Perturbation Of Signaling Pathways Research Articles

Inhibition of microRNA suppression of Dishevelled results in Wnt pathway-associated developmental defects in sea urchin.

MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that regulate gene expressions by binding to the 3' untranslated region of target mRNAs thereby silencing translation. Some miRNAs are key regulators of the Wnt signaling pathways, which impact developmental processes. This study investigates miRNA regulation of different isoforms of Dishevelled (Dvl/Dsh), which encode a key component in the Wnt signaling pathway. The sea urchin Dvl mRNA isoforms have similar spatial distribution in early development, but one isoform is distinctively expressed in the larval ciliary band. We demonstrated that Dvl isoforms are directly suppressed by miRNAs. By blocking miRNA suppression of Dvl isoforms, we observed dose-dependent defects in spicule length, patterning of the primary mesenchyme cells, gut morphology, and cilia. These defects likely result from increased Dvl protein levels, leading to perturbation of Wnt-dependent signaling pathways and additional Dvl-mediated processes. We further demonstrated that overexpression of Dvl isoforms recapitulated some of the Dvl miRNATP-induced phenotypes. Overall, our results indicate that miRNA suppression of Dvl isoforms plays an important role in ensuring proper development and function of primary mesenchyme cells and cilia.

PRMT5 Is Upregulated in Activated T Cells and Is a Novel Therapeutic Target for Acute Gvhd

Introduction: Acute graft-versus-host disease (aGVHD), a T cell-mediated immunological disorder is the leading cause of non-relapse mortality in patients receiving allogeneic bone marrow transplants. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (me2s) of arginine (R) residues on histones (primarily H3R8 and H3R4) and other proteins. PRMT5 is overexpressed in many leukemias and lymphomas, and epigenetic changes driven by PRMT5 lead to repression of tumor suppressors and promote growth and survival of cancer cells. Recently it was shown that T cells are sensitive to R-methylation and PRMT5 promotes activation of memory T helper cells. Here we investigate: 1) mechanisms by which PRMT5 regulates T cell function; and 2) PRMT5 inhibition as a therapeutic strategy for aGVHD.Materials and Methods: Splenic T cells were isolated from lethally irradiated B6D2F1 mice that received either T cell depleted bone marrow (TCD-BM) or TCD-BM with C57/BL6 (B6) allogeneic splenocytes on day 21 post-transplant. In vitro activation of B6 T cells was achieved with CD3/CD28 Dynabeads or co-culture with allogeneic BM-derived dendritic cells. PRMT5 expression (RT-PCR, western blot) and function (H3R8me2s western blot) were evaluated. PRT220, a novel inhibitor of PRMT5, was used to evaluate PRMT5 inhibition on T cell function in vitro and in vivo. We assessed T cell proliferation (Cell Trace Violet, Ki67), apoptosis (Annexin V), cytokine secretion (ELISA, flow cytometry), cell cycle (PI incorporation), and cell signaling (western blot). Lethally irradiated F1 recipients received TCD-BM only (10x106 cells) or TCD-BM + B6 splenocytes (20 x 106). Recipients of allogeneic splenocytes were treated with PRT220 (2mg/kg) or vehicle by oral gavage once weekly starting day 7 post-transplant. Mice were monitored for survival and clinical aGVHD scores.Results: PRMT5 expression and function is upregulated following T cell activation. Inhibition of PRMT5 reduces T cell proliferation and IFN-g secretion. PRMT5 inhibition in CD3/CD28 stimulated T cells results in disruption of multiple histone epigenetic marks, cell-cycle progression (via G1 arrest) and perturbation of ERK-MAPK signaling cascades. Finally, administration of PRT220 resulted in significantly prolonging the survival of allo-transplanted recipient mice (median survival, PRT220 vs. vehicle, 36.5 vs. 26 days, p=0.01). PRT220-treated recipients also exhibited significant lower aGVHD clinical (p<0.05), pathological scores (p<0.05) and lower serum TNF-a (p<0.05) and IFN-g (p<0.05) than vehicle-treated recipients.Conclusions: PRMT5 expression and function are upregulated in activated T cells. Inhibition of PRMT5 function using a novel and specific small-molecule inhibitor, PRT220, down-regulates T cells proliferative and effector response, induces cell-cycle arrest and perturbs signaling pathways. PRT220 shows potent biological activity in vivo by reducing aGVHD clinical severity and significantly prolonging survival in mouse models of aGVHD. Therefore, PRMT5 is a novel and druggable target for aGVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of Casiopeina II-gly secondary targets through a systems pharmacology approach

Casiopeinas are a group of copper-based compounds designed to be used as less toxic, more efficient chemotherapeutic agents. In this study, we analyzed the in vitro effects of Casiopeina II-gly on the expression of canonical biological pathways. Using microarray data from HeLa cell lines treated with Casiopeina II-gly, we identified biological pathways that are perturbed after treatment. We present a novel approach integrating pathway analysis and network theory: The Pathway Crosstalk Network. We constructed a network with deregulated pathways, featuring links between those pathways that crosstalk with each other. We identified modules grouping deregulated pathways that are functionally related. Through this approach, we were able to identify three features of Casiopeina treatment: (a) Perturbation of signaling pathways, related to induction of apoptosis; (b) perturbation of metabolic pathways, and (c) activation of immune responses. These findings can be useful to drive new experimental exploration on their role in adverse effects and efficacy of Casiopeinas.

Homoharringtonine interacts synergistically with bortezomib in NHL cells through MCL-1 and NOXA-dependent mechanisms

BackgroundInteractions between the protein synthesis inhibitor homoharringtonine (HHT) and the proteasome inhibitor bortezomib were investigated in DLBCL and mantle cell lymphoma cells (MCL).MethodsVarious DLBCL and MCL cells were exposed to HHT and bortezomib alone or together after which apoptosis and signaling pathway perturbations were monitored by flow cytometry and Western blot analysis. Xenograft mouse models were used to assess tumor growth and animal survival.ResultsHHT and bortezomib co-administration synergistically induced apoptosis in GC-, ABC- and double-hit DLBCL cells. Similar interactions were observed in MCL cells and in primary lymphoma cells. HHT/bortezomib co-administration diminished binding of MCL-1 to both BAK and NOXA. Knock-down of NOXA significantly diminished lethality whereas MCL-1 knock-down or ectopic NOXA expression increased cell death. Notably, HHT/bortezomib lethality was dramatically reduced in BAK knockout or knockdown cells. Finally, HHT/bortezomib co-administration significantly improved survival compared to single agents in GC- and ABC- xenograft models while exhibiting little toxicity.ConclusionsThese findings indicate that HHT and bortezomib cooperate to kill DLBCL and MCL cells through a process involving MCL-1 down-regulation, NOXA up-regulation, and BAK activation. They also suggest that a strategy combining HHT with bortezomib warrants attention in DLBCL and MCL.

Single-Cell Transcriptomics Characterizes Cell Types in the Subventricular Zone and Uncovers Molecular Defects Impairing Adult Neurogenesis

Neural stem cells (NSCs) contribute to plasticity and repair of the adult brain. Niches harboring NSCs regulate stem cell self-renewal and differentiation. We used comprehensive and untargeted single-cell RNA profiling to generate a molecular cell atlas of the largest germinal region of the adult mouse brain, the subventricular zone (SVZ). We characterized >20 neural and non-neural cell types and gained insights into the dynamics of neurogenesis by predicting future cell states based on computational analysis of RNA kinetics. Furthermore, we applied our single-cell approach to document decreased numbers of NSCs, reduced proliferation activity of progenitors, and perturbations in Wnt and BMP signaling pathways in mice lacking LRP2, an endocytic receptor required for SVZ maintenance. Our data provide a valuable resource to study adult neurogenesis and a proof of principle for the power of single-cell RNA sequencing to elucidate neural cell-type-specific alterations in loss-of-function models.

Cre/lox-controlled spatiotemporal perturbation of FGF signaling in zebrafish.

Spatiotemporal perturbation of signaling pathways in vivo remains challenging and requires precise transgenic control of signaling effectors. Fibroblast growth factor (FGF) signaling guides multiple developmental processes, including body axis formation and cell fate patterning. In zebrafish, mutants and chemical perturbations affecting FGF signaling have uncovered key developmental processes; however, these approaches cause embryo-wide perturbations, rendering assessment of cell-autonomous vs. non-autonomous requirements for FGF signaling in individual processes difficult. Here, we created the novel transgenic line fgfr1-dn-cargo, encoding dominant-negative Fgfr1a with fluorescent tag under combined Cre/lox and heatshock control to perturb FGF signaling spatiotemporally. Validating efficient perturbation of FGF signaling by fgfr1-dn-cargo primed with ubiquitous CreERT2, we established that primed, heatshock-induced fgfr1-dn-cargo behaves similarly to pulsed treatment with the FGFR inhibitor SU5402. Priming fgfr1-dn-cargo with CreERT2 in the lateral plate mesoderm triggered selective cardiac and pectoral fin phenotypes without drastic impact on overall embryo patterning. Harnessing lateral plate mesoderm-specific FGF inhibition, we recapitulated the cell-autonomous and temporal requirement for FGF signaling in pectoral fin outgrowth, as previously inferred from pan-embryonic FGF inhibition. As a paradigm for rapid Cre/lox-mediated signaling perturbations, our results establish fgfr1-dn-cargo as a genetic tool to define the spatiotemporal requirements for FGF signaling in zebrafish. Developmental Dynamics 247:1146-1159, 2018. © 2018 Wiley Periodicals, Inc.

Emerging biomarkers and targeted therapies in urothelial carcinoma.

The use of immunotherapy has revolutionized the management of patients with locally advanced, unresectable, and metastatic urothelial carcinoma (UC); however, platinum-based chemotherapy remains a therapeutic cornerstone both in localized muscle-invasive and advanced UC. There is still no predictive molecular biomarker with clinical utility to help guide treatment and select patients most likely to derive benefit from a particular therapeutic modality or regimen. However, recent research has further characterized the inherent biology and immunology landscapes of UC leading to the development of potential biomarkers and therapeutic targets that could be used upon further validation. Emerging interrogation of The Cancer Genome Atlas (TCGA) and other molecular profiling datasets has led to the identification of distinct molecular subtypes with diverse clinical behaviors with potential sensitivity to various therapies. It has also led to the discovery of multiple frequently altered genes and proteins that could lead to perturbation of intracellular signaling pathways and of the dynamic interactions between tumor cells, their "microenvironment", and the host "macro-environment". The advent of molecular profiling and deeper next-generation sequencing has the potential to change biomarker and "real time" drug sensitivity assessment, introducing and testing the premise of "precision oncology" and personalized medicine. Within this review, we summarize emerging biomarkers that may predict response to cisplatin-based chemotherapy, immunotherapy, emerging targeted therapies, and promising combination strategies. We also highlight a few examples of 'precision medicine' trials aiming to improve outcomes in UC. Since our review is not exhaustive we strongly recommend the readers to follow the continuously changing literature in the very interesting and dynamic field of UC.

Effects of nicotine on the metabolism and gene expression profile of Sprague‑Dawley rat primary osteoblasts.

Smoking is a risk factor associated with bone and oral diseases, particularly periodontitis. Nicotine, the major toxic component of tobacco, is able to affect the quality and quantity of bone. Osteoblasts serve an important role in bone formation. Thus far, the effects of nicotine on metabolism-associated gene and protein expression in osteoblasts have been controversial and the mechanisms remain unclear. The present study assessed alterations in osteogenic activity by performing a Cell Counting kit-8 assay to investigate proliferation, Annexin V-fluorescein isothiocyanate/propidium iodide staining to investigate apoptosis, alizarin red staining to investigate the formation of mineralized nodules, reverse transcription-quantitative polymerase chain reaction and western blotting to investigate the mRNA and protein levels of collagen I, alkaline phosphatase, bone osteocalcin, bone sialoprotein and osteopontin; and mRNA microarray expression analysis, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis to investigate the whole genome expression profile of Sprague-Dawley (SD) rat primary osteoblasts following treatment with different concentrations of nicotine. The results demonstrated that nicotine inhibited proliferation, promoted early apoptosis and inhibited mineralized nodule formation in a dose-dependent manner by regulating alkaline phosphatase activity and the expression of osteoblast metabolism-associated genes and proteins. According to microarray analysis, several genes associated with bone metabolism and genes in the Hedgehog and Notch signaling pathways were downregulated significantly in nicotine-treated osteoblasts. The results of the present study indicated that nicotine may serve an inhibitory, dose-dependent role in SD rat primary osteoblasts that may be caused by the perturbation of genes and signaling pathways associated with bone formation. These results may provide a theoretical basis for future research regarding bone metabolism and targeted treatment of oral diseases associated with smoking.

Transcriptome signatures of p,p´-DDE-induced liver damage in Mus spretus mice

The use of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane) in some countries, although regulated, is contributing to an increased worldwide risk of exposure to this organochlorine pesticide or its derivative p,p’-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene]. Many studies have associated p,p’-DDE exposure to type 2 diabetes, obesity and alterations of the reproductive system, but their molecular mechanisms of toxicity remain poorly understood. We have addressed this issue by using commercial microarrays based on probes for the entire Mus musculus genome to determine the hepatic transcriptional signatures of p,p’-DDE in the phylogenetically close mouse species Mus spretus. High-stringency hybridization conditions and analysis assured reliable results, which were also verified, in part, by qRT-PCR, immunoblotting and/or enzymatic activity. Our data linked 198 deregulated genes to mitochondrial dysfunction and perturbations of central signaling pathways (kinases, lipids, and retinoic acid) leading to enhanced lipogenesis and aerobic glycolysis, inflammation, cell proliferation and testosterone catabolism and excretion. Alterations of transcript levels of genes encoding enzymes involved in testosterone catabolism and excretion would explain the relationships established between p,p´-DDE exposure and reproductive disorders, obesity and diabetes. Further studies will help to fully understand the molecular basis of p,p´-DDE molecular toxicity in liver and reproductive organs, to identify effective exposure biomarkers and perhaps to design efficient p,p’-DDE exposure counteractive strategies.

Chronic stress causes protein kinase C epsilon-aldehyde dehydrogenase 2 signaling pathway perturbation in the rat hippocampus and prefrontal cortex, but not in the myocardium.

Chronic stress is strongly associated with the occurrence and development of depression and cardiovascular disease. Stress can induce altered mitochondrial function and activation of apoptosis in the cardio-cerebral system. However, it is unknown whether the protein kinase C ε (PKCε)-aldehyde dehydrogenase 2 (ALDH2) pathway is altered under chronic stress, and this study sought to address this question. A rat model of depression was established using a chronic unpredictable mild stress (CUMS) protocol. After experiencing CUMS for 4 weeks, the sucrose preference test and the forced swim test verified depressive-like behaviors. Enzyme linked immunosorbent assays showed that ALDH2 activity was decreased in the rat hippocampus and prefrontal cortex, but was not altered in the myocardium. Western blot assays demonstrated reduced levels of ALDH2 and PKCε, but increased levels of 4-hydroxy-2-nonenal (4HNE) adducts. Caspase-3 expression did not obviously alter, but active forms of caspase-3 were increased in the hippocampus and prefrontal cortex. In the myocardium, expression of ALDH2, PKCε and 4HNE adducts did not remarkably alter; while caspase-3 expression was reduced and the active forms of caspase-3 were upregulated. Pearson's correlation test demonstrated that expression of 4HNE adducts was positively correlated with levels of the active forms of caspase-3 in the hippocampus and prefrontal cortex, but not in the myocardium. In conclusion, chronic stress can damage the PKCε-ALDH2 signaling pathway in the hippocampus and prefrontal cortex, but not in the myocardium. Moreover, 4HNE is associated with active forms of caspase-3 in the hippocampus and prefrontal cortex.

Cellular effects of a turmeric root and rosemary leaf extract on canine neoplastic cell lines

BackgroundThe use of nutraceuticals is gaining in popularity in human and canine oncology with a relatively limited understanding of the effects in the vastly different tumor types seen in canine oncology. We have previously shown that turmeric root (TE) and rosemary leaf (RE) extracts can work synergistically to reduce neoplastic cell growth, but the mechanisms are poorly understood and require further elucidation.ResultsThree different canine cell lines (C2 mastocytoma, and CMT-12 mammary carcinoma, D17 osteosarcoma) were treated with 6.3 μg mL−1 extract individually, or 3.1 μg mL−1 of each extract in combination based on studies showing synergy of these two extracts. Apoptosis, antioxidant effects, cellular accumulation of curcumin, and perturbation of signaling pathways were assessed. The TE + RE combination treatment resulted in Caspase 3/7 activation and apoptosis in all cell lines, beyond the effects of TE alone with the CMT-12 cell line being most susceptible. Both extracts had antioxidant effects with RE reducing reactive oxygen species (ROS) by 40–50% and TE reducing ROS by 80–90%. In addition RE treatment enhanced the c-jun N-terminal kinase (JNK) activity in the C2 cell line and TE + RE exposure increased activated JNK by 4–5 times in the CMT-12 cell line. Upon further examination, it was found that RE treatment caused a significant increase in the cellular accumulation of curcumin by approximately 30% in the C2 and D17 cell lines, and by 4.8-fold in the CMT-12 cell line. This increase in intracellular curcumin levels may play a role in the synergy exhibited when using TE and RE in combination.ConclusionsThe use of RE in combination with TE induces a synergistic response to induce apoptosis which is better than either extract alone. This appears to be related to a variable increased TE uptake in cells and activation of pathways involved in the apoptotic response.

Adipogenic Effects and Gene Expression Profiling of Firemaster® 550 Components in Human Primary Preadipocytes.

Background:Exposure to flame retardants has been associated with negative health outcomes including metabolic effects. As polybrominated diphenyl ether flame retardants were pulled from commerce, human exposure to new flame retardants such as Firemaster® 550 (FM550) has increased. Although previous studies in murine systems have shown that FM550 and its main components increase adipogenesis, the effects of FM550 in human models have not been elucidated.Objectives:The objectives of this study were to determine if FM550 and its components are active in human preadipocytes, and to further investigate their mode of action.Methods:Human primary preadipocytes were differentiated in the presence of FM550 and its components. Differentiation was assessed by lipid accumulation and expression of peroxisome proliferator-activated receptor (PPARG), fatty acid binding protein (FABP) 4 and lipoprotein lipase (LPL). mRNA was collected for Poly (A) RNA sequencing and was used to identify differentially expressed genes (DEGs). Functional analysis of DEGs was undertaken in Ingenuity Pathway Analysis.Results:FM550 triphenyl phosphate (TPP) and isopropylated triphenyl phosphates (IPTP), increased adipogenesis in human primary preadipocytes as assessed by lipid accumulation and mRNA expression of regulators of adipogenesis such as , CCAAT enhancer binding protein (C/EBP) and sterol regulatory element binding protein (SREBP) 1 as well as the adipogenic markers FABP4 LPL and perilipin. Poly (A) RNA sequencing analysis revealed potential modes of action including liver X receptor/retinoid X receptor (LXR/RXR) activation, thyroid receptor (TR)/RXR, protein kinase A, and nuclear receptor subfamily 1 group H members activation.Conclusions:We found that FM550, and two of its components, induced adipogenesis in human primary preadipocytes. Further, using global gene expression analysis we showed that both TPP and IPTP likely exert their effects through PPARG to induce adipogenesis. In addition, IPTP perturbed signaling pathways that were not affected by TPP. https://doi.org/10.1289/EHP1318

Novel pan PI3K inhibitor-induced apoptosis in APL cells correlates with suppression of telomerase: An emerging mechanism of action of BKM120

The intertwining between cancer pathogenesis and perturbation of multitude signaling pathways ushered the cancer therapeutic approaches into an unbounded route of targeted therapies. For the nonce and among the plethora of promising inhibitors, intense interest has focused on small molecules targeting different component of PI3K axis. Intrigued by the constant activation of PI3K in leukemia, this study aimed to investigate the effects of BKM120, as the excelled member of pan PI3K inhibitors, in a panel of hematologic malignant cell lines. The resulting data showed that BKM120 exerted a concentration-dependent growth suppressive effect; however, IC50 values varied among the tested cells. Our results outlined that the blockage of PI3K in NB4, as the most sensitive cell line, resulted in a caspase-3-dependent apoptosis probably through NFκB-mediated suppression of c-Myc and hTERT. As far we are aware, to date, there have been no reports of BKM120 effect on enzymatic repression of telomerase, and this study represents for the first time that the anti-proliferative effect of the inhibitor on NB4 is mediated by down-regulation of telomerase; shedding new light on the novel mechanism of action of BKM120.

Anti-angiogenic properties of artemisinin derivatives (Review).

Angiogenesis, the process involving the development of new blood vessels from existing capillaries, is critical for growth and wound healing. However, pathological angiogenesis contributes to the pathogeneses of numerous diseases, including cancer, rheumatoid arthritis, diabetic retinopathy and macular degeneration. Hence, the inhibition of angiogenesis is an effective therapeutic approach for these diseases. Apart from its anti-malarial properties, artemisinin and its derivatives also exhibit potent anti-angiogenic properties. The molecular mechanisms underlying their inhibitory effects on angiogenesis have been studied by several groups. These investigations have revealed that artemisinins inhibit angiogenesis via the perturbations of cellular signaling pathways involved in the regulation of angiogenesis. Along with a brief introduction to artemisinin derivatives, this review provides a detailed summary of the effects of artemisinins on the mitogen-activated protein kinase(MAPK) pathway, the nuclear factor-κB(NF-κB) pathway and the phosphatidylinositide3-kinase(PI3K)/protein kinaseB(Akt)/mammalian target of rapamycin(mTOR) pathway. Due to the multiplicity of their actions on relevant signaling pathways, artemisinins are promising candidates with potential for use as anti-angiogenic agents for the treatment of related diseases or disorders.

Revealing common disease mechanisms shared by tumors of different tissues of origin through semantic representation of genomic alterations and topic modeling

BackgroundCancer is a complex disease driven by somatic genomic alterations (SGAs) that perturb signaling pathways and consequently cellular function. Identifying patterns of pathway perturbations would provide insights into common disease mechanisms shared among tumors, which is important for guiding treatment and predicting outcome. However, identifying perturbed pathways is challenging, because different tumors can have the same perturbed pathways that are perturbed by different SGAs. Here, we designed novel semantic representations that capture the functional similarity of distinct SGAs perturbing a common pathway in different tumors. Combining this representation with topic modeling would allow us to identify patterns in altered signaling pathways.ResultsWe represented each gene with a vector of words describing its function, and we represented the SGAs of a tumor as a text document by pooling the words representing individual SGAs. We applied the nested hierarchical Dirichlet process (nHDP) model to a collection of tumors of 5 cancer types from TCGA. We identified topics (consisting of co-occurring words) representing the common functional themes of different SGAs. Tumors were clustered based on their topic associations, such that each cluster consists of tumors sharing common functional themes. The resulting clusters contained mixtures of cancer types, which indicates that different cancer types can share disease mechanisms. Survival analysis based on the clusters revealed significant differences in survival among the tumors of the same cancer type that were assigned to different clusters.ConclusionsThe results indicate that applying topic modeling to semantic representations of tumors identifies patterns in the combinations of altered functional pathways in cancer.

Cancerous perturbations within the ERK, PI3K/Akt, and Wnt/β-catenin signaling network constitutively activate inter-pathway positive feedback loops.

Perturbations in molecular signaling pathways are a result of genetic or epigenetic alterations, which may lead to malignant transformation of cells. Despite cellular robustness, specific genetic or epigenetic changes of any gene can trigger a cascade of failures, which result in the malfunctioning of cell signaling pathways and lead to cancer phenotypes. The extent of cellular robustness has a link with the architecture of the network such as feedback and feedforward loops. Perturbation in components within feedback loops causes a transition from a regulated to a persistently activated state and results in uncontrolled cell growth. This work represents the mathematical and quantitative modeling of ERK, PI3K/Akt, and Wnt/β-catenin signaling crosstalk to show the dynamics of signaling responses during genetic and epigenetic changes in cancer. ERK, PI3K/Akt, and Wnt/β-catenin signaling crosstalk networks include both intra and inter-pathway feedback loops which function in a controlled fashion in a healthy cell. Our results show that cancerous perturbations of components such as EGFR, Ras, B-Raf, PTEN, and components of the destruction complex cause extreme fragility in the network and constitutively activate inter-pathway positive feedback loops. We observed that the aberrant signaling response due to the failure of specific network components is transmitted throughout the network via crosstalk, generating an additive effect on cancer growth and proliferation.

E2A-PBX1 Remodels Oncogenic Signaling Networks in B-cell Precursor Acute Lymphoid Leukemia.

There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%-7% of pediatric and 50% of pre-B-cell receptor (preBCR+) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR+/E2A-PBX1+ leukemias in vitro and in vivo Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937-49. ©2016 AACR.

Abstract IA27: Perturbation of host cellular regulatory networks by human papillomaviruses

Abstract Human papillomaviruses (HPVs) are small viruses with ~8,000 bp double-stranded, circular genomes. There are over 200 HPV types and they all infect epithelial cells. While most HPV infections are subclinical others cause overt lesions. Most HPV-associated lesions are benign but some “high-risk” HPVs cause lesions that have a propensity for malignant progression and approximately 10% of all human cancers worldwide are caused by high-risk HPV infections. HPV-associated cancers generally represent non-productive infections, i.e. viral proteins are synthesized but no infectious virus is produced. HPV-associated cancers retain expression of two viral genes, E6 and E7, and continued expression of these two viral genes is necessary for cancer cell proliferation and survival. High-risk HPV E6 and E7 encode small, non-enzymatic proteins of ~150 and ~100 amino acids, respectively. These viral proteins exert their oncogenic activities by associating with and functionally reprogramming critical cellular regulatory nodes; indeed, they have been reported to affect the majority of the “Hallmarks of Cancer” as defined by Hanahan and Weinberg (Cell 144:646). In addition to targeting a number of well-known tumor suppressor pathways including TP53 and RB1, E6 and E7 also, directly and indirectly, affect many other cellular signaling pathways. As a result, HPV E6/E7 expressing cells develop unexpected vulnerabilities to perturbation of signaling pathways that are readily tolerated in normal cells. Citation Format: Karl Münger. Perturbation of host cellular regulatory networks by human papillomaviruses. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA27.

Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.

Targeting metabolism in cellular senescence, a role for intervention

Cellular senescence has gained much attention as a contributor to aging and susceptibility to disease. Senescent cells undergo a stable cell cycle arrest and produce pro-inflammatory cytokines. However, an additional feature of the senescence phenotype is an altered metabolic state. Despite maintaining a non-dividing state, senescent cells display a high metabolic rate. Metabolic changes characteristic of replicative senescence include altered mitochondrial function and perturbations in growth signaling pathways, such as the mTORC1-signaling pathway. Recent evidence has raised the possibility that these metabolic changes may be essential for the induction and maintenance of the senescent state. Interventions such as rapamycin treatment and methionine restriction impact key aspects of metabolism and delay cellular senescence to extend cellular lifespan. Here, we review the metabolic changes and potential metabolic regulators of the senescence program. In addition, we will discuss how lifespan-extending regimens prevent metabolic stress that accompanies and potentially regulates the senescence program.