HOXA9 forms a repressive complex with nuclear matrix-associated protein SAFB to maintain acute myeloid leukemia.

HOXA9 is commonly upregulated in acute myeloid leukemia (AML), in which it confers a poor prognosis. Characterizing the protein interactome of endogenous HOXA9 in human AML, we identified a chromatin complex of HOXA9 with the nuclear matrix attachment protein SAFB. SAFB perturbation phenocopied HOXA9 knockout to decrease AML proliferation, increase differentiation and apoptosis in vitro, and prolong survival in vivo. Integrated genomic, transcriptomic, and proteomic analyses further demonstrated that the HOXA9-SAFB (H9SB)-chromatin complex associates with nucleosome remodeling and histone deacetylase (NuRD) and HP1γ to repress the expression of factors associated with differentiation and apoptosis, including NOTCH1, CEBPδ, S100A8, and CDKN1A. Chemical or genetic perturbation of NuRD and HP1γ-associated catalytic activity also triggered differentiation, apoptosis, and the induction of these tumor-suppressive genes. Importantly, this mechanism is operative in other HOXA9-dependent AML genotypes. This mechanistic insight demonstrates the active HOXA9-dependent differentiation block as a potent mechanism of disease maintenance in AML that may be amenable to therapeutic intervention by targeting the H9SB interface and/or NuRD and HP1γ activity.

Single-Cell Lipidomics: An Automated and Accessible Microfluidic Workflow Validated by Capillary Sampling.

We report the first demonstration of a microfluidics-based approach to measure lipids in single living cells using widely available liquid chromatography mass spectrometry (LC-MS) instrumentation. The method enables the rapid sorting of live cells into liquid chambers formed on standard Petri dishes and their subsequent dispensing into vials for analysis using LC-MS. This approach facilitates automated sampling, data acquisition, and analysis and carries the additional advantage of chromatographic separation, aimed at reducing matrix effects present in shotgun lipidomics approaches. We demonstrate that our method detects comparable numbers of features at around 200 lipids in populations of single cells versus established live single-cell capillary sampling methods and with greater throughput, albeit with the loss of spatial resolution. We also show the importance of optimization steps in addressing challenges from lipid contamination, especially in blanks, and demonstrate a 75% increase in the number of lipids identified. This work opens up a novel, accessible, and high-throughput way to obtain single-cell lipid profiles and also serves as an important validation of single-cell lipidomics through the use of different sampling methods.

Single-Cell Untargeted Lipidomics Using Liquid Chromatography and Data-Dependent Acquisition after Live Cell Selection.

We report the development and validation of an untargeted single-cell lipidomics method based on microflow chromatography coupled to a data-dependent mass spectrometry method for fragmentation-based identification of lipids. Given the absence of single-cell lipid standards, we show how the methodology should be optimized and validated using a dilute cell extract. The methodology is applied to dilute pancreatic cancer and macrophage cell extracts and standards to demonstrate the sensitivity requirements for confident assignment of lipids and classification of the cell type at the single-cell level. The method is then coupled to a system that can provide automated sampling of live, single cells into capillaries under microscope observation. This workflow retains the spatial information and morphology of cells during sampling and highlights the heterogeneity in lipid profiles observed at the single-cell level. The workflow is applied to show changes in single-cell lipid profiles as a response to oxidative stress, coinciding with expanded lipid droplets. This demonstrates that the workflow is sufficiently sensitive to observing changes in lipid profiles in response to a biological stimulus. Understanding how lipids vary in single cells will inform future research into a multitude of biological processes as lipids play important roles in structural, biophysical, energy storage, and signaling functions.

Artificial intelligence driven definition of food preference endotypes in UK Biobank volunteers is associated with distinctive health outcomes and blood based metabolomic and proteomic profiles.

BACKGROUND: Specific food preferences can determine an individual's dietary patterns and therefore, may be associated with certain health risks and benefits. METHODS: Using food preference questionnaire (FPQ) data from a subset comprising over 180,000 UK Biobank participants, we employed Latent Profile Analysis (LPA) approach to identify the main patterns or profiles among participants. blood biochemistry across groups/profiles was compared using the non-parametric Kruskal-Wallis test. We applied the Limma algorithm for differential abundance analysis on 168 metabolites and 2923 proteins, and utilized the Database for Annotation, Visualization and Integrated Discovery (DAVID) to identify enriched biological processes and pathways. Relative risks (RR) were calculated for chronic diseases and mental conditions per group, adjusting for sociodemographic factors. RESULTS: Based on their food preferences, three profiles were termed: the putative Health-conscious group (low preference for animal-based or sweet foods, and high preference for vegetables and fruits), the Omnivore group (high preference for all foods), and the putative Sweet-tooth group (high preference for sweet foods and sweetened beverages). The Health-conscious group exhibited lower risk of heart failure (RR = 0.86, 95%CI 0.79-0.93) and chronic kidney disease (RR = 0.69, 95%CI 0.65-0.74) compared to the two other groups. The Sweet-tooth group had greater risk of depression (RR = 1.27, 95%CI 1.21-1.34), diabetes (RR = 1.15, 95%CI 1.01-1.31), and stroke (RR = 1.22, 95%CI 1.15-1.31) compared to the other two groups. Cancer (overall) relative risk showed little difference across the Health-conscious, Omnivore, and Sweet-tooth groups with RR of 0.98 (95%CI 0.96-1.01), 1.00 (95%CI 0.98-1.03), and 1.01 (95%CI 0.98-1.04), respectively. The Health-conscious group was associated with lower levels of inflammatory biomarkers (e.g., C-reactive Protein) which are also known to be elevated in those with common metabolic diseases (e.g., cardiovascular disease). Other markers modulated in the Health-conscious group, ketone bodies, insulin-like growth factor-binding protein (IGFBP), and Growth Hormone 1 were more abundant, while leptin was less abundant. Further, the IGFBP pathway, which influences IGF1 activity, may be significantly enhanced by dietary choices. CONCLUSIONS: These observations align with previous findings from studies focusing on weight loss interventions, which include a reduction in leptin levels. Overall, the Health-conscious group, with preference to healthier food options, has better health outcomes, compared to Sweet-tooth and Omnivore groups.

Evaluation of a proteomic signature coupled with the kidney failure risk equation in predicting end stage kidney disease in a chronic kidney disease cohort.

BACKGROUND: The early identification of patients at high-risk for end-stage renal disease (ESRD) is essential for providing optimal care and implementing targeted prevention strategies. While the Kidney Failure Risk Equation (KFRE) offers a more accurate prediction of ESRD risk compared to static eGFR-based thresholds, it does not provide insights into the patient-specific biological mechanisms that drive ESRD. This study focused on evaluating the effectiveness of KFRE in a UK-based advanced chronic kidney disease (CKD) cohort and investigating whether the integration of a proteomic signature could enhance 5-year ESRD prediction. METHODS: Using the Salford Kidney Study biobank, a UK-based prospective cohort of over 3000 non-dialysis CKD patients, 433 patients met our inclusion criteria: a minimum of four eGFR measurements over a two-year period and a linear eGFR trajectory. Plasma samples were obtained and analysed for novel proteomic signals using SWATH-Mass-Spectrometry. The 4-variable UK-calibrated KFRE was calculated for each patient based on their baseline clinical characteristics. Boruta machine learning algorithm was used for the selection of proteins most contributing to differentiation between patient groups. Logistic regression was employed for estimation of ESRD prediction by (1) proteomic features; (2) KFRE; and (3) proteomic features alongside KFRE. RESULTS: SWATH maps with 943 quantified proteins were generated and investigated in tandem with available clinical data to identify potential progression biomarkers. We identified a set of proteins (SPTA1, MYL6 and C6) that, when used alongside the 4-variable UK-KFRE, improved the prediction of 5-year risk of ESRD (AUC = 0.75 vs AUC = 0.70). Functional enrichment analysis revealed Rho GTPases and regulation of the actin cytoskeleton pathways to be statistically significant, inferring their role in kidney function and the pathogenesis of renal disease. CONCLUSIONS: Proteins SPTA1, MYL6 and C6, when used alongside the 4-variable UK-KFRE achieve an improved performance when predicting a 5-year risk of ESRD. Specific pathways implicated in the pathogenesis of podocyte dysfunction were also identified, which could serve as potential therapeutic targets. The findings of our study carry implications for comprehending the involvement of the Rho family GTPases in the pathophysiology of kidney disease, advancing our understanding of the proteomic factors influencing susceptibility to renal damage.

Quantitative SWATH-based proteomic profiling of urine for the identification of endometrial cancer biomarkers in symptomatic women.

BACKGROUND: A non-invasive endometrial cancer detection tool that can accurately triage symptomatic women for definitive testing would improve patient care. Urine is an attractive biofluid for cancer detection due to its simplicity and ease of collection. The aim of this study was to identify urine-based proteomic signatures that can discriminate endometrial cancer patients from symptomatic controls. METHODS: This was a prospective case-control study of symptomatic post-menopausal women (50 cancers, 54 controls). Voided self-collected urine samples were processed for mass spectrometry and run using sequential window acquisition of all theoretical mass spectra (SWATH-MS). Machine learning techniques were used to identify important discriminatory proteins, which were subsequently combined in multi-marker panels using logistic regression. RESULTS: The top discriminatory proteins individually showed moderate accuracy (AUC > 0.70) for endometrial cancer detection. However, algorithms combining the most discriminatory proteins performed well with AUCs > 0.90. The best performing diagnostic model was a 10-marker panel combining SPRR1B, CRNN, CALML3, TXN, FABP5, C1RL, MMP9, ECM1, S100A7 and CFI and predicted endometrial cancer with an AUC of 0.92 (0.96-0.97). Urine-based protein signatures showed good accuracy for the detection of early-stage cancers (AUC 0.92 (0.86-0.9)). CONCLUSION: A patient-friendly, urine-based test could offer a non-invasive endometrial cancer detection tool in symptomatic women. Validation in a larger independent cohort is warranted.

Proteomic signature associated with chronic kidney disease (CKD) progression identified by data-independent acquisition mass spectrometry.

BACKGROUND: Halting progression of chronic kidney disease (CKD) to established end stage kidney disease is a major goal of global health research. The mechanism of CKD progression involves pro-inflammatory, pro-fibrotic, and vascular pathways, but pathophysiological differentiation is currently lacking. METHODS: Plasma samples of 414 non-dialysis CKD patients, 170 fast progressors (with ∂ eGFR-3 ml/min/1.73 m2/year or worse) and 244 stable patients (∂ eGFR of - 0.5 to + 1 ml/min/1.73 m2/year) with a broad range of kidney disease aetiologies, were obtained and interrogated for proteomic signals with SWATH-MS. We applied a machine learning approach to feature selection of proteins quantifiable in at least 20% of the samples, using the Boruta algorithm. Biological pathways enriched by these proteins were identified using ClueGo pathway analyses. RESULTS: The resulting digitised proteomic maps inclusive of 626 proteins were investigated in tandem with available clinical data to identify biomarkers of progression. The machine learning model using Boruta Feature Selection identified 25 biomarkers as being important to progression type classification (Area Under the Curve = 0.81, Accuracy = 0.72). Our functional enrichment analysis revealed associations with the complement cascade pathway, which is relevant to CKD as the kidney is particularly vulnerable to complement overactivation. This provides further evidence to target complement inhibition as a potential approach to modulating the progression of diabetic nephropathy. Proteins involved in the ubiquitin-proteasome pathway, a crucial protein degradation system, were also found to be significantly enriched. CONCLUSIONS: The in-depth proteomic characterisation of this large-scale CKD cohort is a step toward generating mechanism-based hypotheses that might lend themselves to future drug targeting. Candidate biomarkers will be validated in samples from selected patients in other large non-dialysis CKD cohorts using a targeted mass spectrometric analysis.

Proteomic analysis identifies subgroups of patients with active systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a clinically and biologically heterogenous autoimmune disease. We aimed to investigate the plasma proteome of patients with active SLE to identify novel subgroups, or endotypes, of patients. METHOD: Plasma was collected from patients with active SLE who were enrolled in the British Isles Lupus Assessment Group Biologics Registry (BILAG-BR). The plasma proteome was analysed using a data-independent acquisition method, Sequential Window Acquisition of All theoretical mass spectra mass spectrometry (SWATH-MS). Unsupervised, data-driven clustering algorithms were used to delineate groups of patients with a shared proteomic profile. RESULTS: In 223 patients, six clusters were identified based on quantification of 581 proteins. Between the clusters, there were significant differences in age (p = 0.012) and ethnicity (p = 0.003). There was increased musculoskeletal disease activity in cluster 1 (C1), 19/27 (70.4%) (p = 0.002) and renal activity in cluster 6 (C6) 15/24 (62.5%) (p = 0.051). Anti-SSa/Ro was the only autoantibody that significantly differed between clusters (p = 0.017). C1 was associated with p21-activated kinases (PAK) and Phospholipase C (PLC) signalling. Within C1 there were two sub-clusters (C1A and C1B) defined by 49 proteins related to cytoskeletal protein binding. C2 and C6 demonstrated opposite Rho family GTPase and Rho GDI signalling. Three proteins (MZB1, SND1 and AGL) identified in C6 increased the classification of active renal disease although this did not reach statistical significance (p = 0.0617). CONCLUSIONS: Unsupervised proteomic analysis identifies clusters of patients with active SLE, that are associated with clinical and serological features, which may facilitate biomarker discovery. The observed proteomic heterogeneity further supports the need for a personalised approach to treatment in SLE.

High Throughput LC-MS Platform for Large Scale Screening of Bioactive Polar Lipids in Human Plasma and Serum.

Lipids play a key role in many biological processes, and their accurate measurement is critical to unraveling the biology of diseases and human health. A high throughput HILIC-based (LC-MS) method for the semiquantitative screening of over 2000 lipids, based on over 4000 MRM transitions, was devised to produce an accessible and robust lipidomic screen for phospholipids in human plasma/serum. This methodology integrates many of the advantages of global lipid analysis with those of targeted approaches. Having used the method as an initial "wide class" screen, it can then be easily adapted for a more targeted analysis and quantification of key, dysregulated lipids. Robustness was assessed using 1550 continuous injections of plasma extracts onto a single column and via the evaluation of columns from 5 different batches of stationary phase. Initial screens in positive (239 lipids, 431 MRM transitions) and negative electrospray ionization (ESI) mode (232 lipids, 446 MRM transitions) were assessed for reproducibility, sensitivity, and dynamic range using analysis times of 8 min. The total number of lipids monitored using these screening methods was 433 with an overlap of 38 lipids in both modes. A polarity switching method for accurate quantification, using the same LC conditions, was assessed for intra- and interday reproducibility, accuracy, dynamic range, stability, carryover, dilution integrity, and matrix interferences and found to be acceptable. This polarity switching method was then applied to lipids important in the stratification of human prostate cancer samples.

Data-independent acquisition mass spectrometry in severe rheumatic heart disease (RHD) identifies a proteomic signature showing ongoing inflammation and effectively classifying RHD cases.

BACKGROUND: Rheumatic heart disease (RHD) remains a major source of morbidity and mortality in developing countries. A deeper insight into the pathogenetic mechanisms underlying RHD could provide opportunities for drug repurposing, guide recommendations for secondary penicillin prophylaxis, and/or inform development of near-patient diagnostics. METHODS: We performed quantitative proteomics using Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectrometry (SWATH-MS) to screen protein expression in 215 African patients with severe RHD, and 230 controls. We applied a machine learning (ML) approach to feature selection among the 366 proteins quantifiable in at least 40% of samples, using the Boruta wrapper algorithm. The case-control differences and contribution to Area Under the Receiver Operating Curve (AUC) for each of the 56 proteins identified by the Boruta algorithm were calculated by Logistic Regression adjusted for age, sex and BMI. Biological pathways and functions enriched for proteins were identified using ClueGo pathway analyses. RESULTS: Adiponectin, complement component C7 and fibulin-1, a component of heart valve matrix, were significantly higher in cases when compared with controls. Ficolin-3, a protein with calcium-independent lectin activity that activates the complement pathway, was lower in cases than controls. The top six biomarkers from the Boruta analyses conferred an AUC of 0.90 indicating excellent discriminatory capacity between RHD cases and controls. CONCLUSIONS: These results support the presence of an ongoing inflammatory response in RHD, at a time when severe valve disease has developed, and distant from previous episodes of acute rheumatic fever. This biomarker signature could have potential utility in recognizing different degrees of ongoing inflammation in RHD patients, which may, in turn, be related to prognostic severity.

Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells.

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.

Multi-Omics Reveals Mechanisms of Partial Modulation of COVID-19 Dysregulation by Glucocorticoid Treatment.

Treatments for COVID-19 infections have improved dramatically since the beginning of the pandemic, and glucocorticoids have been a key tool in improving mortality rates. The UK's National Institute for Health and Care Excellence guidance is for treatment to be targeted only at those requiring oxygen supplementation, however, and the interactions between glucocorticoids and COVID-19 are not completely understood. In this work, a multi-omic analysis of 98 inpatient-recruited participants was performed by quantitative metabolomics (using targeted liquid chromatography-mass spectrometry) and data-independent acquisition proteomics. Both 'omics datasets were analysed for statistically significant features and pathways differentiating participants whose treatment regimens did or did not include glucocorticoids. Metabolomic differences in glucocorticoid-treated patients included the modulation of cortisol and bile acid concentrations in serum, but no alleviation of serum dyslipidemia or increased amino acid concentrations (including tyrosine and arginine) in the glucocorticoid-treated cohort relative to the untreated cohort. Proteomic pathway analysis indicated neutrophil and platelet degranulation as influenced by glucocorticoid treatment. These results are in keeping with the key role of platelet-associated pathways and neutrophils in COVID-19 pathogenesis and provide opportunity for further understanding of glucocorticoid action. The findings also, however, highlight that glucocorticoids are not fully effective across the wide range of 'omics dysregulation caused by COVID-19 infections.

The use of missing values in proteomic data-independent acquisition mass spectrometry to enable disease activity discrimination.

MOTIVATION: Data-independent acquisition mass spectrometry allows for comprehensive peptide detection and relative quantification than standard data-dependent approaches. While less prone to missing values, these still exist. Current approaches for handling the so-called missingness have challenges. We hypothesized that non-random missingness is a useful biological measure and demonstrate the importance of analysing missingness for proteomic discovery within a longitudinal study of disease activity. RESULTS: The magnitude of missingness did not correlate with mean peptide concentration. The magnitude of missingness for each protein strongly correlated between collection time points (baseline, 3 months, 6 months; R = 0.95-0.97, confidence interval = 0.94-0.97) indicating little time-dependent effect. This allowed for the identification of proteins with outlier levels of missingness that differentiate between the patient groups characterized by different patterns of disease activity. The association of these proteins with disease activity was confirmed by machine learning techniques. Our novel approach complements analyses on complete observations and other missing value strategies in biomarker prediction of disease activity. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proteomics and Informatics for Understanding Phases and Identifying Biomarkers in COVID-19 Disease.

The emergence of novel coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 coronavirus, has necessitated the urgent development of new diagnostic and therapeutic strategies. Rapid research and development, on an international scale, has already generated assays for detecting SARS-CoV-2 RNA and host immunoglobulins. However, the complexities of COVID-19 are such that fuller definitions of patient status, trajectory, sequelae, and responses to therapy are now required. There is accumulating evidence-from studies of both COVID-19 and the related disease SARS-that protein biomarkers could help to provide this definition. Proteins associated with blood coagulation (D-dimer), cell damage (lactate dehydrogenase), and the inflammatory response (e.g., C-reactive protein) have already been identified as possible predictors of COVID-19 severity or mortality. Proteomics technologies, with their ability to detect many proteins per analysis, have begun to extend these early findings. To be effective, proteomics strategies must include not only methods for comprehensive data acquisition (e.g., using mass spectrometry) but also informatics approaches via which to derive actionable information from large data sets. Here we review applications of proteomics to COVID-19 and SARS and outline how pipelines involving technologies such as artificial intelligence could be of value for research on these diseases.

Proteomic Analysis of an Induced Pluripotent Stem Cell Model Reveals Strategies to Treat Juvenile Myelomonocytic Leukemia.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of early childhood with a poor survival rate, thus there is a requirement for improved treatment strategies. Induced pluripotent stem cells offer the ability to model disease and develop new treatment strategies. JMML is frequently associated with mutations in PTPN11. Children with Noonan syndrome, a development disorder, have an increased incidence of JMML associated with specific germline mutations in PTPN11. We undertook a proteomic assessment of myeloid cells derived from induced pluripotent stem cells obtained from Noonan syndrome patients with PTPN11 mutations, either associated or not associated with an increased incidence of JMML. We report that the proteomic perturbations induced by the leukemia-associated PTPN11 mutations are associated with TP53 and NF-Kκb signaling. We have previously shown that MYC is involved in the differential gene expression observed in Noonan syndrome patients associated with an increased incidence of JMML. Thus, we employed drugs to target these pathways and demonstrate differential effects on clonogenic hematopoietic cells derived from Noonan syndrome patients, who develop JMML and those who do not. Further, we demonstrated these small molecular inhibitors, JQ1 and CBL0137, preferentially extinguish primitive hematopoietic cells from sporadic JMML patients as opposed to cells from healthy individuals.

A consideration of publication-derived immune-related associations in Coronavirus and related lung damaging diseases.

BACKGROUND: The severe acute respiratory syndrome virus SARS-CoV-2, a close relative of the SARS-CoV virus, is the cause of the recent COVID-19 pandemic affecting, to date, over 14 million individuals across the globe and demonstrating relatively high rates of infection and mortality. A third virus, the H5N1, responsible for avian influenza, has caused infection with some clinical similarities to those in COVID-19 infections. Cytokines, small proteins that modulate immune responses, have been directly implicated in some of the severe responses seen in COVID-19 patients, e.g. cytokine storms. Understanding the immune processes related to COVID-19, and other similar infections, could help identify diagnostic markers and therapeutic targets. METHODS: Here we examine data of cytokine, immune cell types, and disease associations captured from biomedical literature associated with COVID-19, Coronavirus in general, SARS, and H5N1 influenza, with the objective of identifying potentially useful relationships and areas for future research. RESULTS: Cytokine and cell-type associations captured from Medical Subject Heading (MeSH) terms linked to thousands of PubMed records, has identified differing patterns of associations between the four corpuses of publications (COVID-19, Coronavirus, SARS, or H5N1 influenza). Clustering of cytokine-disease co-occurrences in the context of Coronavirus has identified compelling clusters of co-morbidities and symptoms, some of which already known to be linked to COVID-19. Finally, network analysis identified sub-networks of cytokines and immune cell types associated with different manifestations, co-morbidities and symptoms of Coronavirus, SARS, and H5N1. CONCLUSION: Systematic review of research in medicine is essential to facilitate evidence-based choices about health interventions. In a fast moving pandemic the approach taken here will identify trends and enable rapid comparison to the literature of related diseases.

hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsa-mir183, the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication.

EVI1 oncoprotein expression and CtBP1-association oscillate through the cell cycle.

Aberrantly high expression of EVI1 in acute myeloid leukaemia (AML) is associated with poor prognosis. For targeted treatment of EVI1 overexpressing AML a more detailed understanding of aspects of spatiotemporal interaction dynamics of the EVI1 protein is important. EVI1 overexpressing SB1690CB AML cells were used for quantification and protein interaction studies of EVI1 and ΔEVI1. Cells were cell cycle-synchronised by mimosine and nocodazole treatment and expression of EVI1 and related proteins assessed by western blot, immunoprecipitation and immunofluorescence. EVI1 protein levels oscillate through the cell cycle, and EVI1 is degraded partly by the proteasome complex. Both EVI1 and ΔEVI1 interact with the co-repressor CtBP1 but dissociate from CtBP1 complexes during mitosis. Furthermore, a large fraction of EVI1, but not ΔEVI1 or CtBP1, resides in the nuclear matrix. In conclusion, EVI1- protein levels and EVI1-CtBP1 interaction dynamics vary though the cell cycle and differ between EVI1 and ΔEVI1. These data ad to the functional characterisation of the EVI1 protein in AML and will be important for the development of targeted therapeutic approaches for EVI1-driven AML.

EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

Pride and prejudice - What can we learn from peer review?

Objectives: Peer review is a powerful tool that steers the education and practice of medical researchers but may allow biased critique by anonymous reviewers. We explored factors unrelated to research quality that may influence peer review reports, and assessed the possibility that sub-types of reviewers exist. Our findings could potentially improve the peer review process.Methods: We evaluated the harshness, constructiveness and positiveness in 596 reviews from journals with open peer review, plus 46 reviews from colleagues' anonymously reviewed manuscripts. We considered possible influencing factors, such as number of authors and seasonal trends, on the content of the review. Finally, using machine-learning we identified latent types of reviewer with differing characteristics.Results: Reviews provided during a northern-hemisphere winter were significantly harsher, suggesting a seasonal effect on language. Reviews for articles in journals with an open peer review policy were significantly less harsh than those with an anonymous review process. Further, we identified three types of reviewers: nurturing, begrudged, and blasé.Conclusion: Nurturing reviews were in a minority and our findings suggest that more widespread open peer reviewing could improve the educational value of peer review, increase the constructive criticism that encourages researchers, and reduce pride and prejudice in editorial processes.