Facile Access to High Solid Content Monodispersed Microspheres via Dual-Component Surfactants Regulation toward High-Performance Colloidal Photonic Crystals.

Monodispersed microspheres play a major role in optical science and engineering, providing ideal building blocks for structural color materials. However, the method toward high solid content (HSC) monodispersed microspheres has remained a key hurdle. Herein, a facile access to harvest monodispersed microspheres based on the emulsion polymerization mechanism is demonstrated, where anionic and nonionic surfactants are employed to achieve the electrostatic and steric dual-stabilization balance in a synergistic manner. Monodispersed poly(styrene-butyl acrylate-methacrylic acid) colloidal latex with 55 wt% HSC is achieved, which shows an enhanced self-assembly efficiency of 280% compared with the low solid content (10 wt%) latex. In addition, Ag-coated colloidal photonic crystal (Ag@CPC) coating with near-zero refractive index is achieved, presenting the characteristics of metamaterials. And an 11-fold photoluminescence emission enhancement of CdSe@ZnS quantum dots is realized by the Ag@CPC metamaterial coating. Taking advantage of high assembly efficiency, easily large-scale film-forming of the 55 wt% HSC microspheres latex, robust Ag@CPC metamaterial coatings could be easily produced for passive cooling. The coating demonstrates excellent thermal insulation performance with theoretical cooling power of 30.4 W m-2, providing practical significance for scalable CPC architecture coatings in passive cooling.

PCA-Plus: Enhanced principal component analysis with illustrative applications to batch effects and their quantitation.

Background: Principal component analysis (PCA), a standard approach to analysis and visualization of large datasets, is commonly used in biomedical research for detecting similarities and differences among groups of samples. We initially used conventional PCA as a tool for critical quality control of batch and trend effects in multi-omic profiling data produced by The Cancer Genome Atlas (TCGA) project of the NCI. We found, however, that conventional PCA visualizations were often hard to interpret when inter-batch differences were moderate in comparison with intra-batch differences; it was also difficult to quantify batch effects objectively. We, therefore, sought enhancements to make the method more informative in those and analogous settings. Results: We have developed algorithms and a toolbox of enhancements to conventional PCA that improve the detection, diagnosis, and quantitation of differences between or among groups, e.g., groups of molecularly profiled biological samples. The enhancements include (i) computed group centroids; (ii) sample-dispersion rays; (iii) differential coloring of centroids, rays, and sample data points; (iii) trend trajectories; and (iv) a novel separation index (DSC) for quantitation of differences among groups. Conclusions: PCA-Plus has been our most useful single tool for analyzing, visualizing, and quantitating batch effects, trend effects, and class differences in molecular profiling data of many types: mRNA expression, microRNA expression, DNA methylation, and DNA copy number. An early version of PCA-Plus has been used as the central graphical visualization in our MBatch package for near-real-time surveillance of data for analysis working groups in more than 70 TCGA, PanCancer Atlas, PanCancer Analysis of Whole Genomes, and Genome Data Analysis Network projects of the NCI. The algorithms and software are generic, hence applicable more generally to other types of multivariate data as well. PCA-Plus is freely available in a down-loadable R package at our MBatch website.

One-Pot Synthesis of Robust Fluorescent Nanocomposite Gel via Frontal Polymerization.

Fluorescent nanocomposite gels have attracted increasing attention due to their excellent optical properties, as well as enhanced mechanical strength originating from the nanoparticles. At present, two-step methods are usually employed, where fluorescent nanoparticles are firstly prepared, followed by mixing with gel precursor to achieve the final products after gelation, which suffer from the disadvantages of a tedious and time-consuming process. Thus, the development of a facile strategy is highly desirable, which still remains an obstacle. Herein, a new one-pot synthesis method towards robust fluorescent nanocomposite gels via frontal polymerization (FP) is proposed, where small molecular precursors (citric acid (CA) and urea, or L-cysteine) and gel precursor (vinyl monomers) are mixed together as co-reactants. During the FP process, a lot of heat release gives rise to the generation of carbonized polymer dots (CPDs). Thus, companying with the propagating of the polymerization, the production of fluorescent CPDs/gel composite is completed. In addition, as a nanofiller, CPDs dramatically enhance the mechanical property of the CPDs/gel composite. This work proposes a new fast and efficient one-pot strategy for the production of CPDs/gel composite, which will guide the development of high-performance polymer nanocomposites through an in situ synchronous reaction fashion.

Focal Adhesion Kinase as a Potential Target in AML and MDS.

Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics (P = 2 × 10-4) and relapse (P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3-ITD (P = 0.0024) or RAS (P = 0.05) mutations and strongly correlated with p-SRC and integrinß3 levels. FAK protein levels were significantly higher in CD34+ (P = 5.42 × 10-20) and CD34+CD38- MDS (P = 7.62 × 10-9) cells compared with normal CD34+ cells. MDS patients with higher FAK in CD34+ cells tended to have better overall survival (P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML. Coculture with mesenchymal stromal cells (MSC) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia-stromal interactions and supports leukemia cell survival; hence, FAK is a potential therapeutic target in myeloid leukemia. Mol Cancer Ther; 16(6); 1133-44. ©2017 AACR.

Low expression of ASH2L protein correlates with a favorable outcome in acute myeloid leukemia.

ASH2L encodes a trithorax group protein that is a core component of all characterized mammalian histone H3K4 methyltransferase complexes, including mixed lineage leukemia (MLL) complexes. ASH2L protein levels in primary leukemia patient samples have not yet been defined. We analyzed ASH2L protein expression in 511 primary AML patient samples using reverse phase protein array (RPPA) technology. We discovered that ASH2L expression is significantly increased in a subset of patients carrying fms-related tyrosine kinase 3 (FLT3) mutations. Furthermore, we observed that low levels of ASH2L are associated with increased overall survival. We also compared ASH2L levels to the expression of 230 proteins previously analyzed on this array. ASH2L expression was inversely correlated with 32 proteins, mostly involved in cell adhesion and cell cycle inhibition, while a positive correlation was observed for 50 proteins, many of which promote cell proliferation. Together, these results indicate that a lower level of ASH2L protein is beneficial to AML patients.

Phosphorylation of GSK3α/ß correlates with activation of AKT and is prognostic for poor overall survival in acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) patients with highly active AKT tend to do poorly. Cell cycle arrest and apoptosis are tightly regulated by AKT via phosphorylation of GSK3α and ß isoforms which inactivates these kinases. In the current study we examine the prognostic role of AKT mediated GSK3 phosphorylation in AML. METHODS: We analyzed GSK3α/ß phosphorylation by reverse phase protein analysis (RPPA) in a cohort of 511 acute myeloid leukemia (AML) patients. Levels of phosphorylated GSK3 were correlated with patient characteristics including survival and with expression of other proteins important in AML cell survival. RESULTS: High levels of p-GSK3α/ß correlated with adverse overall survival and a lower incidence of complete remission duration in patients with intermediate cytogenetics, but not in those with unfavorable cytogenetics. Intermediate cytogenetic patients with FLT3 mutation also fared better respectively when p-GSK3α/ß levels were lower. Phosphorylated GSK3α/ß expression was compared and contrasted with that of 229 related cell cycle arrest and/or apoptosis proteins. Consistent with p-GSK3α/ß as an indicator of AKT activation, RPPA revealed that p-GSK3α/ß positively correlated with phosphorylation of AKT, BAD, and P70S6K, and negatively correlated with ß-catenin and FOXO3A. PKCδ also positively correlated with p-GSK3α/ß expression, suggesting crosstalk between the AKT and PKC signaling pathways in AML cells. CONCLUSIONS: These findings suggest that AKT-mediated phosphorylation of GSK3α/ß may be beneficial to AML cell survival, and hence detrimental to the overall survival of AML patients. Intrinsically, p-GSK3α/ß may serve as an important adverse prognostic factor for a subset of AML patients.

MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199.

Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.

Erk Negative Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia.

Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation of oncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation of Erk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels, we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse models for pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance.

Loss of TRIM62 expression is an independent adverse prognostic factor in acute myeloid leukemia.

BACKGROUND: Tripartite motif (TRIM)-62 is a putative tumor suppressor gene whose role in leukemia is unknown. MATERIALS AND METHODS: We evaluated the effect of TRIM62 protein expression in patients with acute myeloid leukemia (AML). We used reverse-phase protein array methodology to determine TRIM62 levels in leukemia-enriched protein samples from 511 patients newly diagnosed with AML. RESULTS: TRIM62 levels in AML cells were significantly lower than in normal CD34-positive cells, suggesting that TRIM62 loss might be involved in leukemogenesis, but was not associated with specific karyotypic abnormalities or Nucleophosmin (NPM1), Fms-like Tyrosine Kinase-3 (FLT3), or rat sarcoma viral oncogene (RAS) mutational status. Low TRIM62 levels were associated with shorter complete remission duration and significantly shorter event-free and overall survival rates, particularly among patients with intermediate-risk cytogenetics. In that AML subgroup, age and TRIM62 levels were the most powerful independent prognostic factors for survival. TRIM62 protein levels further refined the risk associated with NPM1 and FLT3 mutational status. TRIM62 loss was associated with altered expression of proteins involved in leukemia stem cell homeostasis (ß-catenin and Notch), cell motility, and adhesion (integrin-ß3, ras-related C3 botulinum toxin substrate [RAC], and fibronectin), hypoxia (Hypoxia-inducible factor 1-alpha [HIF1α], egl-9 family hypoxia-inducible factor 1 [Egln1], and glucose-regulated protein, 78 kDa [GRP78]), and apoptosis (B-cell lymphoma-extra large (BclXL) and caspase 9). CONCLUSION: Low TRIM62 levels, consistent with a tumor suppressor role, represent an independent adverse prognostic factor in AML.

Development of a robust classifier for quality control of reverse-phase protein arrays.

MOTIVATION: High-throughput reverse-phase protein array (RPPA) technology allows for the parallel measurement of protein expression levels in approximately 1000 samples. However, the many steps required in the complex protocol (sample lysate preparation, slide printing, hybridization, washing and amplified detection) may create substantial variability in data quality. We are not aware of any other quality control algorithm that is tuned to the special characteristics of RPPAs. RESULTS: We have developed a novel classifier for quality control of RPPA experiments using a generalized linear model and logistic function. The outcome of the classifier, ranging from 0 to 1, is defined as the probability that a slide is of good quality. After training, we tested the classifier using two independent validation datasets. We conclude that the classifier can distinguish RPPA slides of good quality from those of poor quality sufficiently well such that normalization schemes, protein expression patterns and advanced biological analyses will not be drastically impacted by erroneous measurements or systematic variations. AVAILABILITY AND IMPLEMENTATION: The classifier, implemented in the "SuperCurve" R package, can be freely downloaded at http://bioinformatics.mdanderson.org/main/OOMPA:Overview or http://r-forge.r-project.org/projects/supercurve/. The data used to develop and validate the classifier are available at http://bioinformatics.mdanderson.org/MOAR.

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells.

We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.

Age-related epigenetic drift in the pathogenesis of MDS and AML.

The myelodysplastic syndrome (MDS) is a clonal hematologic disorder that frequently evolves to acute myeloid leukemia (AML). Its pathogenesis remains unclear, but mutations in epigenetic modifiers are common and the disease often responds to DNA methylation inhibitors. We analyzed DNA methylation in the bone marrow and spleen in two mouse models of MDS/AML, the NUP98-HOXD13 (NHD13) mouse and the RUNX1 mutant mouse model. Methylation array analysis showed an average of 512/3445 (14.9%) genes hypermethylated in NHD13 MDS, and 331 (9.6%) genes hypermethylated in RUNX1 MDS. Thirty-two percent of genes in common between the two models (2/3 NHD13 mice and 2/3 RUNX1 mice) were also hypermethylated in at least two of 19 human MDS samples. Detailed analysis of 41 genes in mice showed progressive drift in DNA methylation from young to old normal bone marrow and spleen; to MDS, where we detected accelerated age-related methylation; and finally to AML, which markedly extends DNA methylation abnormalities. Most of these genes showed similar patterns in human MDS and AML. Repeat element hypomethylation was rare in MDS but marked the transition to AML in some cases. Our data show consistency in patterns of aberrant DNA methylation in human and mouse MDS and suggest that epigenetically, MDS displays an accelerated aging phenotype.

Reverse-phase protein array analysis to identify biomarker proteins in human pancreatic cancer.

BACKGROUND: Pancreatic cancer is the fourth leading cause of cancer death in the United States. The high mortality rate of patients with pancreatic cancer is primarily due to the difficulty of early diagnosis and a lack of effective therapies. There is an urgent need to discover novel molecular targets for early diagnosis and new therapeutic approaches to improve the clinical outcome of this deadly disease. AIM: We utilized the reverse-phase protein assay (RPPA) to identify differentially expressed biomarker proteins in tumors and matched adjacent, normal-appearing tissue samples from 15 pancreatic cancer patients. METHODS: The antibody panel used for the RPPA included 130 key proteins involved in various cancer-related pathways. The paired t test was used to determine the significant differences between matched pairs, and the false discovery rate-adjusted p values were calculated to take into account the effect of multiple comparisons. RESULTS: After correcting for multiple comparisons, we found 19 proteins that had statistically significant differences in expression between matched pairs. However, only four (AKT, ß-catenin, GAB2, and PAI-1) of them met the conservative criteria (both a q value <0.05 and a fold-change of ≥3/2 or ≤2/3) to be considered differentially expressed. Overexpression of AKT, ß-catenin, and GAB2 in pancreatic cancer tissues identified by RPPA has also been further confirmed by western blot analysis. Further analysis identified several significantly associated canonical pathways and overrepresented network functions. CONCLUSION: GAB2, a newly identified protein in pancreatic cancer, may provide additional insight into this cancer's pathogenesis. Future studies in a larger population are warranted to further confirm our results.

Proteomic profiling identifies distinct protein patterns in acute myelogenous leukemia CD34+CD38- stem-like cells.

Acute myeloid leukemia (AML) is believed to arise from leukemic stem-like cells (LSC) making understanding the biological differences between LSC and normal stem cells (HSC) or common myeloid progenitors (CMP) crucial to understanding AML biology. To determine if protein expression patterns were different in LSC compared to other AML and CD34+ populations, we measured the expression of 121 proteins by Reverse Phase Protein Arrays (RPPA) in 5 purified fractions from AML marrow and blood samples: Bulk (CD3/CD19 depleted), CD34-, CD34+(CMP), CD34+CD38+ and CD34+CD38-(LSC). LSC protein expression differed markedly from Bulk (n =31 cases, 93/121 proteins) and CD34+ cells (n = 30 cases, 88/121 proteins) with 54 proteins being significantly different (31 higher, 23 lower) in LSC than in either Bulk or CD34+ cells. Sixty-seven proteins differed significantly between CD34+ and Bulk blasts (n = 69 cases). Protein expression patterns in LSC and CD34+ differed markedly from normal CD34+ cells. LSC were distinct from CD34+ and Bulk cells by principal component and by protein signaling network analysis which confirmed individual protein analysis. Potential targetable submodules in LSC included the proteins PU.1(SP1), P27, Mcl1, HIF1α, cMET, P53, Yap, and phospho-Stats 1, 5 and 6. Protein expression and activation in LSC differs markedly from other blast populations suggesting that studies of AML biology should be performed in LSC.

Impact of molecular alterations and targeted therapy in appendiceal adenocarcinomas.

UNLABELLED: Appendiceal adenocarcinomas (AAs) are rare and this has limited their molecular understanding. The purpose of our study was to characterize the molecular profile of AA and explore the role of targeted therapy against cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR). PATIENTS AND METHODS: We performed a retrospective review of 607 patients with AA at a single institution. A total of 149 patients underwent molecular testing for at least one of the following: activating mutations in KRAS, BRAF, cKIT, EGFR, or PI3K; protein expression of c-KIT or COX-2; or microsatellite instability (MSI) status by immunohistochemistry. Kaplan-Meier product limit method and log-rank test were used to estimate overall survival (OS) and to determine associations among OS, COX-2 expression, KRAS mutations, and other characteristics. RESULTS: Age, grade, stage, signet ring cells, mucinous histology, and completeness of cytoreduction score correlated with survival outcomes. COX-2 expression, KRAS, PI3K, and BRAF mutations were seen in 61%, 55%, 17%, and 4% of patients, respectively. High MSI was seen in 6% of patients. KRAS mutation was strongly associated with well differentiated or moderately differentiated AA (p < .01). COX-2 expression (p = .33) and the presence of KRAS mutation (p = .91) had no impact on OS. The use of celecoxib in patients whose tumors expressed COX-2 (p = .84) and the use of cetuximab or panitumumab in patients with KRAS wild-type tumors (p = .83) also had no impact on OS. CONCLUSION: In this cohort, we demonstrated that COX-2 expression and KRAS mutations were frequently seen in AA, although neither exhibited any prognostic significance. MSI was infrequent in AA. Targeted therapy against COX-2 and EGFR appeared to provide no clinical benefit. Well and moderately differentiated AA were molecularly distinct from poorly differentiated AA.

Detailed genome-wide SNP analysis of major salivary carcinomas localizes subtype-specific chromosome sites and oncogenes of potential clinical significance.

The molecular genetic alterations underlying the development and diversity of salivary gland carcinomas are largely unknown. To characterize these events, comparative genomic hybridization analysis was performed, using a single-nucleotide polymorphism microarray platform, of 60 fresh-frozen specimens that represent the main salivary carcinoma types: mucoepidermoid carcinoma (MEC), adenoid cystic carcinoma (ACC), and salivary duct carcinoma (SDC). The results were correlated with the clinicopathologic features and translocation statuses to characterize the genetic alterations. The most commonly shared copy number abnormalities (CNAs) in all types were losses at chromosomes 6q23-26 and the 9p21 region. Subtype-specific CNAs included a loss at 12q11-12 in ACC and a gain at 17q11-12 in SDC. Focal copy number losses included 1p36.33-p36-22 in ACC, 9p13.2 in MEC, and 3p12.3-q11-2, 6q21-22.1, 12q14.1, and 12q15 in SDC. Tumor-specific amplicons were identified at 11q23.3 (PVRL1) in ACC, 11q13.3 (NUMA1) in MEC, and 6p21.1 (CCND3), 9p13.2 (PAX5), 12q15 (CNOT2/RAB3IP), 12q21.1 (GLIPR1L1), and 17q12 (ERBB2/CCL4) in SDC. A comparative CNA analysis of fusion-positive and fusion-negative ACCs and MECs revealed relatively lower CNAs in fusion-positive tumors than in fusion-negative tumors in both tumor types. An association between CNAs and high grade and advanced stage was observed in MECs only. These findings support the pathogenetic segregation of these entities and define novel chromosomal sites for future identification of biomarkers and therapeutic targets.

Prognostic impact and targeting of CRM1 in acute myeloid leukemia.

Chromosomal region maintenance 1 (CRM1) is a nuclear export receptor recognizing proteins bearing a leucine-rich nuclear export signal. CRM1 is involved in nuclear export of tumor suppressors such as p53. We investigated the prognostic significance of CRM1 in acute myeloid leukemia (AML) and effects of a novel small-molecule selective inhibitor of CRM1. CRM1 protein expression was determined in 511 newly diagnosed AML patients and was correlated with mouse double minute 2 (MDM2) and p53 levels. High CRM1 expression was associated with short survival of patients and remained an adverse prognostic factor in multivariate analysis. CRM1 inhibitor KPT-185 induced mainly full-length p53 and apoptosis in a p53-dependent manner, whereas inhibition of proliferation was p53 independent. Patient samples with p53 mutations showed low sensitivity to KPT-185. Nuclear retention of p53 induced by CRM1 inhibition synergized with increased levels of p53 induced by MDM2 inhibition in apoptosis induction. KPT-185 and Nutlin-3a, alone and in combination, induced synergistic apoptosis in patient-derived CD34(+)/CD38(-) AML, but not in normal progenitor cells. Data suggest that CRM1 exerts an antiapoptotic function and is highly prognostic in AML. We propose a novel combinatorial approach for the therapy of AML, aimed at maximal activation of p53-mediated apoptosis by concomitant MDM2 and CRM1 inhibition.

Transglutaminase 2 expression in acute myeloid leukemia: association with adhesion molecule expression and leukemic blast motility.

Acute myeloid leukemia (AML) is a heterogenous disease with differential oncogene association, outcome and treatment regimens. Treatment strategies for AML have improved outcome but despite increased molecular biological information AML is still associated with poor prognosis. Proteomic analysis on the effects of a range of leukemogenic oncogenes showed that the protein transglutaminase 2 (TG2) is expressed at greater levels as a consequence of oncogenic transformation. Further analysis of this observation was performed with 511 AML samples using reverse phase proteomic arrays, demonstrating that TG2 expression was higher at relapse than diagnosis in many cases. In addition elevated TG2 expression correlated with increased expression of numerous adhesion proteins and many apoptosis regulating proteins, two processes related to leukemogenesis. TG2 has previously been linked to drug resistance in cancer and given the negative correlation between TG2 levels and peripheral blasts observed increased TG2 levels may lead to the protection of the leukemic stem cell due to increased adhesion/reduced motility. TG2 may therefore form part of a network of proteins that define poor outcome in AML patients and potentially offer a target to sensitize AML stem cells to drug treatment.

Metabolic shifts induced by fatty acid synthase inhibitor orlistat in non-small cell lung carcinoma cells provide novel pharmacodynamic biomarkers for positron emission tomography and magnetic resonance spectroscopy.

PURPOSE: Abnormal fatty acid (FA) synthesis is one of the common features of cancer. Fatty acid synthase (FASN), a multifunctional enzyme playing a key role in biosynthesis of FA, is up-regulated in prostate, breast, and lung carcinomas. Orlistat is a FDA-approved anti-obesity drug that inhibits the thioesterase domain of FASN, interferes with cellular FA synthesis, can arrest tumor cell proliferation, and induces tumor cell apoptosis. The current study was aimed to investigate the metabolic changes associated with FASN inhibition by orlistat and to understand the molecular mechanisms behind the observed metabolic changes in non-small cell lung carcinoma (NSCLC) cell lines. PROCEDURES: Changes in metabolite pools in four NSCLC cell lines (H441, H1975, H3255, and PC14) with different mutational profiles were studied using NMR spectroscopy before and after in vitro incubation with sub-toxic concentration of orlistat and [1-(13)C]D-glucose or [1,2-(13)C2]choline. In vitro radiotracer accumulation assays in cells were performed with [(3)H]acetate, [(14)C]fluoroacetate, and 2-deoxy-2-[(18)F]fluoro-D-glucose. In parallel, microarray profiling of genes involved in the regulation of carbohydrate and lipid metabolism was performed. RESULTS: In orlistat-treated NSCLC cells, FASN inhibition results in characteristic changes in intermediary metabolites (FAs, choline, phospholipids, and TCA cycle metabolites) as observed by magnetic resonance spectroscopy. Further, FASN inhibition by orlistat induces multiple adaptive changes in FA synthetic pathway and associated metabolic pathways, including induction of ketone metabolism and glutaminolysis, as well as the up-regulation of 5' adenosine monophosphate-activated protein kinase. CONCLUSIONS: These observed changes in metabolic pools in orlistat-treated cells demonstrate the critical role of fatty acid de novo synthesis and metabolism for cellular energy production, especially in tumor cells with low glycolytic activity, which goes beyond the widely accepted concept that FA synthesis is important for cell membrane biosynthesis in rapidly proliferating tumor cells.

Prognostically relevant gene signatures of high-grade serous ovarian carcinoma.

Because of the high risk of recurrence in high-grade serous ovarian carcinoma (HGS-OvCa), the development of outcome predictors could be valuable for patient stratification. Using the catalog of The Cancer Genome Atlas (TCGA), we developed subtype and survival gene expression signatures, which, when combined, provide a prognostic model of HGS-OvCa classification, named "Classification of Ovarian Cancer" (CLOVAR). We validated CLOVAR on an independent dataset consisting of 879 HGS-OvCa expression profiles. The worst outcome group, accounting for 23% of all cases, was associated with a median survival of 23 months and a platinum resistance rate of 63%, versus a median survival of 46 months and platinum resistance rate of 23% in other cases. Associating the outcome prediction model with BRCA1/BRCA2 mutation status, residual disease after surgery, and disease stage further optimized outcome classification. Ovarian cancer is a disease in urgent need of more effective therapies. The spectrum of outcomes observed here and their association with CLOVAR signatures suggests variations in underlying tumor biology. Prospective validation of the CLOVAR model in the context of additional prognostic variables may provide a rationale for optimal combination of patient and treatment regimens.