Acute Lymphoblastic Leukemia Is a Bcl-2 Dependent Disease: Proteomic Profiling and Pre-Clinical Efficacy Of a Selective Bcl-2 Antagonist ABT-199

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3919-3919 ◽  
Author(s):  
Marina Konopleva ◽  
Juliana M Benito ◽  
Karine G. Harutyunyan ◽  
Isabel Marzo ◽  
LaKiesha Debose ◽  
...  
Keyword(s):  
Family Members ◽  
Burkitt’S Lymphoma ◽  
Black Box ◽  
Burkitt's Lymphoma ◽  
Differentially Expressed ◽  
Bimolecular Fluorescence Complementation ◽  
Differentially Expressed Proteins ◽  
Proteomic Profiling ◽  
Two Samples ◽  
Apoptotic Proteins

Abstract The expression of Bcl-2 family proteins is perturbed in multiple types of cancers, including leukemias, and is associated with disease progression and resistance to chemotherapy. ABT-199 (GDC-0199) is a new BH3 mimetic that was developed to specifically target Bcl-2 while sparing Bcl-XL, hence avoiding thrombocytopenia intrinsic to 1st generation BH3 mimetics like ABT-737 (Souers et al., Nat Med, 2013). In this study, we report proteomic profiling of Bcl-2 family members in a large series of ALL patients (pts) and pre-clinical activity of ABT-199. Expression of 20 pro- and anti-apoptotic proteins was studied in 186 newly diagnosed ALL using reverse phase protein arrays (RPPA). Supervised clustering demonstrated distinct differences in 11 proteins in ALL with different cytogenetic and FAB characteristics (Fig. 1, p<0.005, false discovery rate <0.2%). Among these, pts with Burkitt's leukemia/lymphoma (n=9) expressed low levels of Bcl-2 and Bax while maintaining high expression of Bim, caspases and PARP. In contrast, t(4;11) pts expressed higher levels of Bcl-2, Bax and Bim. No significant differences in Bcl-XL or Mcl-1 levels were found in different ALL subtypes. Figure 1 RPPA profiling of apoptosis regulators in ALL. Heatmap of differentially expressed proteins based on cytogenetics and immunophenotype. Black box, Burkitt's leukemia; red box, t(4;11). Figure 1. RPPA profiling of apoptosis regulators in ALL. Heatmap of differentially expressed proteins based on cytogenetics and immunophenotype. Black box, Burkitt's leukemia; red box, t(4;11). The potential of ABT-199 to disrupt interactions between Bcl-2 and different pro-apoptotic proteins was studied using Bimolecular Fluorescence Complementation (BiFC, J Biol Chem 288:4935, 2013). The coding sequences for human Bcl-2, Bim, Bak, Bax and Noxa were subcloned into BiFC plasmids containing Venus fragments and transfected into HeLa cells. Approximately 60-70% of transfected cells were positive for Venus fluorescence due to association between Bcl-2 and Bim, Noxa, Bax or Bak. ABT-199 (2.5 µM, 24 hrs) significantly reduced Venus signal, indicating an inhibition of the interactions of Bcl-2 with these proteins, most potently with the multidomain proteins Bax and Bak (95%±18% and 85%±15% inhibition, respectively). ABT-199 rapidly induced apoptotic cell death in ALL cell lines and in primary ALL samples. Pre-B ALL cells (Nalm-6, REH, SEMK2 and RS4;11) were sensitive to ABT-199 and ABT-737 (IC50 0.007-1.4µM (199) and 0.035-0.7µM (737)). Notably, ABT-199 was more cytotoxic than ABT-737 against MLL-rearranged SEMK2 and RS4;11 cells, consistent with the notion of the greater Bcl-2 dependency of these cells. Lentiviral silencing of Bcl-XL sensitized REH cells to apoptosis by ABT-199 and ABT-737. T-ALL cells (PF-382, MOLT-4, P-12) expressed lower levels of Bcl-2 and were uniformly less sensitive to ABT-199 compared to ABT-737 (IC50 3.7±1.1µM vs 0.7±0.3µM, p=0.01). Burkitt's lymphoma cells Ramos and Raji had low Bcl-2 and high Mcl-1 expression, and were resistant to both agents (IC50>4µM). Next, the cytotoxic activity of ABT-199 was tested against a panel of 12 genetically diverse primary ALL samples, including 6 from pts with relapsed or refractory disease. Ten out of twelve samples (83%) were exquisitely sensitive to both agents, with IC50 values of 0.0001-0.14µM for ABT-199 and 0.0004-0.3µM for ABT-737. One of the four Ph+ samples was resistant to both agents, and one of the two T-ALL was less sensitive to ABT-199 compared to ABT-737. Two samples with t(4;11) were highly sensitive to ABT-199. All primary ALL samples tested (n=7) expressed high levels of Bcl-2, and no significant correlation between sensitivity and expression of Bcl-2 family members was found. Importantly, three human-derived xenografts from pediatric pre-B-ALL samples (1345, 1809, 0398) were very sensitive to ABT-199 (IC50 3nM, 0.1nM and 2.3nM, respectively). Finally, anti-leukemia activity of ABT-199 was tested in MLL-rearranged patient-derived xenograft NSG mice. Treatment with ABT-199 at 100mg/kg/d by oral gavage days 13-23 significantly reduced leukemia tumor burden as determined by bioluminescence imaging (average 70% reduction in BLI signal in 4 ABT-treated mice compared to 4 control animals at 9 weeks, p=0.03). In summary, proteomic profiling and patterns of sensitivity to Bcl-2 inhibition indicate that ALL, with exception of Burkitt's lymphoma, represents a Bcl-2 dependent disease. These results provide strong rationale for introducing ABT-199, which recently showed impressive efficacy in CLL trials, into the clinical armamentarium of ALL therapy. Disclosures: Konopleva: AbbVie, Inc: Research Funding. Leverson:AbbVie, Inc.: Employment, Equity Ownership.

scholarly journals Proteomic profiling reveals differentially expressed proteins associated with amylose accumulation during rice grain filling

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Hengdong Zhang ◽  
Jiana Chen ◽  
Shuanglü Shan ◽  
Fangbo Cao ◽  
Guanghui Chen ◽  
...  
Keyword(s):  
Amylose Content ◽  
Starch Synthesis ◽  
Grain Filling ◽  
Adenosine Diphosphate ◽  
Differentially Expressed ◽  
Rice Grain ◽  
Rice Cultivars ◽  
Differentially Expressed Proteins ◽  
Proteomic Profiling ◽  
Rice Grains

Abstract Background Amylose accumulation in rice grains is controlled by genetic and environmental factors. Amylose content is a determinant factor of rice quality in terms of cooking and eating. Great variations in amylose content in indica rice cultivars have been observed. The current study was to identify differentially expressed proteins in starch and sucrose metabolism and glycolysis/gluconeogenesis pathways and their relationships to amylose synthesis using two rice cultivars possess contrasting phenotypes in grain amylose content. Results Synthesis and accumulation of amylose in rice grains significantly affected the variations between rice cultivars in amylose contents. The high amylose content cultivar has three down-regulated differentially expressed proteins, i.e., LOC_Os01g62420.1, LOC_Os02g36600.1, and LOC_Os08g37380.2 in the glycolysis/gluconeogenesis pathway, which limit the glycolytic process and decrease the glucose-1-phosphate consumption. In the starch and sucrose metabolic pathway, an up-regulated protein, i.e., LOC_Os06g04200.1 and two down-regulated proteins, i.e., LOC_Os05g32710.1 and LOC_Os04g43360.1 were identified (Figure 4). Glucose-1-phosphate is one of the first substrates in starch synthesis and glycolysis that are catalyzed to form adenosine diphosphate glucose (ADPG), then the ADPG is catalyzed by granule-bound starch synthase I (GBSS I) to elongate amylose. Conclusions The results indicate that decreasing the consumption of glucose-1-phosphate in the glycolytic process is essential for the formation of ADPG and UDPG, which are substrates for amylose synthesis. In theory, amylose content in rice can be regulated by controlling the fate of glucose-1-phosphate.

scholarly journals Plasma proteomic profiles of healthy and mastitic cows – host responses to bovine mastitis

2013 ◽  
Vol 56 (1) ◽  
pp. 980-987 ◽  
Author(s):  
L.L. Niu ◽  
C.H. Wei ◽  
L.X. Du
Keyword(s):  
Dairy Cows ◽  
Acute Phase Proteins ◽  
Bovine Mastitis ◽  
Economic Loss ◽  
Differentially Expressed ◽  
Host Responses ◽  
Common Disease ◽  
Differentially Expressed Proteins ◽  
Proteomic Profiling ◽  
Two Dimensional Gel Electrophoresis

Abstract. Mastitis is the most common disease in dairy cows and has resulted in a tremendous economic loss in dairy industry. In the present study, differentially expressed proteins (DEP) were identified among healthy, moderate and severe mastitic cows by proteomic profiling. The health status of cows was closely determined by the somatic cell count (SCC). Differentially expressed proteins were resolved using the two-dimensional gel electrophoresis (2-DE) with the pH 4–7 non-linear DryStrips. Subsequently, 8 protein spots, which altered more than 3-fold, were isolated and identified with the matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF/TOF MS). The identified spots were split into four proteins: α-2-HS-glycoprotein, serum albumin, transthyretin (TTR) and haptoglobin, respectively. Compared with the healthy cows, the expression of haptoglobin was up-regulated in mastitic cows, and the others were down-regulated. Moreover, the proteomic data were consistent with the results of Western blot. All of the identified DEPs were acute phase proteins, which acted together and represented the consequence of serial cascades after mastitic infection. More importantly, the α-2-HS-glycoprotein was novel identified corresponding to the bovine mastitis in Chinese Holstein dairy cows. Taken together, our results indicate that the host responses may play an important role in the pathogenesis of mastitis and provide the potential diagnostic indicator of the underlying mastitis in dairy cows.

scholarly journals Improving Sperm Cryopreservation With Type III Antifreeze Protein: Proteomic Profiling of Cynomolgus Macaque (Macaca fascicularis) Sperm

2021 ◽  
Vol 12 ◽  
Author(s):  
Bingbing Chen ◽  
Shengnan Wang ◽  
Briauna Marie Inglis ◽  
Hao Ding ◽  
Angbaji Suo ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Sperm Motility ◽  
Cynomolgus Macaque ◽  
Antifreeze Protein ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Proteomic Profiling ◽  
Freezing And Thawing ◽  
Proteomic Change ◽  
Acrosomal Integrity

Antifreeze protein III (AFP III) is used for the cryopreservation of germ cells in various animal species. However, the exact mechanism of its cryoprotection is largely unknown at the molecular level. In this study, we investigated the motility, acrosomal integrity, and mitochondrial membrane potential (MMP), as well as proteomic change, of cynomolgus macaque sperm after cryopreservation. Sperm motility, acrosomal integrity, and MMP were lower after cryopreservation (p &lt; 0.001), but significant differences in sperm motility and MMP were observed between the AFP-treated sperm sample (Cryo+AFP) and the non-treated sample (Cryo–AFP) (p &lt; 0.01). A total of 141 and 32 differentially expressed proteins were, respectively, identified in cynomolgus macaque sperm cryopreserved without and with 0.1 μg/ml AFP III compared with fresh sperm. These proteins were mainly involved in the mitochondrial production of reactive oxygen species (ROS), glutathione (GSH) synthesis, and cell apoptosis. The addition of AFP III in the sperm freezing medium resulted in significant stabilization of cellular molecular functions and/or biological processes in sperm, as illustrated by the extent of proteomic changes after freezing and thawing. According to the proteomic change of differentially expressed proteins, we hypothesized a novel molecular mechanism for cryoprotection that AFP III may reduce the release of cytochrome c and thereby reduce sperm apoptosis by modulating the production of ROS in mitochondria. The molecular mechanism that AFP III acts with sperm proteins for cellular protection against cryoinjuries needs further study.

scholarly journals Proteomic profiling of von Hippel–Lindau syndrome and multiple endocrine neoplasia type 2 pheochromocytomas reveals different expression of chromogranin B

2007 ◽  
Vol 14 (2) ◽  
pp. 463-471 ◽  
Author(s):  
Frederieke M Brouwers ◽  
Sven Gläsker ◽  
Amanda F Nave ◽  
Alexander O Vortmeyer ◽  
Irina Lubensky ◽  
...  
Keyword(s):  
Multiple Endocrine Neoplasia ◽  
Multiple Endocrine Neoplasia Type ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Proteomic Profiling ◽  
Chromogranin B ◽  
Von Hippel Lindau ◽  
Endocrine Neoplasia ◽  
Von Hippel Lindau Syndrome

Pheochromocytomas are catecholamine-producing tumors that can occur in the context of von Hippel–Lindau syndrome (VHL) and multiple endocrine neoplasia type 2 (MEN2). Pheochromocytomas in these two syndromes differ in histopathological features, catecholamine metabolism, and clinical phenotype. To further investigate the nature of these differences, we compared the global protein expressions of 8 MEN2A-associated pheochromocytomas with 11 VHL-associated pheochromocytomas by two-dimensional gel electrophoresis proteomic profiling followed by sequencing and identification of differentially expressed proteins. Although both types of pheochromocytoma shared similarities in their protein expression patterns, the expression of several proteins was distinctly different between VHL- and MEN2A-associated pheochromocytomas. We identified several of these differentially expressed proteins. One of the proteins with higher expression in MEN2-associated tumors was chromogranin B, of which the differential expression was confirmed by western blot analysis. Our results expand the evidence for proteomic differences between these two tumor entities, and suggest that VHL-associated pheochromocytomas may be deficient in fundamental machinery for catecholamine storage. In light of these new findings, as well as existing evidence for differences between both types of pheochromocytomas, we propose that these tumors may have different developmental origins.

scholarly journals Proteomic profiling identifies outcome-predictive markers in patients with peripheral T-cell lymphoma, not otherwise specified

2018 ◽  
Vol 2 (19) ◽  
pp. 2533-2542 ◽  
Author(s):  
Maja Ludvigsen ◽  
Martin Bjerregård Pedersen ◽  
Kristina Lystlund Lauridsen ◽  
Tim Svenstrup Poulsen ◽  
Stephen Jacques Hamilton-Dutoit ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Expression ◽  
Lymphoid Tissue ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Proteomic Profiling ◽  
Peripheral T Cell Lymphoma ◽  
Not Otherwise Specified

Abstract Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) constitutes a heterogeneous category of lymphomas, which do not fit into any of the specifically defined T-cell lymphoma entities. Both the pathogenesis and tumor biology in PTCL-NOS are poorly understood. Protein expression in pretherapeutic PTCL-NOS tumors was analyzed by proteomics. Differentially expressed proteins were compared in 3 distinct scenarios: (A) PTCL-NOS tumor tissue (n = 18) vs benign lymphoid tissue (n = 8), (B) clusters defined by principal component analysis (PCA), and (C) tumors from patients with chemosensitive vs refractory PTCL-NOS. Selected differentially expressed proteins identified by proteomics were correlated with clinico-pathological features and outcome in a larger cohort of patients with PTCL-NOS (n = 87) by immunohistochemistry (IHC). Most proteins with altered expression were identified comparing PTCL-NOS vs benign lymphoid tissue. PCA of the protein profile defined 3 distinct clusters. All benign samples clustered together, whereas PTCL-NOS tumors separated into 2 clusters with different patient overall survival rates (P = .001). Differentially expressed proteins reflected large biological diversity among PTCL-NOS, particularly associated with alterations of “immunological” pathways. The 2 PTCL-NOS subclusters defined by PCA showed disturbance of “stress-related” and “protein metabolic” pathways. α-Enolase 1 (ENO1) was found differentially expressed in all 3 analyses, and high intratumoral ENO1 expression evaluated by IHC correlated with poor outcome (hazard ratio, 2.09; 95% confidence interval, 1.17-3.73; P = .013). High expression of triosephosphate isomerase (TPI1) also showed a tendency to correlate with poor survival (P = .057). In conclusion, proteomic profiling of PTCL-NOS provided evidence of markedly altered protein expression and identified ENO1 as a novel potential prognostic marker.

scholarly journals Proteomic profiling analysis reveals that glutathione system plays important roles responding to osmotic stress in wheat (Triticum aestivumL.) roots

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2334 ◽  
Author(s):  
Jianhui Ma ◽  
Wen Dong ◽  
Daijing Zhang ◽  
Xiaolong Gao ◽  
Lina Jiang ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Protein Expression ◽  
Absolute Quantification ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Glutathione System ◽  
Proteomic Profiling ◽  
Wheat Roots ◽  
Factors Affecting ◽  
Isobaric Tag

Wheat is one of the most important crops in the world, and osmotic stress has become one of the main factors affecting wheat production. Understanding the mechanism of the response of wheat to osmotic stress would be greatly significant. In the present study, isobaric tag for relative and absolute quantification (iTRAQ) was used to analyze the changes of protein expression in the wheat roots exposed to different osmotic stresses. A total of 2,228 expressed proteins, including 81 differentially expressed proteins, between osmotic stress and control, were found. The comprehensive analysis of these differentially expressed proteins revealed that osmotic stress increased the variety of expressed proteins and suppressed the quantity of expressed proteins in wheat roots. Furthermore, the proteins for detoxifying and reactive oxygen species scavenging, especially the glutathione system, played important roles in maintaining organism balance in response to osmotic stress in wheat roots. Thus, the present study comprehensively describes the protein expression changes in wheat roots in response to osmotic stress, providing firmer foundation to further study the mechanism of osmotic resistance in wheat.

Deep Proteomic Profiling Predicts Differential Chemosensitivity In Anaplastic Large Cell Lymphoma Cell Lines

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1670-1670
Author(s):  
Surendra Dasari ◽  
Kenneth L Johnson ◽  
Carrie J. H. Hepplemann ◽  
Ariel J Caride ◽  
Jason D Theis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Proteomic Profiling ◽  
Spectral Counts ◽  
Large Cell Lymphoma ◽  
Alk Positive ◽  
Druggable Targets

Abstract Background The lymphoma proteome is the phenotypic representation of the underlying genetic and epigenetic makeup of each individual patient's tumor. The proteome is rich in druggable targets and offers a unique opportunity for the hematologist to personalize therapy. We developed a deep proteomic profiling method using ion exchange fractionation and tandem mass spectrometry. In this pilot study, we applied this method to detect differentially expressed proteins in anaplastic large cell lymphoma (ALCL) cell lines with previously known (ALK-positive) and unknown (ALK-negative) therapeutic targets. We then asked whether integrative informatic analysis of these data could be used to predict drug sensitivity in each of the cell lines. Methods To examine reproducibility of our method, proteins were extracted independently from 4 pellets each of FE-PD (ALK-negative) and Karpas 299 (ALK-positive) ALCL cells, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Resulting peptides were separated into 6 fractions using strong anion exchange (SAX) chromatography. Peptides in each fraction were analyzed via shotgun proteomics on a QExactive mass spectrometer. Peptide mass spectra (MS/MS) were matched against a RefSeq human protein sequence database using MyriMatch software. Reversed sequences were added to the database to measure identification false discovery rates (FDRs). IDPicker filtered the peptide identifications at 2% FDR. Proteins with at least two unique peptide identifications and five MS/MS matches were considered to be present in the sample. Filtered protein identifications and corresponding spectral counts were used as input to QuasiTel software, which was configured to use proteins with at least one spectrum per biological replicate. Proteins that were significantly differentially expressed (quasi p-value < 0.05) with an absolute log2 fold-change of at least 0.5 fold were loaded into the Ingenuity Pathway Analysis (IPA) software, and a master list of drugs and corresponding gene targets was assembled using PharmGKB database and the Drug-Gene Interaction Database (DGID). The resulting drug-gene target list was merged with the differentially expressed protein identifications. Candidate targets were validated by Western blot and candidate drugs were assessed in viability assays. Results The SAX-LC-MS/MS method identified 10,111 proteins from all replicate analyses of FE-PD and Karpas 299 samples, and 93% of the identified proteome was detected in all 4 replicate analyses. The detected proteome was well represented by key transcription factors, phophatases, kinases, translation regulators and transmembrane regulators. There were 1369 proteins differentially expressed between the 2 cell lines, 709 up regulated in Karpas 299 cell line and 673 up regulated in FE-PD. Differentially expressed proteins also showed consistent expression across the biological replicates (Figure 1). Our integrated approach to identify candidate targets and drugs “rediscovered” ALK in Karpas299 and unexpectedly identified relative overexpression of the IL2-IL2RA-STAT5A-STAT5B network in ALK-negative FE-PD cells. Western blot confirmed these findings. As expected, 50-100 nM crizotinib (ALK inhibitor) decreased Karpas 299 viability (p =0.016) but had no effect on FE-PD (Figure 2). In contrast, 50-100 nM of the experimental STAT5 inhibitor 573108 (EMD Millipore) decreased FE-PD viability (p=0.002) but had no effect on Karpas 299 (Figure 2). Conclusion The lymphoma proteome is complex with 10,000 proteins and contains druggable targets that can be reproducibly identified using SAX-LC-MS/MS. These targets vary across samples and integrated informatic analysis can predict target-drug combinations that have efficacy in an experimental model. These data suggest that SAX-LC-MS/MS could be used to personalize treatment regimens in lymphoma patients. Figure 1: Spectral counts of 1369 differentially expressed proteins between Karpas299 and FE-PD cell lines were normalized and plotted in a heat map. Red color indicates down regulation and yellow color indicates up regulation. Disclosures: No relevant conflicts of interest to declare.

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