Complete miRNA-15/16 loss in mice promotes hematopoietic progenitor expansion and a myeloid-biased hyperproliferative state.

Dysregulated apoptosis and proliferation are fundamental properties of cancer, and microRNAs (miRNA) are critical regulators of these processes. Loss of miR-15a/16-1 at chromosome 13q14 is the most common genomic aberration in chronic lymphocytic leukemia (CLL). Correspondingly, the deletion of either murine miR-15a/16-1 or miR-15b/16-2 locus in mice is linked to B cell lymphoproliferative malignancies. However, unexpectedly, when both miR-15/16 clusters are eliminated, most double knockout (DKO) mice develop acute myeloid leukemia (AML). Moreover, in patients with CLL, significantly reduced expression of miR-15a, miR-15b, and miR-16 associates with progression of myelodysplastic syndrome to AML, as well as blast crisis in chronic myeloid leukemia. Thus, the miR-15/16 clusters have a biological relevance for myeloid neoplasms. Here, we demonstrate that the myeloproliferative phenotype in DKO mice correlates with an increase of hematopoietic stem and progenitor cells (HSPC) early in life. Using single-cell transcriptomic analyses, we presented the molecular underpinning of increased myeloid output in the HSPC of DKO mice with gene signatures suggestive of dysregulated hematopoiesis, metabolic activities, and cell cycle stages. Functionally, we found that multipotent progenitors (MPP) of DKO mice have increased self-renewing capacities and give rise to significantly more progeny in the granulocytic compartment. Moreover, a unique transcriptomic signature of DKO MPP correlates with poor outcome in patients with AML. Together, these data point to a unique regulatory role for miR-15/16 during the early stages of hematopoiesis and to a potentially useful biomarker for the pathogenesis of myeloid neoplasms.

Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis.

BACKGROUND: Patients with endometriosis suffer with chronic pelvic pain and infertility, and from the lack of pharmacologic therapies that consistently halt disease progression. Differences in the endometrium of patients with endometriosis vs. unaffected controls are well-documented. Specifically, shed endometrial tissues (delivered to the pelvic cavity via retrograde menstruation) reveal that a subset of stromal cells exhibiting pro-inflammatory, pro-fibrotic, and pro-senescence-like phenotypes is enhanced in endometriosis patients compared to controls. Additionally, cultured biopsy-derived endometrial stromal cells from endometriosis patients exhibit impaired decidualization, a defined differentiation process required for human embryo implantation and pregnancy. Quercetin, a senolytic agent, shows therapeutic potential for pulmonary fibrosis, a disorder attributed to senescent pulmonary fibroblasts. In rodent models of endometriosis, quercetin shows promise, and quercetin improves decidualization in vitro. However, the exact mechanisms are not completely understood. Therefore, we investigated the effects of quercetin on menstrual effluent-derived endometrial stromal cells from endometriosis patients and unaffected controls to define the signaling pathways underlying quercetin's effects on endometrial stromal cells. METHODS: Menstrual effluent-derived endometrial stromal cells were collected and cultured from unaffected controls and endometriosis patients and then, low passage cells were treated with quercetin (25 µM) under basal or standard decidualization conditions. Decidualization responses were analyzed by measuring the production of IGFBP1 and PRL. Also, the effects of quercetin on intracellular cAMP levels and cellular oxidative stress responses were measured. Phosphokinase arrays, western blotting, and flow cytometry methods were performed to define the effects of quercetin on various signaling pathways and the potential mechanistic roles of quercetin. RESULTS: Quercetin significantly promotes decidualization of control- and endometriosis-endometrial stromal cells. Quercetin substantially reduces the phosphorylation of multiple signaling molecules in the AKT and ERK1/2 pathways, while enhancing the phosphorylation of p53 and total p53 levels. Furthermore, p53 inhibition blocks decidualization while p53 activation promotes decidualization. Finally, we provide evidence that quercetin increases apoptosis of endometrial stromal cells with a senescent-like phenotype. CONCLUSIONS: These data provide insight into the mechanisms of action of quercetin on endometrial stromal cells and warrant future clinical trials to test quercetin and other senolytics for treating endometriosis.

Inherited cancer predisposing mutations in patients with therapy-related myeloid neoplasms.

Some patients with therapy-related myeloid neoplasms (t-MN) may have unsuspected inherited cancer predisposition syndrome (CPS). We propose a set of clinical criteria to identify t-MN patients with high risk of CPS (HR-CPS). Among 225 t-MN patients with an antecedent non-myeloid malignancy, our clinical criteria identified 52 (23%) HR-CPS patients. Germline whole-exome sequencing identified pathogenic or likely pathogenic variants in 10 of 27 HR-CPS patients compared to 0 of 9 low-risk CPS patients (37% vs. 0%, p = 0.04). These simple clinical criteria identify t-MN patients most likely to benefit from genetic testing for inherited CPS.

Single-cell analysis of menstrual endometrial tissues defines phenotypes associated with endometriosis.

BACKGROUND: Endometriosis is a common, complex disorder which is underrecognized and subject to prolonged delays in diagnosis. It is accompanied by significant changes in the eutopic endometrial lining. METHODS: We have undertaken the first single-cell RNA-sequencing (scRNA-Seq) comparison of endometrial tissues in freshly collected menstrual effluent (ME) from 33 subjects, including confirmed endometriosis patients (cases) and controls as well as symptomatic subjects (who have chronic symptoms suggestive of endometriosis but have not been diagnosed). RESULTS: We identify a unique subcluster of proliferating uterine natural killer (uNK) cells in ME-tissues from controls that is almost absent from endometriosis cases, along with a striking reduction of total uNK cells in the ME of cases (p < 10-16). In addition, an IGFBP1+ decidualized subset of endometrial stromal cells are abundant in the shed endometrium of controls when compared to cases (p < 10-16) confirming findings of compromised decidualization of cultured stromal cells from cases. By contrast, endometrial stromal cells from cases are enriched in cells expressing pro-inflammatory and senescent phenotypes. An enrichment of B cells in the cases (p = 5.8 × 10-6) raises the possibility that some may have chronic endometritis, a disorder which predisposes to endometriosis. CONCLUSIONS: We propose that characterization of endometrial tissues in ME will provide an effective screening tool for identifying endometriosis in patients with chronic symptoms suggestive of this disorder. This constitutes a major advance, since delayed diagnosis for many years is a major clinical problem in the evaluation of these patients. Comprehensive analysis of ME is expected to lead to new diagnostic and therapeutic approaches to endometriosis and other associated reproductive disorders such as female infertility.

Maternal oxytocin administration modulates gene expression in the brains of perinatal mice.

OBJECTIVES: Oxytocin (OXT) is widely used to facilitate labor. However, little is known about the effects of perinatal OXT exposure on the developing brain. We investigated the effects of maternal OXT administration on gene expression in perinatal mouse brains. METHODS: Pregnant C57BL/6 mice were treated with saline or OXT at term (n=6-7/group). Dams and pups were euthanized on gestational day (GD) 18.5 after delivery by C-section. Another set of dams was treated with saline or OXT (n=6-7/group) and allowed to deliver naturally; pups were euthanized on postnatal day 9 (PND9). Perinatal/neonatal brain gene expression was determined using Illumina BeadChip Arrays and real time quantitative PCR. Differential gene expression analyses were performed. In addition, the effect of OXT on neurite outgrowth was assessed using PC12 cells. RESULTS: Distinct and sex-specific gene expression patterns were identified in offspring brains following maternal OXT administration at term. The microarray data showed that female GD18.5 brains exhibited more differential changes in gene expression compared to male GD18.5 brains. Specifically, Cnot4 and Frmd4a were significantly reduced by OXT exposure in male and female GD18.5 brains, whereas Mtap1b, Srsf11, and Syn2 were significantly reduced only in female GD18.5 brains. No significant microarray differences were observed in PND9 brains. By quantitative PCR, OXT exposure reduced Oxtr expression in female and male brains on GD18.5 and PND9, respectively. PC12 cell differentiation assays revealed that OXT induced neurite outgrowth. CONCLUSIONS: Prenatal OXT exposure induces sex-specific differential regulation of several nervous system-related genes and pathways with important neural functions in perinatal brains.

Phenotypic and functional characteristics of murine CD11c+ B cells which is suppressed by metformin.

Since the description of age-associated or autoimmune-associated B cells (ABCs), there has been a growing interest in the role of these cells in autoimmunity. ABCs are differently defined depending on the research group and are heterogenous subsets. Here, we sought to characterize ABCs in Sle1/2/3 triple congenic (TC) mice, which is a well accepted mouse model of lupus. Compared to follicular (FO) B cells, ABCs have many distinct functional properties, including antigen presentation. They express key costimulatory molecules for T cell activation and a distinct profile of cytokines. Moreover, they exhibit an increased capacity for antigen uptake. ABCs were also compared with germinal center (GC) B cells, which are antigen activated B cell population. There are several phenotypic similarities between ABCs and GC B cells, but GC B cells do not produce proinflammatory cytokines or take up antigen. While T cell proliferation and activation is induced by both FO B and ABCs in an antigen-dependent manner, ABCs induce stronger T cell receptor signaling in naïve CD4+ T cells and preferentially induce differentiation of T follicular helper (Tfh) cells. We found that ABCs exhibit a distinct transcriptomic profile which is focused on metabolism, cytokine signaling and antigen uptake and processing. ABCs exhibit an increase in both glycolysis and oxidative phosphorylation compared to FO B cells. Treatment of ABCs with metformin suppresses antigen presentation by decreasing antigen uptake, resulting in decreased Tfh differentiation. Taken together, these findings define a fundamental connection between metabolism and function within ABCs.

Antigen-presenting aged neutrophils induce CD4+ T cells to exacerbate inflammation in sepsis.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow-derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture-induced sepsis but not in CIRP-/- mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non-APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ-mediated hyper-NETosis.

Molecular dynamics analysis of conserved hydrophobic and hydrophilic bond-interaction networks in ErbB family kinases.

The EGFR (epidermal growth factor receptor)/ErbB/HER (human EGFR) family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signalling pathways. To elucidate the atomistic mechanisms of dimerization-dependent activation in the ErbB family, we have performed molecular dynamics simulations of the intracellular kinase domains of three members of the ErbB family (those with known kinase activity), namely EGFR, ErbB2 (HER2) and ErbB4 (HER4), in different molecular contexts: monomer against dimer and wild-type against mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to correspondence of specific bond-interaction networks and collective dynamical modes. We find that in the active conformation of the ErbB kinases, key subdomain motions are co-ordinated through conserved hydrophilic interactions: activating bond-networks consisting of hydrogen bonds and salt bridges. The inactive conformations also demonstrate conserved bonding patterns (albeit less extensive) that sequester key residues and disrupt the activating bond network. Both conformational states have distinct hydrophobic advantages through context-specific hydrophobic interactions. We show that the functional (activating) asymmetric kinase dimer interface forces a corresponding change in the hydrophobic and hydrophilic interactions that characterize the inactivating bond network, resulting in motion of the αC-helix through allostery. Several of the clinically identified activating kinase mutations of EGFR act in a similar fashion to disrupt the inactivating bond network. The present molecular dynamics study reveals a fundamental difference in the sequence of events in EGFR activation compared with that described for the Src kinase Hck.

Predictive molecular biomarkers for determining neoadjuvant chemosensitivity in muscle invasive bladder cancer.

INTRODUCTION: Identifying neoadjuvant chemotherapy (NAC) response in patients with muscle invasive bladder cancer (MIBC) has had limited success based on clinicopathological features and molecular subtyping. Identification of chemotherapy responsive cohorts would facilitate delivery to those most likely to benefit. OBJECTIVE: Develop a molecular signature that can identify MIBC NAC responders (R) and non-responders (NR) using a cohort of known NAC response phenotypes, and better understand differences in molecular pathways and subtype classifications between NAC R and NR. MATERIALS AND METHODS: Presented are the messenger RNA (mRNA) and microRNA (miRNA) differential expression profiles from initial transurethral resection of bladder tumor (TURBT) specimens of a discovery cohort of MIBC patients consisting of 7 known NAC R and 11 NR, and a validation cohort consisting of 3 R and 5 NR. Pathological response at time of cystectomy after NAC was used to classify initial TURBT specimens as R (pT0) versus NR (≥pT2). RNA and miRNA from FFPE blocks were sequenced using RNAseq and qPCR, respectively. RESULTS: The discovery cohort had 2309 genes, while the validation cohort had 602 genes and 13 miRNA differentially expressed between R and NR. Gene set enrichment analysis identified mitochondrial gene expression, DNA replication initiation, DNA unwinding in the R discovery cohort and positive regulation of vascular associated smooth muscle cell proliferation in the NR discovery cohort. Canonical correlation (CC) analysis was applied to differentiate R versus NR. 3 CCs (CC13, CC16, and CC17) had an AUC >0.65 in the discovery and validation dataset. Gene ontology enrichment showed CC13 as nucleoside triphosphate metabolic process, CC16 as cell cycle and cellular response to DNA damage, CC17 as DNA packaging complex. All patients were classified using established molecular subtypes: Baylor, UNC, CIT, Lund, MD Anderson, TCGA, and Consensus Class. The MD Anderson p53-like subtype, CIT MC4 subtype and Consensus Class stroma rich subtype had the strongest correlation with a NR phenotype, while no subtype had a strong correlation with the R phenotype. CONCLUSIONS: Our results identify molecular signatures that can be used to differentiate MIBC NAC R versus NR, salient molecular pathway differences, and highlight the utility of molecular subtyping in relation to NAC response.

Proteomic and Single-Cell Transcriptomic Dissection of Human Plasmacytoid Dendritic Cell Response to Influenza Virus.

Plasmacytoid dendritic cells [pDCs] represent a rare innate immune subset uniquely endowed with the capacity to produce substantial amounts of type-I interferons. This function of pDCs is critical for effective antiviral defenses and has been implicated in autoimmunity. While IFN-I and select cytokines have been recognized as pDC secreted products, a comprehensive agnostic profiling of the pDC secretome in response to a physiologic stimulus has not been reported. We applied LC-MS/MS to catalogue the repertoire of proteins secreted by pDCs in the unperturbed condition and in response to challenge with influenza H1N1. We report the identification of a baseline pDC secretome, and the repertoire of virus-induced proteins including most type-I interferons, various cytokines, chemokines and granzyme B. Additionally, using single-cell RNA-seq [scRNA-seq], we perform multidimensional analyses of pDC transcriptional diversity immediately ex vivo and following stimulation. Our data evidence preexisting pDC heterogeneity, with subsequent highly specialized roles within the pDC population upon stimulation ranging from dedicated cytokine super-producers to cells with APC-like traits. Dynamic expression of transcription factors and surface markers characterize subclusters within activated pDCs. Integrating the proteomic and transcriptomic datasets confirms the pDC-subcluster origin of the proteins identified in the secretome. Our findings represent the most comprehensive molecular characterization of primary human pDCs at baseline, and in response to influenza virus, reported to date.

Investigations into SCAMP5, a candidate lupus risk gene expressed in plasmacytoid dendritic cells.

OBJECTIVE: We have investigated the molecular function of SCAMP5, a candidate risk gene for SLE exclusively expressed in plasmacytoid dendritic cells (pDCs) among peripheral leucocytes. METHODS: We tested the independence of the association in SCAMP5 with SLE by performing conditional analyses. We profiled the expression pattern of SCAMP5 among circulating leucocytes at the transcript and protein levels. Using lentiviral vectors, we localised the subcellular distribution of SCAMP5 alongside the interferon secretory pathway. We analysed pDCs for the expression of SCAMP5 and interferon production capacity by SCAMP5 genotype. Finally, we examined pDC-specific SCAMP5 isoforms by total RNAseq analysis and examined for genotype-associated quantitative differences therein. RESULTS: A conditional analysis revealed evidence of an independent genetic association of SCAMP5 with SLE. Among circulating leucocytes, SCAMP5 is uniquely expressed in pDCs at the transcript and protein levels, with main presence in the Golgi apparatus and minor presence at the cell periphery. In live cells, SCAMP5 displayed dynamic Golgi-cell surface trafficking and localised with the interferon secretory pathway. SCAMP5 did not differ in expression levels in pDCs between genotyped donors; however, a transient interferon secretory defect was noted in pDCs from donors carrying the risk genotype. CONCLUSIONS: SCAMP5 constitutes a novel SLE risk gene on the basis of genomic data and expression in a cell type widely implicated in SLE pathogenesis. While we could not find evidence of quantitative expression differences in SCAMP5 between genotyped donors, SCAMP5 remains an attractive gene to explore given its highly restricted expression pattern and colocalisation with interferon secretion.

Prognostic Molecular Signatures for Metastatic Potential in Clinically Low-Risk Stage I and II Clear Cell Renal Cell Carcinomas.

Introduction: For patients with localized node-negative (Stage I and II) clear cell renal cell carcinomas (ccRCC), current clinicopathological staging has limited predictive capability because of their low risk. Analyzing molecular signatures at the time of nephrectomy can aid in understanding future metastatic potential. Objective: Develop a molecular signature that can stratify patients who have clinically low risk ccRCC, but have high risk genetic changes driving an aggressive metastatic phenotype. Patients, Materials, and Methods: Presented is the differential expression of mRNA and miRNA in 44 Stage I and Stage II patients, 21 who developed metastasis within 5 years of nephrectomy, compared to 23 patients who remained disease free for more than 5 years. Extracted RNA from nephrectomy specimens preserved in FFPE blocks was sequenced using RNAseq. MiRNA expression was performed using the TaqMan OpenArray qPCR protocol. Results: One hundred thirty one genes and 2 miRNA were differentially expressed between the two groups. Canonical correlation (CC) analysis was applied and four CCs (CC32, CC20, CC9, and CC7) have an AUC > 0.65 in our dataset with similar predictive power in the TCGA-KIRC dataset. Gene set enrichment showed CC9 as kidney development/adhesion, CC20 as oxidative phosphorylation pathway, CC32 as RNA binding/spindle and CC7 as immune response. In a multivariate Cox model, the four CCs were able to identify high/low risk groups for metastases in the TCGA-KIRC (p < 0.05) with odds ratios of CC32 = 5.7, CC20 = 4.4, CC9 = 3.6, and CC7 = 2.7. Conclusion: These results identify molecular signatures for more aggressive tumors in clinically low risk ccRCC patients who have a higher potential of metastasis than would be expected.

Menstrual Effluent Provides a Novel Diagnostic Window on the Pathogenesis of Endometriosis.

Endometriosis is a chronic inflammatory disorder characterized by the presence of endometrial-like tissue growing outside of the uterus. Although the cause is unknown, retrograde menstruation leads to deposition of endometrial cells into the peritoneal cavity. Lack of disease recognition and long diagnostic delays (6-10 years) lead to substantial personal, social and financial burdens, as well as delayed treatment. A non-invasive diagnostic for endometriosis is a major unmet clinical need. Here, we assessed whether differences in menstrual effluent-derived stromal fibroblast cells (ME-SFCs) from women with and without endometriosis provide the basis for a non-invasive diagnostic for endometriosis. In addition, we investigated whether treatment of control ME-SFCs with inflammatory cytokines (TNF and IL-1ß) could induce an endometriosis-like phenotype. ME-SFCs from laparoscopically diagnosed endometriosis patients exhibit reduced decidualization capacity, measured by IGFBP1 production after exposure to cAMP. A receiver operating characteristic (ROC) curve developed using decidualization data from controls and endometriosis subjects yielded an area under the curve of 0.92. In addition, a significant reduction in ALDH1A1 gene expression and increased podoplanin surface expression were also observed in endometriosis ME-SFCs when compared to control ME-SFCs. These endometriosis-like phenotypes can be reproduced in control ME-SFCs by exposure to inflammatory cytokines (TNF and IL-1ß) and are associated with increased cell migration. These results are consistent with the hypothesis that chronic intrauterine inflammation influences the development of endometriosis lesions following retrograde menstruation. In conclusion, the analysis of ME-SFCs can provide an accurate, rapid, and non-invasive diagnostic for endometriosis and insight into disease pathogenesis.

Correction: Identification of grade and origin specific cell populations in serous epithelial ovarian cancer by single cell RNA-seq.

[This corrects the article DOI: 10.1371/journal.pone.0206785.].

Identification of grade and origin specific cell populations in serous epithelial ovarian cancer by single cell RNA-seq.

Here we investigated different cell populations within ovarian cancer using single-cell RNA seq: fourteen samples from nine patients with differing grades (high grade, low grade and benign) as well as different origin sites (primary and metastatic tumor site, ovarian in origin and fallopian in origin). We were able to identify sixteen distinct cell populations with specific cells correlated to high grade tumors, low grade tumors, benign and one population unique to a patient with a breast cancer relapse. Furthermore the proportion of these populations changes from primary to metastatic in a shift from mainly epithelial cells to leukocytes with few cancer epithelial cells in the metastases. Differential gene expression shows myeloid lineage cells are the primary cell group expressing soluble factors in primary samples while fibroblasts do so in metastatic samples. The leukocytes that were captured did not seem to be suppressed through known pro-tumor cytokines from any of the cell populations. Single cell RNA-seq is necessary to de-tangle cellular heterogeneity for better understanding of ovarian cancer progression.

Analysis of Somatic Mutations in Cancer: Molecular Mechanisms of Activation in the ErbB Family of Receptor Tyrosine Kinases.

The ErbB/EGFR/HER family of kinases consists of four homologous receptor tyrosine kinases which are important regulatory elements in many cellular processes, including cell proliferation, differentiation, and migration. Somatic mutations in, or over-expression of, the ErbB family is found in many cancers and is correlated with a poor prognosis; particularly, clinically identified mutations found in non-small-cell lung cancer (NSCLC) of ErbB1 have been shown to increase its basal kinase activity and patients carrying these mutations respond remarkably to the small tyrosine kinase inhibitor gefitinib. Here, we analyze the potential effects of the currently catalogued clinically identified mutations in the ErbB family kinase domains on the molecular mechanisms of kinase activation. Recently, we identified conserved networks of hydrophilic and hydrophobic interactions characteristic to the active and inactive conformation, respectively. Here, we show that the clinically identified mutants influence the kinase activity in distinctive fashion by affecting the characteristic interaction networks.

A multiscale modeling approach to investigate molecular mechanisms of pseudokinase activation and drug resistance in the HER3/ErbB3 receptor tyrosine kinase signaling network.

Multiscale modeling provides a powerful and quantitative platform for investigating the complexity inherent in intracellular signaling pathways and rationalizing the effects of molecular perturbations on downstream signaling events and ultimately, on the cell phenotype. Here we describe the application of a multiscale modeling scheme to the HER3/ErbB3 receptor tyrosine kinase (RTK) signaling network, which regulates critical cellular processes including proliferation, migration and differentiation. The HER3 kinase is a topic of current interest and investigation, as it has been implicated in mechanisms of resistance to tyrosine kinase inhibition (TKI) of EGFR and HER2 in the treatment of many human malignancies. Moreover, the commonly regarded status of HER3 as a catalytically inactive 'pseudokinase' has recently been challenged by our previous study, which demonstrated robust residual kinase activity for HER3. Through our multiscale model, we investigate the most significant molecular interactions that contribute to potential mechanisms of HER3 activity and the physiological relevance of this activity to mechanisms of drug resistance in an ErbB-driven tumor cell in silico. The results of our molecular-scale simulations support the characterization of HER3 as a weakly active kinase that, in contrast to its fully-active ErbB family members, depends upon a unique hydrophobic interface to coordinate the alignment of specific catalytic residues required for its activity. Translating our molecular simulation results of the uniquely active behavior of the HER3 kinase into a physiologically relevant environment, our HER3 signaling model demonstrates that even a weak level of HER3 activity may be sufficient to induce AKT signaling and TKI resistance in the context of an ErbB signaling-dependent tumor cell, and therefore therapeutic targeting of HER3 may represent a superior treatment strategy for specific ErbB-driven cancers.

Cancer Cell: Linking Oncogenic Signaling to Molecular Structure.

A multiscale strategy is presented for constructing models of intracellular signaling networks in which the oncogenic behavior of the network is encoded through alternate parameterization of the kinetic and structural properties of mutant oncoproteins. The approach uses molecular dynamics and docking simulations to quantify altered topologies of interactions as well as to provide the missing parameters for network models of both wild-type and oncogenic signaling. Through simulation of the resulting signaling networks, the global behavior of these networks may then be compared and functional roles may be assigned to the mutant oncoproteins. An example of this approach is presented in which structural alterations found in a mutant form of the epidermal growth factor receptor are represented as kinetic perturbations in a model of growth factor signaling. Based on network parameters estimated from molecular-level simulations, simulations at the network level show that small perturbations in molecular structure can lead to profoundly altered cellular phenotype.

Molecular systems biology of ErbB1 signaling: bridging the gap through multiscale modeling and high-performance computing.

The complexity in intracellular signaling mechanisms relevant for the conquest of many diseases resides at different levels of organization with scales ranging from the subatomic realm relevant to catalytic functions of enzymes to the mesoscopic realm relevant to the cooperative association of molecular assemblies and membrane processes. Consequently, the challenge of representing and quantifying functional or dysfunctional modules within the networks remains due to the current limitations in our understanding of mesoscopic biology, i.e., how the components assemble into functional molecular ensembles. A multiscale approach is necessary to treat a hierarchy of interactions ranging from molecular (nm, ns) to signaling (microm, ms) length and time scales, which necessitates the development and application of specialized modeling tools. Complementary to multiscale experimentation (encompassing structural biology, mechanistic enzymology, cell biology, and single molecule studies) multiscale modeling offers a powerful and quantitative alternative for the study of functional intracellular signaling modules. Here, we describe the application of a multiscale approach to signaling mediated by the ErbB1 receptor which constitutes a network hub for the cell's proliferative, migratory, and survival programs. Through our multiscale model, we mechanistically describe how point-mutations in the ErbB1 receptor can profoundly alter signaling characteristics leading to the onset of oncogenic transformations. Specifically, we describe how the point mutations induce cascading fragility mechanisms at the molecular scale as well as at the scale of the signaling network to preferentially activate the survival factor Akt. We provide a quantitative explanation for how the hallmark of preferential Akt activation in cell-lines harboring the constitutively active mutant ErbB1 receptors causes these cell-lines to be addicted to ErbB1-mediated generation of survival signals. Consequently, inhibition of ErbB1 activity leads to a remarkable therapeutic response in the addicted cell lines.