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Pediatric acute myeloid leukemia (AML) remains a fatal disease for at least 30% of patients, stressing the need for improved therapies and better risk stratification. As proteins are the unifying feature of (epi)genetic and environmental alterations, and are often targeted by novel chemotherapeutic agents, we studied the proteomic landscape of pediatric AML. Protein expression and activation levels were measured in 500 bulk leukemic patients' samples and 30 control CD34+ cell samples, using reverse phase protein arrays with 296 strictly validated antibodies. The multistep MetaGalaxy analysis methodology was applied and identified nine protein expression signatures (PrSIG), based on strong recurrent protein expression patterns. PrSIG were associated with cytogenetics and mutational state, and with favorable or unfavorable prognosis. Analysis based on treatment (i.e., ADE vs. ADE plus bortezomib) identified three PrSIG that did better with ADE plus bortezomib than with ADE alone. When PrSIG were studied in the context of cytogenetic risk groups, PrSIG were independently prognostic after multivariate analysis, suggesting a potential value for proteomics in combination with current classification systems. Proteins with universally increased (n=7) or decreased (n=17) expression were observed across PrSIG. Certain proteins significantly differentially expressed from normal could be identified, forming a hypothetical platform for personalized medicine.
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Leucemia Mieloide Aguda , Proteômica , Bortezomib , Criança , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Prognóstico , Análise Serial de Proteínas , ProteínasRESUMO
Calcific aortic valve disease (CAVD) is the most common heart valve disease requiring intervention. Most research on CAVD has focused on inflammation, ossification, and cellular phenotype transformation. To gain a broader picture into the wide range of cellular and molecular mechanisms involved in this disease, we compared the total protein profiles between calcified and non-calcified areas from 5 human valves resected during surgery. The 1413 positively identified proteins were filtered down to 248 proteins present in both calcified and non-calcified segments of at least 3 of the 5 valves, which were then analyzed using Ingenuity Pathway Analysis. Concurrently, the top 40 differentially abundant proteins were grouped according to their biological functions and shown in interactive networks. Finally, the abundance of selected osteogenic proteins (osteopontin, osteonectin, osteocalcin, osteoprotegerin, and RANK) was quantified using ELISA and/or immunohistochemistry. The top pathways identified were complement system, acute phase response signaling, metabolism, LXR/RXR and FXR/RXR activation, actin cytoskeleton, mineral binding, nucleic acid interaction, structural extracellular matrix (ECM), and angiogenesis. There was a greater abundance of osteopontin, osteonectin, osteocalcin, osteoprotegerin, and RANK in the calcified regions than the non-calcified ones. The osteogenic proteins also formed key connections between the biological signaling pathways in the network model. In conclusion, this proteomic analysis demonstrated the involvement of multiple signaling pathways in CAVD. The interconnectedness of these pathways provides new insights for the treatment of this disease.
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Estenose da Valva Aórtica , Calcinose , Valva Aórtica/metabolismo , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/cirurgia , Calcinose/metabolismo , Humanos , Osteogênese/fisiologia , Proteoma/metabolismo , ProteômicaRESUMO
Acute myeloid leukemia (AML) is a heterogeneous disease with variable responses to therapy. Cytogenetic and genomic features are used to classify AML patients into prognostic and treatment groups. However, these molecular characteristics harbor significant patient-to-patient variability and do not fully account for AML heterogeneity. RNA-based classifications have also been applied in AML as an alternative approach, but transcriptomic grouping is strongly associated with AML morphologic lineages. We used a training cohort of newly diagnosed AML patients and conducted unsupervised RNA-based classification after excluding lineage-associated genes. We identified three AML patient groups that have distinct biological pathways associated with outcomes. Enrichment of inflammatory pathways and downregulation of HOX pathways were associated with improved outcomes, and this was validated in 2 independent cohorts. We also identified a group of AML patients who harbored high metabolic and mTOR pathway activity, and this was associated with worse clinical outcomes. Using a comprehensive reverse phase protein array, we identified higher mTOR protein expression in the highly metabolic group. We also identified a positive correlation between degree of resistance to venetoclax and mTOR activation in myeloid and lymphoid cell lines. Our approach of integrating RNA, protein, and genomic data uncovered lineage-independent AML patient groups that share biologic mechanisms and can inform outcomes independent of commonly used clinical and demographic variables; these groups could be used to guide therapeutic strategies.
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BACKGROUND: Galectin 3 (LGALS3) gene expression is associated with poor survival in acute myeloid leukemia (AML) but the prognostic impact of LGALS3 protein expression in AML is unknown. LGALS3 supports diverse survival pathways including RAS mediated cascades, protein expression and stability of anti-apoptotic BCL2 family members, and activation of proliferative pathways including those mediated by beta Catenin. CD74 is a positive regulator of CD44 and CXCR4 signaling and this molecule may be critical for AML stem cell function. At present, the role of LGALS3 and CD74 in AML is unclear. In this study, we examine protein expression of LGALS3 and CD74 by reverse phase protein analysis (RPPA) and identify new protein networks associated with these molecules. In addition, we determine prognostic potential of LGALS3, CD74, and their protein networks for clinical correlates in AML patients. METHODS: RPPA was used to determine relative expression of LGALS3, CD74, and 229 other proteins in 231 fresh AML patient samples and 205 samples were from patients who were treated and evaluable for outcome. Pearson correlation analysis was performed to identify proteins associated with LGALS3 and CD74. Progeny clustering was performed to generate protein networks. String analysis was performed to determine protein:protein interactions in networks and to perform gene ontology analysis. Kaplan-Meir method was used to generate survival curves. FINDINGS: LGALS3 is highest in monocytic AML patients and those with elevated LGALS3 had significantly shorter remission duration compared to patients with lower LGALS3 levels (median 21.9 vs 51.3â¯weeks, pâ¯=â¯0.016). Pearson correlation of LGALS3 with 230 other proteins identifies a distinct set of 37 proteins positively correlated with LGALS3 expression levels with a high representation of proteins involved in AKT and ERK signaling pathways. Thirty-one proteins were negatively correlated with LGALS3 including an AKT phosphatase. Pearson correlation of proteins associated with CD74 identified 12 proteins negatively correlated with CD74 and 16 proteins that are positively correlated with CD74. CD74 network revealed strong association with CD44 signaling and a high representation of apoptosis regulators. Progeny clustering was used to build protein networks based on LGALS3 and CD74 associated proteins. A strong relationship of the LGALS3 network with the CD74 network was identified. For AML patients with both the LGALS3 and CD74 protein cluster active, median overall survival was only 24.3â¯weeks, median remission duration was 17.8â¯weeks, and no patient survived beyond one year. INTERPRETATION: The findings from this study identify for the first time protein networks associated with LGALS3 and CD74 in AML. Each network features unique pathway characteristics. The data also suggest that the LGALS3 network and the CD74 network each support AML cell survival and the two networks may cooperate in a novel high risk AML population. FUND: Leukemia Lymphoma Society provided funds to SMK for RPPA study of AML patient population. Texas Leukemia provided funds to PPR and SMK to study CD74 and LGALS3 expression in AML patients using RPPA. No payment was involved in the production of this manuscript.
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Biomarcadores Tumorais , Ligante CD27/metabolismo , Galectina 3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Adulto , Idoso , Proteínas Sanguíneas , Ligante CD27/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , Feminino , Galectina 3/genética , Galectinas , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Transdução de SinaisRESUMO
DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.
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Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Bactérias/metabolismo , Instabilidade Cromossômica/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Instabilidade Genômica , Humanos , Proteínas de Membrana Transportadoras/fisiologia , Mutagênese , Mutação , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: Acute promyelocytic leukemia (APL) is the most prognostically favorable subtype of Acute myeloid leukemia (AML). Defining the features that allow identification of APL patients likely to relapse after therapy remains challenging. EXPERIMENTAL DESIGN: Proteomic profiling is performed on 20 newly diagnosed APL, 205 non-APL AML, and 10 normal CD34+ samples using Reverse Phase Protein Arrays probed with 230 antibodies. RESULTS: Comparison between APL and non-APL AML samples identifies 8.3% of the proteins to be differentially expressed. Proteins higher expressed in APL are involved in the pro-apoptotic pathways or are linked to higher proliferation. The "MetaGalaxy" approach that considers proteins in relation to other assayed proteins stratifies the APL patients into two protein signatures. All of the relapse patients (n = 4/4) are in protein signature 2 (S2). Comparison of proteins between the signatures shows significant differences in relative expression for 38 proteins. Protein expression summary plots suggest less translational activity in combination with a less proliferative character for S2 compared to signature 1. CONCLUSIONS AND CLINICAL RELEVANCE: This study provides a potential proteomic-based classification of APL patients that may be useful for risk stratification and therapeutic guidance. Validation in a larger independent cohort is required.
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Perfilação da Expressão Gênica , Leucemia Promielocítica Aguda/sangue , Proteínas de Neoplasias/sangue , Análise Serial de Proteínas , Proteômica , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Although cure rates for acute leukemia have steadily improved over the past decades, leukemia remains a deadly disease. Enhanced risk stratification and new therapies are needed to improve outcome. Extensive genetic analyses have identified many mutations that contribute to the development of leukemia. However, most mutations occur infrequently and most gene alterations have been difficult to target. Most patients have more than one driver mutation in combination with secondary mutations, that result in a leukemic transformation via the alteration of proteins. The proteomics of acute leukemia could more directly identify proteins to facilitate risk stratification, predict chemoresistance and aid selection of therapy. Areas covered: This review discusses aberrantly expressed proteins identified by mass spectrometry and reverse phase protein arrays and their relationship to survival. In addition, we will discuss proteins in the context of functionally related protein groups. Expert commentary: Proteomics is a powerful tool to analyze protein abundance and functional alterations simultaneously for large numbers of patients. In the forthcoming years, validation of tools to quickly assess protein levels to enable routine rapid profiling of proteins with differential abundance and functional activation may be used as adjuncts to aid in therapy selection and to provide additional prognostic insights.
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Leucemia/metabolismo , Proteômica/métodos , Pesquisa Translacional Biomédica , Animais , Biomarcadores Tumorais/metabolismo , Humanos , Leucemia/diagnóstico , Transdução de SinaisRESUMO
Melanoma, one of the toughest tumors to treat, features high metastasis and high lethality. Pinocembrin is a natural flavanone with versatile biological and pharmacological activities. Here, we evaluated the anti-tumor effects of pinocembrin against melanoma in vitro and in vivo. In vitro, pinocembrin inhibited the proliferation of melanoma cells (B16F10 and A375) in a dose-dependent manner. It induced endoplasmic reticulum stress via IRE1α/Xbp1 pathway and triggered caspase-12/-4 mediated apoptosis in both cell lines. Furthermore, we discovered that pinocembrin suppressed autophagy through the activation of PI3K/Akt/mTOR pathway, which serves as a dual mechanism to enhance the pro-death effect of pinocembrin. In vivo, pinocembrin inhibited the growth of B16F10 by inducing apoptosis. Taken together, our results demonstrated that pinocembrin can induce ER stress mediated apoptosis and suppress autophagy in melanoma, indicating its application potential for melanoma therapy.
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Apoptose , Autofagia , Estresse do Retículo Endoplasmático , Flavanonas/farmacologia , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Ativação Enzimática , Células HEK293 , Humanos , Melanoma Experimental , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Heterogeneity in the genetic landscape of pediatric acute myeloid leukemia (AML) makes personalized medicine challenging. As genetic events are mediated by the expression and function of proteins, recognition of recurrent protein patterns could enable classification of pediatric AML patients and could reveal crucial protein dependencies. This could help to rationally select combinations of therapeutic targets. To determine whether protein expression levels could be clustered into functionally relevant groups, custom reverse-phase protein arrays were performed on pediatric AML (n = 95) and CD34+ normal bone marrow (n = 10) clinical specimens using 194 validated antibodies. To analyze proteins in the context of other proteins, all proteins were assembled into 31 protein functional groups (PFG). For each PFG, an optimal number of protein clusters was defined that represented distinct transition states. Block clustering analysis revealed strong correlations between various protein clusters and identified the existence of 12 protein constellations stratifying patients into 8 protein signatures. Signatures were correlated with therapeutic outcome, as well as certain laboratory and demographic characteristics. Comparison of acute lymphoblastic leukemia specimens from the same array and AML pediatric patient specimens demonstrated disease-specific signatures, but also identified the existence of shared constellations, suggesting joint protein deregulation between the diseases.Implication: Recognition of altered proteins in particular signatures suggests rational combinations of targets that could facilitate stratified targeted therapy. Mol Cancer Res; 16(8); 1275-86. ©2018 AACRSee related article by Hoff et al., p. 1263.
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Perfilação da Expressão Gênica/métodos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Proteínas/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/patologia , Masculino , Adulto JovemRESUMO
Pediatric acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, and the second leading cause of pediatric cancer-related death in developed countries. While the cure rate for newly diagnosed ALL is excellent, the genetic heterogeneity and chemoresistance of leukemia cells at relapse makes individualized curative treatment plans difficult. We hypothesize that genetic events would coalesce into a finite number of protein signatures that could guide the design of individualized therapy. Custom reverse-phase protein arrays were produced from pediatric ALL (n = 73) and normal CD34+ (n = 10) samples with 194 validated antibodies. Proteins were allocated into 31 protein functional groups (PFG) to analyze them in the context of other proteins, based on known associations from the literature. The optimal number of protein clusters was determined for each PFG. Protein networks showed distinct transition states, revealing "normal-like" and "leukemia-specific" protein patterns. Block clustering identified strong correlation between various protein clusters that formed 10 protein constellations. Patients that expressed similar recurrent combinations of constellations comprised 7 distinct signatures, correlating with risk stratification, cytogenetics, and laboratory features. Most constellations and signatures were specific for T-cell ALL or pre-B-cell ALL; however, some constellations showed significant overlap. Several signatures were associated with Hispanic ethnicity, suggesting that ethnic pathophysiologic differences likely exist. In addition, some constellations were enriched for "normal-like" protein clusters, whereas others had exclusively "leukemia-specific" patterns.Implications: Recognition of proteins that have universally altered expression, together with proteins that are specific for a given signature, suggests targets for directed combinatorial inhibition or replacement to enable personalized therapy. Mol Cancer Res; 16(8); 1263-74. ©2018 AACRSee related article by Hoff et al., p. 1275.
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Perfilação da Expressão Gênica/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Proteínas/genética , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
Posttranslational histone tail modifications are known to play a role in leukemogenesis and are therapeutic targets. A global analysis of the level and patterns of expression of multiple histone-modifying proteins (HMP) in acute myeloid leukemia (AML) and the effect of different patterns of expression on outcome and prognosis has not been investigated in AML patients. Here we analyzed 20 HMP by reverse phase protein array (RPPA) in a cohort of 205 newly diagnosed AML patients. Protein levels were correlated with patient and disease characteristics, including survival and mutational state. We identified different protein clusters characterized by higher (more on) or lower (more off) expression of HMP, relative to normal CD34+ cells. On state of HMP was associated with poorer outcome compared to normal-like and a more off state. FLT3 mutated AML patients were significantly overrepresented in the more on state. DNA methylation related mutations showed no correlation with the different HMP states. In this study, we demonstrate for the first time that HMP form recurrent patterns of expression and that these significantly correlate with survival in newly diagnosed AML patients.
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Regulação Leucêmica da Expressão Gênica , Código das Histonas , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Metilação de DNA , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise Serial de Proteínas , Mapas de Interação de Proteínas , Análise de SobrevidaRESUMO
BACKGROUND: Many common clustering algorithms require a two-step process that limits their efficiency. The algorithms need to be performed repetitively and need to be implemented together with a model selection criterion. These two steps are needed in order to determine both the number of clusters present in the data and the corresponding cluster memberships. As biomedical datasets increase in size and prevalence, there is a growing need for new methods that are more convenient to implement and are more computationally efficient. In addition, it is often essential to obtain clusters of sufficient sample size to make the clustering result meaningful and interpretable for subsequent analysis. RESULTS: We introduce Shrinkage Clustering, a novel clustering algorithm based on matrix factorization that simultaneously finds the optimal number of clusters while partitioning the data. We report its performances across multiple simulated and actual datasets, and demonstrate its strength in accuracy and speed applied to subtyping cancer and brain tissues. In addition, the algorithm offers a straightforward solution to clustering with cluster size constraints. CONCLUSIONS: Given its ease of implementation, computing efficiency and extensible structure, Shrinkage Clustering can be applied broadly to solve biomedical clustering tasks especially when dealing with large datasets.
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Algoritmos , Encéfalo/metabolismo , Neoplasias da Mama/diagnóstico , Análise por Conglomerados , Bases de Dados Factuais , Feminino , Regulação da Expressão Gênica , Humanos , Neoplasias/classificação , Neoplasias/genética , Neoplasias/patologiaRESUMO
It remains unclear whether causal, rather than merely correlational, relationships in molecular networks can be inferred in complex biological settings. Here we describe the HPN-DREAM network inference challenge, which focused on learning causal influences in signaling networks. We used phosphoprotein data from cancer cell lines as well as in silico data from a nonlinear dynamical model. Using the phosphoprotein data, we scored more than 2,000 networks submitted by challenge participants. The networks spanned 32 biological contexts and were scored in terms of causal validity with respect to unseen interventional data. A number of approaches were effective, and incorporating known biology was generally advantageous. Additional sub-challenges considered time-course prediction and visualization. Our results suggest that learning causal relationships may be feasible in complex settings such as disease states. Furthermore, our scoring approach provides a practical way to empirically assess inferred molecular networks in a causal sense.
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Causalidade , Redes Reguladoras de Genes , Neoplasias/genética , Mapeamento de Interação de Proteínas/métodos , Software , Biologia de Sistemas , Algoritmos , Biologia Computacional , Simulação por Computador , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Estimating the optimal number of clusters is a major challenge in applying cluster analysis to any type of dataset, especially to biomedical datasets, which are high-dimensional and complex. Here, we introduce an improved method, Progeny Clustering, which is stability-based and exceptionally efficient in computing, to find the ideal number of clusters. The algorithm employs a novel Progeny Sampling method to reconstruct cluster identity, a co-occurrence probability matrix to assess the clustering stability, and a set of reference datasets to overcome inherent biases in the algorithm and data space. Our method was shown successful and robust when applied to two synthetic datasets (datasets of two-dimensions and ten-dimensions containing eight dimensions of pure noise), two standard biological datasets (the Iris dataset and Rat CNS dataset) and two biological datasets (a cell phenotype dataset and an acute myeloid leukemia (AML) reverse phase protein array (RPPA) dataset). Progeny Clustering outperformed some popular clustering evaluation methods in the ten-dimensional synthetic dataset as well as in the cell phenotype dataset, and it was the only method that successfully discovered clinically meaningful patient groupings in the AML RPPA dataset.