scholarly journals Multi-omics analysis of AML cells treated with azacitidine reveals highly variable cell surface proteome remodeling

2018 ◽  
Author(s):  
Kevin K Leung ◽  
Aaron Nguyen ◽  
Tao Shi ◽  
Lin Tang ◽  
Xiaochun Ni ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Protein Level ◽  
Dna Methyltransferase ◽  
Immune Defense ◽  
Surface Proteins ◽  
Gene Set Enrichment Analysis ◽  
Surface Proteome ◽  
The Impact ◽  
Cell Surface Proteome

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are diseases of abnormal hematopoietic differentiation with aberrant epigenetic alterations. Azacitidine (AZA) is a DNA methyltransferase inhibitor (DNMTi) widely used to treat MDS and AML, yet the impact of AZA on the cell surface proteome has not been defined. To identify potential therapeutic targets for use in combination with AZA in AML patients, we investigated the effects of AZA treatment on four AML cell lines (KG1a, HL60, HNT34, and AML193), representing different stages of differentiation. The effect of AZA treatment on these cell lines was characterized at three levels: the DNA methylome (methylation array), the transcriptome (gene expression array), and the cell surface proteome (glycoprotein capture with SILAC labeling). Untreated AML cell lines showed substantial overlap in their methylomes, transcriptomes, and cell surface proteomes. AZA treatment globally reduced DNA methylation in all cell lines, but changes in the transcriptome and surface proteome were subtle and differed among the cell lines. Transcriptome analysis identified five commonly up-regulated coding genes upon AZA treatment in all four cell lines, TRPM4 being the only gene encoding a surface protein, and surface proteomics analysis found no commonly regulated proteins. Gene Set Enrichment Analysis (GSEA) of differentially-regulated RNA and surface proteins showed a decrease in metabolism pathways and an increase in immune defense response pathways. As such, AZA treatment in four AML cell lines had diverse effects at the individual gene and protein level, but converged to regulation of metabolism and immune response at the pathway level. Given the heterogeneous response of AZA in the four cell lines at the gene and protein level, we discuss potential therapeutic strategies for combinations with AZA.

scholarly journals Multiomics of azacitidine-treated AML cells reveals variable and convergent targets that remodel the cell-surface proteome

2018 ◽  
Vol 116 (2) ◽  
pp. 695-700 ◽  
Author(s):  
Kevin K. Leung ◽  
Aaron Nguyen ◽  
Tao Shi ◽  
Lin Tang ◽  
Xiaochun Ni ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Immune Defense ◽  
Surface Proteins ◽  
Gene Set Enrichment Analysis ◽  
Individual Gene ◽  
Surface Proteome ◽  
The Impact ◽  
Cell Surface Proteome

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are diseases of abnormal hematopoietic differentiation with aberrant epigenetic alterations. Azacitidine (AZA) is a DNA methyltransferase inhibitor widely used to treat MDS and AML, yet the impact of AZA on the cell-surface proteome has not been defined. To identify potential therapeutic targets for use in combination with AZA in AML patients, we investigated the effects of AZA treatment on four AML cell lines representing different stages of differentiation. The effect of AZA treatment on these cell lines was characterized at three levels: the DNA methylome, the transcriptome, and the cell-surface proteome. Untreated AML cell lines showed substantial overlap at all three omics levels; however, while AZA treatment globally reduced DNA methylation in all cell lines, changes in the transcriptome and surface proteome were subtle and differed among the cell lines. Transcriptome analysis identified five commonly up-regulated coding genes upon AZA treatment in all four cell lines, TRPM4 being the only gene encoding a surface protein, and surface proteome analysis found no commonly regulated proteins. Gene set enrichment analysis of differentially regulated RNA and surface proteins showed a decrease in metabolic pathways and an increase in immune defense response pathways. As such, AZA treatment led to diverse effects at the individual gene and protein levels but converged to common responses at the pathway level. Given the heterogeneous responses in the four cell lines, we discuss potential therapeutic strategies for AML in combination with AZA.

scholarly journals Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy

2021 ◽  
Vol 118 (4) ◽  
pp. e2018861118
Author(s):  
Wentao Chen ◽  
Kurt Yun Mou ◽  
Paige Solomon ◽  
Rahul Aggarwal ◽  
Kevin K. Leung ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Surface Proteins ◽  
Cell Type ◽  
Hematological Cancers ◽  
A Cell ◽  
Surface Proteome ◽  
The Impact ◽  
Cell Surface Proteome

MYC is a powerful transcription factor overexpressed in many human cancers including B cell and prostate cancers. Antibody therapeutics are exciting opportunities to attack cancers but require knowledge of surface proteins that change due to oncogene expression. To identify how MYC overexpression remodels the cell surface proteome in a cell autologous fashion and in different cell types, we investigated the impact of MYC overexpression on 800 surface proteins in three isogenic model cell lines either of B cell or prostate cell origin engineered to have high or low MYC levels. We found that MYC overexpression resulted in dramatic remodeling (both up- and down-regulation) of the cell surfaceome in a cell type-dependent fashion. We found systematic and large increases in distinct sets of >80 transporters including nucleoside transporters and nutrient transporters making cells more sensitive to toxic nucleoside analogs like cytarabine, commonly used for treating hematological cancers. Paradoxically, MYC overexpression also increased expression of surface proteins driving cell turnover such as TNFRSF10B, also known as death receptor 5, and immune cell attacking signals such as the natural killer cell activating ligand NCR3LG1, also known as B7-H6. We generated recombinant antibodies to these two targets and verified their up-regulation in MYC overexpression cell lines and showed they were sensitive to bispecific T cell engagers (BiTEs). Our studies demonstrate how MYC overexpression leads to dramatic bidirectional remodeling of the surfaceome in a cell type-dependent but functionally convergent fashion and identify surface targets or combinations thereof as possible candidates for cytotoxic metabolite or immunotherapy.

scholarly journals Mapping the High-Risk Multiple Myeloma Cell Surface Proteome Identifies T-Cell Inhibitory Receptors for Immune Targeting

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 265-265
Author(s):  
Francesco Di Meo ◽  
Christina Yu ◽  
Annamaria Cesarano ◽  
Aljoufi Arafat ◽  
Silvia Marino ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Cell Surface ◽  
Cell Lines ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Surface Proteome ◽  
Cell Surface Proteome

Abstract Multiple myeloma (MM) is an incurable malignancy of mature plasma cells. Despite major advances in the therapeutic armamentarium of MM, only 50% of patients survive more than 5 years after diagnosis, with significantly lower rates (21%) for high-risk patients. Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA (B-cell maturation antigen) shows high response rates in relapsed/refractory patients. However, most patients have disease remission that lasts less than 18 months, prompting the search for additional and synergistic therapeutic approaches. We unbiasedly mapped the cell surface proteome of MM by integrating Mass-Spectrometry (MS) and RNA-seq analyses from 7 MM cell lines and 904 primary MM patient samples bearing high-risk cytogenetics. To identify cell surface proteins, we ran a pool of 4,761 proteins and 16,000+ transcripts through five repositories. An integrated scoring database was developed by scoring each ID based on the number of databases (0-5) it was identified in, with 0 if the molecule was not found in any and 5 if the protein was found in all five. We identified 402 proteins with a surface score of 3 or higher in MM cell lines and patient samples by transcriptomics and proteomics. We prioritized the 326 candidates that were more highly expressed in patients. Based on functional enrichment analyses, we found the proteins formed three main networks with immune mechanisms representing the largest cluster (227 out of 326 cell surface proteins) followed by transporters and adhesion proteins.Based on a pipeline we previously established (1), we further selected 97 candidates minimally expressed in normal tissues. This list included current therapeutic targets such as BCMA, SLAMF7, ITGB7 and LY9. Validation in primary patient samples by western blot and flow-cytometric analyses, enabled the identification of 10 top candidates (CCR1, CD320, FCRL3, IL12RB1, ITGA4, LAX1, LILRB4, LRRC8D, SEMA4A, SLAMF6) that resulted most frequently and highly expressed. We found that LAX1, LILRB4 and SEMA4A significantly impact myeloma patient overall survival based on Kaplan-Meier analysis in the MM Research Foundation (MMRF) cohort (2). CCR1, IL12RB1, LILRB4 and SEMA4A were upregulated by the treatment with Bortezomib or Venetoclax that conversely, decreased BCMA expression in MM U266 cells. By stratifying the patient population, we found that the SEMA4A and LAX1 were up-regulated in patients with t(4;14) compared to patients with no cytogenetic abnormality; LILRB4 in patients with t(14;16) and CCR1 patients with t(14;16) and t(14;20). By calculating co-expression levels CCR1-LILRB4 and CCR1-FCRL3 resulted co-expressed in 100% of patients. For safety purposes (3), we excluded candidates with high (>55%) protein abundance in highly-purified normal hematopoietic stem cells and activated T-cells, narrowing down the list to 6 top candidates (CCR1, FCRL3, IL12RB1, LILRB4, LRRC8D, SEMA4A). To define the function of this group of promising cell surface targets, we used a CRISPR/Cas9 inducible system in KMS11 MM cells. We found that knock-out of CCR1, LRRC8D and SEMA4A individually reduces the MM cell growth by ~60%, 50% and 50% respectively, and almost completely abrogates MM cell migration through porous chambers by >80%. By co-culturing irradiated KO and control MM cells with healthy donor T-cells we also found that lack of CCR1 increased T-cell proliferation by 50% compared to controls and enhanced killing of MM cells, suggesting that CCR1 may suppress T-cell mediated immune responses in addition to play a role in MM cell survival and migration. This study suggests the contribution of an altered MM surfaceome to disease development and may lead to potential novel immunotherapeutic approaches for high-risk MM. References 1. Perna F et al., Cancer Cell 2017 3. Dong C et al., in press Oncogene 2021 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Antibody‐based cell‐surface proteome profiling of metastatic breast cancer primary explants and cell lines

2018 ◽  
Vol 93 (4) ◽  
pp. 448-457 ◽  
Author(s):  
Vera S. Donnenberg ◽  
Jayce Jieming Zhang ◽  
Erika Moravcikova ◽  
Ernest Michael Meyer ◽  
Haihui Lu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Cell Surface ◽  
Cell Lines ◽  
Metastatic Breast ◽  
Proteome Profiling ◽  
Primary Explants ◽  
Surface Proteome ◽  
Cell Surface Proteome

scholarly journals AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

2021 ◽  
Author(s):  
Antonescu Costin ◽  
Eden Ross ◽  
Rehman Ata ◽  
Thanusi Thavarajah ◽  
Sergei Medvedev ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Cell Surface ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Metabolic Sensor ◽  
Acute Changes ◽  
Surface Proteome ◽  
Amp Activated Protein Kinase ◽  
Cell Surface Proteome

The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute regulation by treatment with A-769662, at least some of which is mediated by the metabolic sensor AMPK.

scholarly journals Discovery and validation of surface N-glycoproteins in MM cell lines and patient samples uncovers immunotherapy targets

2020 ◽  
Vol 8 (2) ◽  
pp. e000915
Author(s):  
Robyn A A Oldham ◽  
Mary L Faber ◽  
Theodore R Keppel ◽  
Amanda R Buchberger ◽  
Matthew Waas ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Multiple Myeloma ◽  
Cell Surface ◽  
Cell Lines ◽  
Patient Outcomes ◽  
Plasma Cells ◽  
Clonal Expansion ◽  
Valuable Resource ◽  
Surface Proteome ◽  
Cell Surface Proteome

BackgroundMultiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow. While recent advances in treatment for MM have improved patient outcomes, the 5-year survival rate remains ~50%. A better understanding of the MM cell surface proteome could facilitate development of new directed therapies and assist in stratification and monitoring of patient outcomes.MethodsIn this study, we first used a mass spectrometry (MS)-based discovery-driven cell surface capture (CSC) approach to map the cell surface N-glycoproteome of MM cell lines. Next, we developed targeted MS assays, and applied these to cell lines and primary patient samples to refine the list of candidate tumor markers. Candidates of interest detected by MS on MM patient samples were further validated using flow cytometry (FCM).ResultsWe identified 696 MM cell surface N-glycoproteins by CSC, and developed 73 targeted MS detection assays. MS-based validation using primary specimens detected 30 proteins with significantly higher abundance in patient MM cells than controls. Nine of these proteins were identified as potential immunotherapeutic targets, including five that were validated by FCM, confirming their expression on the cell surface of primary MM patient cells.ConclusionsThis MM surface N-glycoproteome will be a valuable resource in the development of biomarkers and therapeutics. Further, we anticipate that our targeted MS assays will have clinical benefit for the diagnosis, stratification, and treatment of MM patients.

scholarly journals Exploring the surfaceome of Ewing sarcoma identifies a new and unique therapeutic target

2016 ◽  
Vol 113 (13) ◽  
pp. 3603-3608 ◽  
Author(s):  
Jennifer Town ◽  
Helio Pais ◽  
Sally Harrison ◽  
Lucy F. Stead ◽  
Carole Bataille ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ewing Sarcoma ◽  
Somatic Tissue ◽  
Surface Proteins ◽  
Tumor Type ◽  
Rna Seq ◽  
Genes Encoding ◽  
A Cell ◽  
Surface Proteome ◽  
Cell Surface Proteome

The cell surface proteome of tumors mediates the interface between the transformed cells and the general microenvironment, including interactions with stromal cells in the tumor niche and immune cells such as T cells. In addition, the cell surface proteome of individual cancers defines biomarkers for that tumor type and potential proteins that can be the target of antibody-mediated therapy. We have used next-generation deep RNA sequencing (RNA-seq) coupled to an in-house database of genes encoding cell surface proteins (herein referred to as the surfaceome) as a tool to define a cell surface proteome of Ewing sarcoma compared with progenitor mesenchymal stem cells. This subtractive RNA-seq analysis revealed a specific surfaceome of Ewing and showed unexpectedly that the leucine-rich repeat and Ig domain protein 1 (LINGO1) is expressed in over 90% of Ewing sarcoma tumors, but not expressed in any other somatic tissue apart from the brain. We found that the LINGO1 protein acts as a gateway protein internalizing into the tumor cells when engaged by antibody and can carry antibody conjugated with drugs to kill Ewing sarcoma cells. Therefore, LINGO1 is a new, unique, and specific biomarker and drug target for the treatment of Ewing sarcoma.

scholarly journals AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

2021 ◽  
Author(s):  
Antonescu Costin ◽  
Eden Ross ◽  
Rehman Ata ◽  
Thanusi Thavarajah ◽  
Sergei Medvedev ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Cell Surface ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Metabolic Sensor ◽  
Acute Changes ◽  
Surface Proteome ◽  
Amp Activated Protein Kinase ◽  
Cell Surface Proteome

The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute regulation by treatment with A-769662, at least some of which is mediated by the metabolic sensor AMPK.

scholarly journals CIRFESS: An interactive resource for querying the set of theoretically detectable peptides for cell surface and extracellular enrichment proteomic studies

2020 ◽  
Author(s):  
Matthew Waas ◽  
Jack Littrell ◽  
Rebekah L. Gundry
Keyword(s):  
Cell Surface ◽  
Critical Role ◽  
Affinity Reagents ◽  
Extracellular Proteome ◽  
Online Resource ◽  
Health And Disease ◽  
Limited Mass ◽  
Multiple Prediction ◽  
Surface Proteome ◽  
Cell Surface Proteome

AbstractCell surface transmembrane, extracellular, and secreted proteins are high value targets for immunophenotyping, drug development, and studies related to intercellular communication in health and disease. As the number of specific and validated affinity reagents that target this subproteome are limited, mass spectrometry (MS)-based approaches will continue to play a critical role in enabling discovery and quantitation of these molecules. Given the technical considerations that make MS-based cell surface proteome studies uniquely challenging, it can be difficult to select an appropriate experimental approach. To this end, we have integrated multiple prediction strategies and annotations into a single online resource, Compiled Interactive Resource for Extracellular and Surface Studies (CIRFESS). CIRFESS enables rapid interrogation of the human proteome to reveal the cell surface proteome theoretically detectable by current approaches and highlights where current prediction strategies provide concordant and discordant information. We applied CIRFESS to identify the percentage of various subsets of the proteome which are expected to be captured by targeted enrichment strategies, including two established methods and one that is possible but not yet demonstrated. These results will inform the selection of available proteomic strategies and development of new strategies to enhance coverage of the cell surface and extracellular proteome. CIRFESS is available at www.cellsurfer.net/cirfess.

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