The human cytomegalovirus-encoded G protein- coupled receptor UL33 exhibits oncomodulatory properties

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein- coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies

High Accuracy Mutation Detection in Leukemia on a Selected Panel of Cancer Genes

<div><p>With the advent of whole-genome and whole-exome sequencing, high-quality catalogs of recurrently mutated cancer genes are becoming available for many cancer types. Increasing access to sequencing technology, including bench-top sequencers, provide the opportunity to re-sequence a limited set of cancer genes across a patient cohort with limited processing time. Here, we re-sequenced a set of cancer genes in T-cell acute lymphoblastic leukemia (T-ALL) using Nimblegen sequence capture coupled with Roche/454 technology. First, we investigated how a maximal sensitivity and specificity of mutation detection can be achieved through a benchmark study. We tested nine combinations of different mapping and variant-calling methods, varied the variant calling parameters, and compared the predicted mutations with a large independent validation set obtained by capillary re-sequencing. We found that the combination of two mapping algorithms, namely <em>BWA-SW</em> and <em>SSAHA2</em>, coupled with the variant calling algorithm <em>Atlas-SNP2</em> yields the highest sensitivity (95%) and the highest specificity (93%). Next, we applied this analysis pipeline to identify mutations in a set of 58 cancer genes, in a panel of 18 T-ALL cell lines and 15 T-ALL patient samples. We confirmed mutations in known T-ALL drivers, including PHF6, NF1, FBXW7, NOTCH1, KRAS, NRAS, PIK3CA, and PTEN. Interestingly, we also found mutations in several cancer genes that had not been linked to T-ALL before, including JAK3. Finally, we re-sequenced a small set of 39 candidate genes and identified recurrent mutations in TET1, SPRY3 and SPRY4. In conclusion, we established an optimized analysis pipeline for Roche/454 data that can be applied to accurately detect gene mutations in cancer, which led to the identification of several new candidate T-ALL driver mutations.</p> </div

Incorporating robustness requirements into antiwindup design

This paper treats the problem of synthesizing antiwindup compensators that are able to handle plant uncertainty in addition to controller saturation. The uncertainty considered is of the frequency-weighted additive type, often encountered in linear robust control theory, and representative of a wide variety of uncertainty encountered in practice. The main results show how existing linear matrix inequality based antiwindup synthesis algorithms can be modified to produce compensators that accommodate uncertainty better. Embedded within these results is the ever-present performance - robustness tradeoff. A remarkable feature is that the often criticized internal model control antiwindup solution emerges as an ldquooptimally robustrdquo solution. A simple example demonstrates the effectiveness of the modified algorithms

G protein involvement in US28-mediated Tcf-Lef activation.

<p>A, HEK293T cells were co-transfected with the Tcf-Lef reporter gene construct, a US28-expressing construct or empty plasmid control (mock) and various constructs expressing Gα-proteins as indicated, Gα_q-11 shRNA construct or a construct expressing regulator of G protein signaling 2 (RGS2), known to specifically interfere with Gα_q signaling. Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%. B, HEK293T cells were co-transfected with the Tcf-Lef reporter gene construct, US28-expressing construct or empty plasmid control (mock) and an shRNA construct to decrease protein levels of Gα_q. Total cell extracts were analysed on Western blot using antibodies recognizing Gα_q or actin (insert). Bars represent level of Gα_q protein level compared to the actin levels, with the ratio in non-treated mock cells set at 100%. C, HEK293T cells were co-transfected with the Tcf-Lef reporter gene construct, a US28-expressing construct or empty plasmid control (mock) and various constructs expressing Gα₁₃, a constitutive active (CA) Gα₁₃ or Lsc-RGS, encoding the RGS domain of the Rho GTPase guanine nucleotide exchange factor (Rho-GEF) Lsc, known to specifically interfere with transmembrane signaling mediated by activated Gα_12/13. Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%. D, HEK293T cells co-transfected with the Tcf-Lef reporter gene construct, a US28-expressing construct or empty plasmid control (mock) were treated (overnight) with various concentrations of the ROCK inhibitor Y27632 as indicated.</p

HCMV-infected cells stimulate activation of ß-catenin in a US28 dependent manner.

<p>A. HFF cells were infected with HCMV-WT or HCMV-ΔUS28 with a M.O.I of 1 on IBIDI slides. Cells were fixed 24 hours post-infection (hpi) and stained with antibodies recognizing the HCMV immediate early antigen (IEA) and activated ß-catenin respectively. B. U373-MG cells transfected with Tcf-Lef reporter gene were either infected with HCMV-WT or HCMV-ΔUS28 with a M.O.I. of 2, or left uninfected (mock). Luciferase activity was measured 48 h post-infection.</p

Classical Wnt/Frizzled/ß-catenin

<p><b>signaling is not involved in US28-mediated Tcf-Lef activation.</b> A, Western blot analysis of total cell extracts of NIH-3T3 cells, stably expressing US28 or an empty plasmid (mock) which were treated with Wnt3a- (overnight, 200 ng/ml) and vehicle-treated mock cells. The blot was probed with antibodies recognizing the non-phosphorylated (active ß-catenin, total ß-catenin and actin. A representative blot is shown and normalized quantifications of (active) ß-catenin of independent experiments are shown below the blot. B, Western blot analysis of total cell extracts of NIH-3T3 cells stably expressing US28, the non G-protein coupling US28 mutant R¹²⁹A or an empty plasmid (mock) and Wnt3a-treated mock cells. The blot was probed with antibodies recognizing Lrp6-phospho-ser¹⁴⁹⁰ and actin. A representative blot is shown and normalized quantifications of Lrp6-phospho-ser¹⁴⁹⁰ of independent experiments are shown below the blot. C, HEK293T cells co-transfected with the Tcf-Lef reporter gene construct and either US28-expressing or an empty control plasmid (mock) exposed to Wnt3a (overnight, 200 ng/ml). Luciferase activity was measured 24 hr after transfection and is displayed here as the percentage of the non-treated mock control that is set at 100%. D, HEK293T cells co- transfected with the Tcf-Lef reportergene and an US28-expressing construct or empty plasmid control (mock) were exposed to various concentrations (ON, 10–25 µM) of the COX2 inhibitor celecoxib (Cxb). Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%.</p

Schematic representation of the classic Wnt signaling pathway and model of US28-mediated activation of ß-catenin signaling pathway.

<p>The left side of the model indicates components of the classic Wnt/Frizzled mediated activation of ß-catenin. In this pathway the disruption complex (Axin, APC) that enables GSK3ß- and Caseine kinase 1 (CK1)-mediated phosphorylation of ß-catenin leading to its degradation, is disrupted in a Dishevelled-mediated way upon Wnt binding to Frizzled/LRP. US28 activates ß-catenin signaling in a ligand-dependent and independent manner, involving respectively Gα_12/13 and Gα_q proteins and respective RhoGEFs, converging at RhoA/ROCK, resulting in increased Tcf-Lef transcriptional activation.</p