identified based on the hypothesis that a region of especially high amino acid similarity between RhaS and RhaR (another AraC family member) might contact the nearly identical DNA sequences in one major groove of their half-sites. We first made targeted, random mutations and then made alanine substitutions within this region of RhaS. Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding. A genetic loss-of-contact approach was used to identify whether any of the RhaS amino acids in the first or second contacting motif make base-specific DNA contacts. In motif 1, we found that Arg202 and Arg206 both make specific contacts with bp −65 and −67 in rhaI 1, and that Arg202 contacts −46 and Arg206 contacts −48 inrhaI 2. In motif 2, we found that Asp250 and Asn252 both contact the bp −79 in rhaI 1. Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.