RNA-Expression and Proteome Analysis Identify Complementary but Not Identical Molecular Targets in Enzastaurin-Treated Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1915-1915
Author(s):  
Marc Weinkauf ◽  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Elena Hartmann ◽  
Andreas Rosenwald ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lymphoma ◽  
Interaction Network ◽  
Proteome Analysis ◽  
Rna Expression ◽  
Array Analysis ◽  
Expression Array ◽  
2D Page ◽  
Mantle Cell ◽  
Functional Pathways

Abstract Abstract 1915 Poster Board I-938 Introduction: Protein kinase C beta (PKCbeta), a pivotal enzyme in B-cell signaling and survival, is over-expressed in most cases of mantle cell lymphoma (MCL) and results in activation of PI3K/AKT pathway. Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signaling through PKCbeta/PI3K/AKT pathways, induces apoptosis, reduces proliferation, and suppresses tumor-induced angiogenesis. Aim: To optimize the treatment options with this promising inhibitor the goal of this study was to elucidate the molecular pathways altered by Enzastaurin treatment in MCL. Methods: Four documented MCL cell lines (Granta 519, HBL-2, Jeko-1, Rec-1) were harvested after 2-8h Enzastaurin exposure at a previously defined dose of 10μM and analyzed by RNA-array and proteome analysis as previously described (2D-polyacrylamide-gel-electrophoresis (2D-PAGE); Weinkauf 2009). Regulated molecules were mapped in a functional interaction network and candidates representing different pathways were verified by Western blotting. Results: Enzastaurin exposure led to significant reduction of cell viability in all cell lines (15-20%). This was also reflected in distinct alterations of the observed protein patterns in 2D-PAGE after enzastaurin exposure. Of a total of 977 concurrent protein spots 115 (12%) spots exhibited significantly (>3fold) altered protein levels after 4h of enzastaurin exposure. Mass spectrometry of 62 protein spots (39 increase; 23 decreased) identified 108 different candidate proteins, which were used to create a protein interaction network identifying the affected functional pathways. The results of the 2D-PAGE analysis were verified by Western blot in selected candidate proteins of apoptosis (VIM, PLEC1), DNA-repair (RAD50, PCNA, RFC1) and gene expression (EEF1D, SMC1A) pathways. In parallel, RNA-expression array analysis identified 180 different genes regulated early after enzastaurin treatment. Again these genes were mapped to an interaction network, highlighting enzastaurin involvement in e.g. NFkB-, apoptosis and B-cell- and death receptor signaling pathways. Interestingly, the involved genes complemented the regulated proteins in the functional pathways. Network analysis of both screenings (2D-PAGE-based proteomics and RNA-expression array) classified the candidate molecules in functional groups, including DNA repair and replication (e.g. RAD50, PCNA), apoptosis (e.g.PSMC4, VIM), signal transduction (e.g. GRB2, EF1D) and gene expression/mRNA processing (e.g.EEF1D, SFRS7). Thus, combined analysis of both screening methods resulted in a more comprehensive network than each respective analysis. Conclusion: Enzastaurin-treatment affects three main cellular control mechanisms as highlighted by two independent screening approaches (proteomics and expression array analysis). Interestingly, these two layers of molecular response (protein and RNA respectively) resulted in minimal overlap of identified molecules at this early (4h) time, but indicated common pathways nonetheless. Ongoing experiments now incorporate this knowledge to select optimal combination partners of enzastaurin. Disclosures: Dreyling: Lilly Deutschland GmbH: Research Funding.

Integrated CGH- and Epression Array Profiling of Mantle Cell Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2252-2252
Author(s):  
Margit Schraders ◽  
Pedro Jares ◽  
Silvia Bea ◽  
Eric F.P.M. Schoenmakers ◽  
Joannes H.J.M. Van Krieken ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Copy Number ◽  
Array Cgh ◽  
Cell Lymphoma ◽  
Gene Dosage ◽  
Rna Expression ◽  
Array Analysis ◽  
Expression Array ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is characterized by the translocation t(11;14)(q13;q32), which leads to the overexpression of cyclin D1. The cyclin D1-overexpression by itself is however not sufficient for lymphoma development. To identify other genes that are deregulated in MCL, we have performed integrated profiling of high-resolution array-based comparative genomic hybridization (array-CGH) with RNA-expression array analysis on the same set of MCL cases. DNA-alterations of MCL cases were assessed using a 3.6k BAC array with a resolution of 800 kb. On the same cases, RNA-expression array analysis was performed using the GeneChip® Human Genome U133 Plus 2.0 Array from Affymetrix. Integration of genomic-and transcription profiling data was performed using statistical tools. With array-CGH, we identified regions of chromosomal gain or loss and determined minimal common regions (mcr’s) by identifying the smallest region of overlap present in at least 36% of the cases. Integration of array-CGH and expression profiling was performed for genes located within the mcr’s and unsupervised clustering of gene-expression values within each mcr was performed. For several mcr’s, e.g. 1p22.1–31.1, 6q23.2–27 and 11q22.3–23.3, the clustering tree showed two groups, one with the chromosomal aberration and one without. Also, we observed that only a subset of genes located within a cytogenetic anomaly has a concomitant change in mRNA expression level. These genes are regulated by DNA-copy number (gene dosage). Amongst these, we identified several “hypothetical genes” and genes, which encode proteins involved in mitochondrial protein synthesis, the DNA damage repair pathway and the cAMP regulated pathway. This study shows the potential of the integrated profiling approach for identifying genes that are regulated by gene dosage (DNA-copy number). We anticipate that the genes we identified are important for MCL and it’s characteristic features like the low apoptosis rate and the chemotherapy-resistance.

Combined RNA-expression and 2D-PAGE-screening identifies comprehensive interaction networks affected after bortezomib or enzastaurin exposure of mantle cell lymphoma

Talanta ◽  
2010 ◽  
Vol 80 (4) ◽  
pp. 1539-1544 ◽  
Author(s):  
Marc Weinkauf ◽  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Elena Hartmann ◽  
Andreas Rosenwald ◽  
...  
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Interaction Networks ◽  
Rna Expression ◽  
2D Page ◽  
Mantle Cell

scholarly journals Solar simulated ultraviolet radiation inactivates HCoV-NL63 and SARS-CoV-2 coronaviruses at environmentally relevant doses

2021 ◽  
Author(s):  
Georg T. Wondrak ◽  
Jana Jandova ◽  
Spencer J. Williams ◽  
Dominik Schenten
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Uv Light ◽  
Gene Expression Array ◽  
Array Analysis ◽  
Expression Array ◽  
Response Gene ◽  
Full Spectrum ◽  
Solar Uv ◽  
Stress Response Gene

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.

CD10-positive mantle cell lymphoma: biologically distinct entity or an aberrant immunophenotype? Insight, through gene expression profile in a unique case series

2015 ◽  
Vol 68 (10) ◽  
pp. 844-848 ◽  
Author(s):  
Ariz Akhter ◽  
Etienne Mahe ◽  
Lesley Street ◽  
Payam Pournazari ◽  
Marco Perizzolo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mantle Cell Lymphoma ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
Case Series ◽  
Convincing Evidence ◽  
Minimal Impact ◽  
Mantle Cell

BackgroundMantle cell lymphoma (MCL) is an aggressive disease with genetic heterogeneity and discrete clinical subtypes. MCL is rarely CD10 positive. These cases raise the question whether a subset of MCL may be germinal centre (GC) derived, and have distinct clinicopathological characteristics.Aims and methodsA series of nine CD10-positive MCL cases is described herein. The clinicopathological and immunophenotypic features, immunoglobulin somatic hypermutation (SHM) status and gene expression profile (GEP) data are detailed. These features were compared with two independent sets (n=20, each) of CD10-negative MCL cases (controls), which were randomly selected from our institutional registry.ResultsGEP showed distinct expression of a GC signature in CD10-positive MCL cases with minimal impact on downstream signalling pathways. There were no significant differences in the clinicopathological features or clinical outcome between our CD10-positive and CD10-negative MCL cases. The frequency of SHM was comparable with established data.ConclusionsThis study provides convincing evidence that CD10 expression is related to a distinct GC signature in MCL cases, but without clinical or biological implications.

Farnesyltransferase Inhibitor Tipifarnib (Zarnestra) in Refractory Mantle Cell Lymphoma (MCL): A Therapeutic Target Only for Specific Patients Categorized by the 2-Gene Classifier for Predictive Response to Tipifarnib.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1561-1561
Author(s):  
Delphine Rolland ◽  
Vincent Ribrag ◽  
Corinne Haioun ◽  
Herve Ghesquieres ◽  
Fabrice Jardin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Therapeutic Strategies ◽  
Prediction Of Response ◽  
Expression Ratio ◽  
Tumor Biopsy ◽  
Gene Expression Ratio ◽  
Mantle Cell ◽  
Initial Tumor

Abstract Background. Mantle cell lymphoma is one of the most refractory B-cell lymphomas. Despite the recent improvement of novel therapeutic strategies including high dose therapy and the introduction of immunotherapy, MCL patients are not cured by conventional therapeutic strategies. The increasing understanding of the MCL cell biology leads to the development of new drugs targeting to molecular mechanisms of the disease. Among the specific genes found to be overexpressed in MCL by gene expression profiling analysis, farnesyltransferase (FTase) encodes for an enzyme essential in the post- translational modifications of proteins that require prenylation for conversion to mature forms, allowing their participation in various signaling pathways regulating growth and survival. We recently demonstrated that inhibition of FTase by tipifarnib (Zarnestra) is associated in vitro with growth inhibition and apoptosis of MCL cell lines and in vivo with tumor xenograft stability. To determine the efficacy, the safety profile and the toxicity of tipifarnib in MCL, we conducted a phase II trial in patients with refractory MCL, and we evaluated the response considering published molecular predictors. Methods. Primary endpoint was the efficacy measured by the evaluation of the overall response rate (ORR) at 4 cycles, and, in case of response, at 6 cycles. Planned sample size of the study was 27 evaluable patients enrolled for an analysis based on an optimal two-stage design comprising 11 patients for the first stage and 16 for the second stage, under the hypothesis of an ORR of 35% to conclude for an effective drug and an ORR of 10% to conclude for an ineffective drug. Tipifarnib was administered at 300 mg orally twice daily for the first 21 days of each 28-days cycle for 4 to 6 cycles. Prediction of response was retrospectively evaluated in the initial tumor biopsy by the analysis of the 2-gene classifier, the RASGRP1/APTX gene expression ratio, and the AKAP13 expression level. Results. Eleven patients with refractory MCL were included in the analysis. Median number of lines of therapy before tipifarnib was 2.5 (range 1–7). Median age was 71 (range 66–79). All patients presented with stage IV disease and with good performance status. At 4 cycles of tipifarnib, 1 patient presented a complete response and 10 patients were in progressive disease. Two patients progressed during or after the first cycle, three patients after 3 cycles, and five patients after 4 cycles. No grade III-IV hematological toxicities were recorded. One patient presented an unrelated neurological symptom after the first dose administered. Evaluation of the molecular prediction of response to tipifarnib was realized for 3 patients: one responder patient and two non-responder patients. Results showed an increase in the RASGRP1/APTX gene expression ratio and a decrease in AKAP13 expression in the responder patient, and a decrease in the RASGRP1/APTX gene expression ratio and an increase in AKAP13 expression in the non-responder patients. This corresponds to the expected results of the response prediction to tipifarnib. Conclusion. Tipifarnib in refractory MCL was beneficial for only one patient. Response could be exactly predicted by specific molecular predictors of response evaluated in the initial tumor biopsy. These results demonstrate the necessity of categorizing molecularly the patients when targeted therapies are proposed to select those patients that might respond to.

Biology and Treatment of Mantle Cell Lymphoma in the Genomic Era.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-28-SCI-28
Author(s):  
Adrian Wiestner
Keyword(s):  
Gene Expression ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Cell Lymphoma ◽  
Genetic Alterations ◽  
Diagnostic Methods ◽  
Genetic Changes ◽  
Mantle Cell

Abstract Abstract SCI-28 The t11;14 translocation, the genetic hallmark of MCL, drives cyclin D1 expression in the tumor cells and historically facilitated the separation of MCL into a distinct entity. FISH cytogenetics are part of the workup and can be particularly helpful to separate leukemic MCL from CLL. Morphology and clinical course of MCL are heterogeneous and might have suggested the presence of different entities. Gene expression profiling answered this concern and provided several important insights: 1. despite the clinical heterogeneity, MCL has a characteristic gene expression profile supporting the accuracy of current diagnostic methods; 2. cyclin D1 negative MCL has the same diagnostic pathologic and gene expression features as cyclin D1 positive MCL, and 3. a gene expression based measure of tumor proliferation is a potent predictor of outcome and identifies patients with survival probabilities ranging from less than 1 year (highly proliferative tumors) to more than 6 years (low proliferation).1 Biologically, the gene expression based proliferation score integrates several acquired genetic changes in the tumor that include deletions of the INK4a/ARF locus encoding the tumor suppressors p14 and p16, amplification of BMI1, and secondary changes in the cyclin D1 locus. Mutations and deletions that alter the structure of the 3'UTR can enhance cyclin D1 mRNA stability,2 and the loss of miR binding sites in this region can enhance protein translation.3 These changes increase cyclin D1 protein resulting in increased proliferation. Additional genetic lesions such as deletions of ATM and p53 affect DNA damage responses pathways. The high frequency of secondary genetic changes in MCL cells may indicate genomic instability and the presence of additional chromosomal aberrations and certain genetic alterations hold prognostic information.4 With the continued refinement of whole genome genetic approaches the goal of identifying crucial pathways and possible driver genes in the pathogenesis of MCL may be within reach. MCL characterized by an antiapoptotic phenotype combined with features of aggressive lymphomas remains an incurable disease and having the worst outcome among all B-cell lymphomas. Biologic markers that predict treatment response and that could give way to targeted therapy have remained elusive. Several new drugs could help overcome treatment resistance and new analytic tools when incorporated into clinical trials may help dissect mechanisms of drug action and resistance. Our approach has been to incorporate gene expression profiling into a clinical trial of bortezomib to directly monitor the effects of the treatment on tumor biology in vivo. We identified an integrated stress response to bortezomib in sensitive tumors that may yield clinically usefully predictors of sensitivity and that could guide the development of improved therapies. 1. Rosenwald A, Wright G, Wiestner A, et al. The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma. Cancer Cell. 2003;3:185-197. 2. Wiestner A, Tehrani M, Chiorazzi M, et al. Point mutations and genomic deletions in Cyclin D1 create stable truncated mRNAs that are associated with increased proliferation rate and shorter survival in mantle cell lymphoma. Blood. 2007. 3. Chen RW, Bemis LT, Amato CM, et al. Truncation in CCND1 mRNA alters miR-16-1 regulation in mantle cell lymphoma. Blood. 2008;112:822-829. 4. Salaverria I, Espinet B, Carrio A, et al. Multiple recurrent chromosomal breakpoints in mantle cell lymphoma revealed by a combination of molecular cytogenetic techniques. Genes Chromosomes Cancer. 2008;47:1086-1097. Disclosures Off Label Use: Bortezomib in previously untreated patients with MCL.

Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2893-2893
Author(s):  
Marc Weinkauf ◽  
Grit Hutter ◽  
Yvonne Zimmermann ◽  
Malte Rieken ◽  
Alessandro Pastore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Signal Pathways ◽  
Rna Expression ◽  
Cellular Functions ◽  
Mantle Cell ◽  
Canonical Pathways

Abstract Abstract 2893 Background: The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis. Methods: Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment. Results: Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling', (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis' (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling' (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer' (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation' (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death' (BCL6, EEF1D, PAK6, RAD50), ‘cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), ‘cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression' (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways. Conclusion: In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL. Disclosures: Dreyling: Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.

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