Insight into Mantle Cell Lymphoma Pathobiology, Diagnosis, and Treatment Using Network-Based and Drug-Repurposing Approaches.

Mantle cell lymphoma (MCL) is a rare, incurable, and aggressive B-cell non-Hodgkin lymphoma (NHL). Early MCL diagnosis and treatment is critical and puzzling due to inter/intra-tumoral heterogeneity and limited understanding of the underlying molecular mechanisms. We developed and applied a multifaceted analysis of selected publicly available transcriptomic data of well-defined MCL stages, integrating network-based methods for pathway enrichment analysis, co-expression module alignment, drug repurposing, and prediction of effective drug combinations. We demonstrate the "butterfly effect" emerging from a small set of initially differentially expressed genes, rapidly expanding into numerous deregulated cellular processes, signaling pathways, and core machineries as MCL becomes aggressive. We explore pathogenicity-related signaling circuits by detecting common co-expression modules in MCL stages, pointing out, among others, the role of VEGFA and SPARC proteins in MCL progression and recommend further study of precise drug combinations. Our findings highlight the benefit that can be leveraged by such an approach for better understanding pathobiology and identifying high-priority novel diagnostic and prognostic biomarkers, drug targets, and efficacious combination therapies against MCL that should be further validated for their clinical impact.

Interruption of p53-MDM2 Interaction by Nutlin-3a in Human Lymphoma Cell Models Initiates a Cell-Dependent Global Effect on Transcriptome and Proteome Level.

In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and proteomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin-3a, on three lymphoma cell models following p53 activation. Our analysis revealed a system-wide nutlin-3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activation effects, involving transcription, translation, or degradation of protein components of pathways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and glycolysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mitochondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin-3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis.

Invariable Ribosome Stoichiometry During Murine Erythroid Differentiation: Implications for Understanding Ribosomopathies.

Heterogeneity of the main ribosomal composition represents an emerging, yet debatable, mechanism of gene expression regulation with a purported role in ribosomopathies, a group of disorders caused by mutations in ribosomal protein genes (RPs). Ribosomopathies, mysteriously relate with tissue-specific symptoms (mainly anemia and cancer predisposition), despite the ubiquitous expression and necessity of the associated RPs. An outstanding question that may shed light into disease pathogenicity and provide potential pharmacological interventions, is whether and how the ribosomal composition is modified during, the highly affected by RP mutations, process of erythroid differentiation. To address this issue, we analyzed ribosome stoichiometry using an established model of erythroid differentiation, through sucrose gradient ultracentrifugation and quantitative proteomics. We found that differentiation associates with an extensive reprogramming of the overall ribosomal levels, characterized by an increase in monosomes and a decrease in polysomes. However, by calculating a stoichiometry score for each independent ribosomal protein, we found that the main ribosomal architecture remained invariable between immature and differentiated cells. In total, none of the 78 Ribosomal Proteins (RPs- 74 core RPs, Rack1, Fau and 2 paralogs) detected was statistically different between the samples. This data was further verified through antibody-mediated quantification of 6 representative RPs. Moreover, bioinformatic analysis of whole cell proteomic data derived out of 4 additional models of erythropoiesis revealed that RPs were co-regulated across these cell types, too. In conclusion, ribosomes maintain an invariant protein stoichiometry during differentiation, thus excluding ribosome heterogeneity from a potential mechanism of toxicity in ribosomopathies and other erythroid disorders.

Subtyping on Live Lymphoma Cell Lines by Raman Spectroscopy.

Raman spectroscopy is a well-defined spectroscopic technique sensitive to the molecular vibrations of materials, since it provides fingerprint-like information regarding the molecular structure of the analyzed samples. It has been extensively used for non-destructive and label-free cell characterization, particularly in the qualitative and quantitative estimation of amino acids, lipids, nucleic acids, and carbohydrates. Lymphoma cell classification is a crucial task for accurate and prompt lymphoma diagnosis, prognosis, and treatment. Currently, it is mostly based on limited information and requires costly and time-consuming approaches. In this work, we are proposing a fast characterization and differentiation methodology of lymphoma cell subtypes based on Raman spectroscopy. The study was performed in the temperature range of 15-37 °C to identify the best cell measurement conditions. The proposed methodology is fast, accurate, and requires minimal sample preparation, resulting in a potentially promising, non-invasive strategy for early and accurate cell lymphoma characterization.

Proteomics and Drug Repurposing in CLL towards Precision Medicine.

CLL is a hematological malignancy considered as the most frequent lymphoproliferative disease in the western world. It is characterized by high molecular heterogeneity and despite the available therapeutic options, there are many patient subgroups showing the insufficient effectiveness of disease treatment. The challenge is to investigate the individual molecular characteristics and heterogeneity of these patients. Proteomics analysis is a powerful approach that monitors the constant state of flux operators of genetic information and can unravel the proteome heterogeneity and rewiring into protein pathways in CLL patients. This review essences all the available proteomics studies in CLL and suggests the way these studies can be exploited to find effective therapeutic options combined with drug repurposing approaches. Drug repurposing utilizes all the existing knowledge of the safety and efficacy of FDA-approved or investigational drugs and anticipates drug alignment to crucial CLL therapeutic targets, leading to a better disease outcome. The drug repurposing studies in CLL are also discussed in this review. The next goal involves the integration of proteomics-based drug repurposing in precision medicine, as well as the application of this procedure into clinical practice to predict the most appropriate drugs combination that could ensure therapy and the long-term survival of each CLL patient.

Towards analyzing the potential of exosomes to deliver microRNA therapeutics.

Exosome selectivity mechanisms underlying exosome-target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome-based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC-5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo-transmission electron microscopy; (c) cellular uptake studies of rhodamine-labeled exosomes in normal and cancer cells, providing to exosomes either "autologous" or "heterologous" cellular delivery environments; and (d) loading exogenous Alexa Fluor 488-labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC-5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising "bio-shuttles," being pharmacologically exploitable in the context of theranostic applications.

Migration of Type III Secretion System Transcriptional Regulators Links Gene Expression to Secretion.

Many plant-pathogenic bacteria of considerable economic importance rely on type III secretion systems (T3SSs) of the Hrc-Hrp 1 family to subvert their plant hosts. T3SS gene expression is regulated through the HrpG and HrpV proteins, while secretion is controlled by the gatekeeper HrpJ. A link between the two mechanisms was so far unknown. Here, we show that a mechanistic coupling exists between the expression and secretion cascades through the direct binding of the HrpG/HrpV heterodimer, acting as a T3SS chaperone, to HrpJ. The ternary complex is docked to the cytoplasmic side of the inner bacterial membrane and orchestrates intermediate substrate secretion, without affecting early substrate secretion. The anchoring of the ternary complex to the membranes potentially keeps HrpG/HrpV away from DNA. In their multiple roles as transcriptional regulators and gatekeeper chaperones, HrpV/HrpG provide along with HrpJ potentially attractive targets for antibacterial strategies.IMPORTANCE On the basis of scientific/economic importance, Pseudomonas syringae and Erwinia amylovora are considered among the top 10 plant-pathogenic bacteria in molecular plant pathology. Both employ type III secretion systems (T3SSs) of the Hrc-Hrp 1 family to subvert their plant hosts. For Hrc-Hrp 1, no functional link was known between the key processes of T3SS gene expression and secretion. Here, we show that a mechanistic coupling exists between expression and secretion cascades, through formation of a ternary complex involving the T3SS proteins HrpG, HrpV, and HrpJ. Our results highlight the functional and structural properties of a hitherto-unknown complex which orchestrates intermediate T3SS substrate secretion and may lead to better pathogen control through novel targets for antibacterial strategies.

Deciphering lymphoma pathogenesis via state-of-the-art mass spectrometry-based quantitative proteomics.

Mass spectrometry-based quantitative proteomics specifically applied to comprehend the pathogenesis of lymphoma has incremental value in deciphering the heterogeneity in complex deregulated molecular mechanisms/pathways of the lymphoma entities, implementing the current diagnostic and therapeutic strategies. Essential global, targeted and functional differential proteomics analyses although still evolving, have been successfully implemented to shed light on lymphoma pathogenesis to discover and explore the role of potential lymphoma biomarkers and drug targets. This review aims to outline and appraise the present status of MS-based quantitative proteomic approaches in lymphoma research, introducing the current state-of-the-art MS-based proteomic technologies, the opportunities they offer in biological discovery in human lymphomas and the related limitation issues arising from sample preparation to data evaluation. It is a synopsis containing information obtained from recent research articles, reviews and public proteomics repositories (PRIDE). We hope that this review article will aid, assimilate and assess all the information aiming to accelerate the development and validation of diagnostic, prognostic or therapeutic targets for an improved and empowered clinical proteomics application in lymphomas in the nearby future.

c-JUN N-terminal kinase (JNK) is activated and contributes to tumor cell proliferation in classical Hodgkin lymphoma.

c-JUN N-terminal Kinase (JNK) is activated/phosphorylated by upstream MAPK kinases (MKK), and, in turn, phosphorylates and activates its major substrate c-JUN, a member of the activator protein-1 (AP-1) transcription factors. c-JUN is overexpressed and activated in Hodgkin and Reed Sternberg cells (HRS) of classical Hodgkin lymphoma (cHL), however, the mechanism of its activation remains unknown. JNK activation was immunohistochemically assessed in 60 cases of HL and in a control group of 151 B-cell non-Hodgkin lymphomas. The biologic effects of JNK activation in cultured HRS cells were investigated using colony formation, cell growth and viability assays and cell cycle analysis by flow cytometry. Western blotting was used to assess protein levels. p-JNK was expressed in 90% of HL, 83% of Burkitt lymphomas, 28% of mantle cell lymphomas, 23% of diffuse large B-cell lymphomas, 19% of follicular lymphomas, and 18% of extranodal marginal zone lymphomas of MALT type. None of the 48 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma and 18 cases of plasma cell myeloma showed JNK phosphorylation (P < 001, Kruskall-Wallis test). Pharmacological inhibition of JNK activity in cultured HRS cells resulted in a significant decrease of cell growth, which was associated with cell cycle arrest at the G2/M phase. The cell cycle effects were linked to deactivation of c-JUN and upregulation of its known target, the cyclin-dependent kinase inhibitor p21. JNK is highly activated in HRS cells, and may contribute to uncontrolled cell cycle progression and proliferation of tumor cells in cHL.