Maria Hernandez-Valladares

Freezing effects on the acute myeloid leukemia cell proteome and phosphoproteome revealed using optimal quantitative workflows.

UNLABELLED MS-based proteomic studies aiming for the discovery of acute myeloid leukemia (AML) biomarkers require sample processing that can assure an optimal proteome coverage and identification of PTMs. We evaluated different in-solution and filter-aided sample preparation (FASP) proteomic workflows and different enrichment strategies of phosphorylated peptides. The FASP protocols in the label-free and SILAC (stable isotope labelling with amino acids in cell culture) approaches were selected for producing the highest number of quantified proteins with reduced number of missed cleavages. The IMAC method was selected for producing the highest number of quantified phosphopeptides from SILAC-labelled peptides prepared with FASP. Using these selected workflows, we studied the effect of liquid nitrogen storage on the proteome and phosphoproteome of four AML patients. Our results showed that although there was not a major global proteome and phosphoproteome change when compared to their freshly processed counterparts, the freezing appeared to influence the abundance of mitochondrial proteins involved in the respiratory chain transport and affect the phosphorylation of apoptosis related proteins, cell surface interactors, ERK/MAPK targets and proteins involved in thrombin signalling. Our results encourage the assessment of current procedures of AML sample collection and preservation that could be used in future AML biomarker discovery studies. BIOLOGICAL SIGNIFICANCE Proteomic studies aiming to identify potential cancer biomarkers need to utilize the best sample preparation workflows on the samples of interest to achieve maximal proteome coverage. We have tested the most popular and recent proteomic and phosphoproteomic methods on cell lysates from patients with AML and systematically evaluated their performance. Our study shows the relevance of selecting the patient sample procedure giving the highest protein and PTM coverage. Moreover, we assessed how the proteome and phosphoproteome were affected by the conventional liquid nitrogen storage compared to cell lysis of fresh material, using the methods that worked best in our hands. For potential biomarkers that could be used for AML diagnostic and prognostic, it is of great importance to study the behaviour during sample conservation in order to avoid artefactual findings. Our results recommend caution in data interpretation when using different protocols of sample collection and conservation for proteomic and phosphosproteomic research.

Global Cell Proteome Profiling, Phospho-signaling and Quantitative Proteomics for Identification of New Biomarkers in Acute Myeloid Leukemia Patients.

The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging.

Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.

Therapeutic targeting of leukemic stem cells in acute myeloid leukemia – the biological background for possible strategies

ABSTRACT Introduction: Acute myeloid leukemia (AML) is an aggressive malignancy, caused by the accumulation of immature leukemic blasts in blood and bone marrow. There is a relatively high risk of chemoresistant relapse even for the younger patients who can receive the most intensive antileukemic treatment. Treatment directed against the remaining leukemic and preleukemic stem cells will most likely reduce the risk of later relapse. Areas covered: Relevant publications were identified through literature searches. The authors searched for original articles and recent reviews describing (i) the characteristics of leukemic/preleukemic stem cells; (ii) the importance of the bone marrow stem cell niches in leukemogenesis; and (iii) possible therapeutic strategies to target the preleukemic/leukemic stem cells. Expert opinion: Leukemia relapse/progression seems to be derived from residual chemoresistant leukemic or preleukemic stem cells, and a more effective treatment directed against these cells will likely be important to improve survival both for patients receiving intensive treatment and leukemia-stabilizing therapy. Several possible strategies are now considered, including the targeting of the epigenetic regulation of gene expression, proapoptotic intracellular signaling, cell metabolism, telomere activity and the AML-supporting effects by neighboring stromal cells. Due to disease heterogeneity, the most effective stem cell-directed therapy will probably differ between individual patients.

Rethinking the role of osteopontin in human acute myeloid leukemia.

Osteopontin is a secreted glycoprotein that binds to specific receptors and influences the functions of a wide range of cells. It acts both as chemoattractant cytokine and as an extracellular component, and its bone marrow expression in acute myeloid leukemia (AML) has been suggested to have a prognostic impact for patients receiving intensive treatment.[1–3] These studies have mainly examined osteopontin mRNA expression of bone marrow cells [2,3] or protein levels in marrow plasma.[1,2] Osteopontin is thus involved in communication between cells. No previous studies have quantified the constitutive release of osteopontin by primary human AML cells, the variation of this release among patients and its possible prognostic impact. Primary AML cells derived from 79 patients (median age 67 years; range 18–87 years; 34 females and 45 males) were cultured in vitro either (i) alone in serum-free Stem Span medium (Stem Cells Technology, Vancouver, Canada) for 48 h;[4] or (ii) in transwell co-cultures (0.4 lm pore size) with normal bone marrow mesenchymal stem cells (MSCs) for 72 h before supernatant levels were measured by ELISA (R&D Systems, Abingdon, UK).[5] Our cohort represents a group of unselected patients with high numbers of circulating leukemia blasts in peripheral blood (> 15 10/L). As discussed previously, highly enriched AML cell populations can then be prepared by density gradient separation alone,[4,6] and the cells were stored in liquid nitrogen until use. Osteopontin mRNA levels were determined from global gene expression analyses.[7] The protein levels were quantified by applying the label-free proteomic methodology as described in detail elsewhere.[8] The constitutive osteopontin protein release for AML cells cultured alone varied, and detectable levels (>10 pg/mL) were seen only for 28 of the 79 patients (Figure 1(A)). Detectable constitutive release showed significant associations (Mann–Whitney U-test) with age 40 years (p1⁄4 .018), monocytic differentiation (FAB-M4/M5; p1⁄4 .002), and favorable cytogenetics (p< .001). The significances remained also when the two patients with acute promyelocytic leukemia (APL) were excluded. No significant associations with sample storage time or cell viability after 48 h of culturing were detected. We compared the overall survival for patients with and without detectable constitutive osteopontin release. The survival analysis included only those 31 patients (median age 55 years, range 18–75; 13 females and 18 males) that completed intensive induction chemotherapy (7 d of cytarabine infusion combined with 3 d anthracycline treatment) followed by intensive consolidation treatment (either high-dose cytarabine alone in single doses of 3 g/m or combination therapy based on cytarabine single doses of 1 g/m) and potentially allogeneic stem cell transplantation. The long-term overall survival was significantly increased for patients with detectable constitutive osteopontin release (i.e. supernatant levels >10 pg/mL) compared with patients showing undetectable levels (Figures 1(A/B)), p1⁄4 .002). An additional analysis where patients were classified into three groups – undetectable (osteopontin level <10 pg/mL, 16 patients), intermediate (10 pg/mL–1 ng/mL, 6 patients), and high release (> 1 ng/mL, 9 patients) – also reached statistical significance (p1⁄4 .003). Univariate analysis revealed Flt3-ITD (HR1⁄4 3.926; p1⁄4 .008) and undetectable osteopontin levels (HR1⁄4 4.407; p1⁄4 .005) as risk factors for reduced survival. However, in a multivariate Cox-regression analysis including the parameters age, FAB, Flt3-ITD, NPM1-mutations, CD34 expression, disease etiology, and osteopontin release – favorable cytogenetics were excluded due to few patients (only five non-APL patients received/completed intensive treatment, and two of them were lost from follow-up), undetectable osteopontin expression emerged as an independent factor for reduced survival (HR1⁄4 4.537; p1⁄4 .024). We then compared the osteopontin levels for AML cells cultured alone to those cells co-cultured with bone marrow MSCs from healthy donors. The constitutive release by AML cells alone (cultured in co-culture wells) showed significant correlation with the corresponding

How should quality of life assessment be integrated in the evaluation of patients with acute myeloid leukemia

ABSTRACT Introduction: Recent studies in acute myeloid leukemia (AML) suggest that self-reported health status (including quality of life) should be a part of the pretherapy evaluation especially of elderly and unfit patients. However, there is also a need for additional studies to clarify the long-term effects of various antileukemic therapies. Areas covered: We searched for original articles in the PubMed database by the following combinations of terms: (i) acute myeloid leukemia combined with quality of life, geriatric assessment, or quality of life + allogeneic stem cell transplantation; or (ii) acute myeloid leukemia combined with either elderly, unfit, low-dose cytarabine or azacitidine. Expert commentary: We review and discuss the results from studies of quality of life for AML patients treated with conventional chemotherapy, autologous and allogeneic stem cell transplantation, and patients with acute promyelocytic leukemia. Self-reported health status (including quality of life) should be a part of the pretherapy evaluation especially of elderly and unfit AML patients together with performance status, comorbidity scoring and geriatric assessment. The risk of chemoresistance to intensive treatment should also be included. All these aspects should be considered when evaluation the risk for treatment-related mortality and deciding the intensity of the antileukemic therapy.

Proteogenomics approaches for studying cancer biology and their potential in the identification of acute myeloid leukemia biomarkers

ABSTRACT Introduction: Mass spectrometry (MS)-based proteomics has become an indispensable tool for the characterization of the proteome and its post-translational modifications (PTM). In addition to standard protein sequence databases, proteogenomics strategies search the spectral data against the theoretical spectra obtained from customized protein sequence databases. Up to date, there are no published proteogenomics studies on acute myeloid leukemia (AML) samples. Areas covered: Proteogenomics involves the understanding of genomic and proteomic data. The intersection of both datatypes requires advanced bioinformatics skills. A standard proteogenomics workflow that could be used for the study of AML samples is described. The generation of customized protein sequence databases as well as bioinformatics tools and pipelines commonly used in proteogenomics are discussed in detail. Expert commentary: Drawing on evidence from recent cancer proteogenomics studies and taking into account the public availability of AML genomic data, the interpretation of present and future MS-based AML proteomic data using AML-specific protein sequence databases could discover new biological mechanisms and targets in AML. However, proteogenomics workflows including bioinformatics guidelines can be challenging for the wide AML research community. It is expected that further automation and simplification of the bioinformatics procedures might attract AML investigators to adopt the proteogenomics strategy.

Two acute myeloid leukemia patient subsets are identified based on the constitutive PI3K-Akt-mTOR signaling of their leukemic cells; a functional, proteomic, and transcriptomic comparison

ABSTRACT Objectives: Constitutive signaling through the phosphatidylinositol-3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) pathway is present in acute myeloid leukemia (AML) cells. The aim of the study was to compare constitutive PI3K-Akt-mTOR activation of primary AML cells for a large group of unselected patients. Methods: We investigated expression and phosphorylation of 18 mediators in the PI3K-Akt-mTOR main track by flow cytometry for AML cells derived from 77 patients, and compared this with global gene expression profiles, proteomic, and transcriptomic profiles, and susceptibility to antileukemic agents. Results: Patients were divided into two main subsets showing generally high or low constitutive pathway activation. The high activation subset was characterized by decreased frequency of cells showing monocytic differentiation, increased frequency of adverse karyotypes, decreased constitutive cytokine release, and increased expression of certain integrins. Finally, the two groups differed in their expression of genes encoding regulators of protein phosphorylation, whereas phosphoproteomic analyses showed differences especially with regard to transcriptional regulation. Antiproliferative effects of pathway inhibition were generally stronger for the low phosphorylation subset. Conclusion: The constitutive PI3K-Akt-mTOR activation differed between patients; this difference appears to be a part of complex phenotypic differences including cell communication, intracellular signaling through other pathways, and transcriptional regulation.

Insulin-Like Growth Factor-I as an Effector Element of the Cytokine IL-4 in the Development of a Leishmania major Infection

Certain cytokines modulate the expression of insulin-like growth factor- (IGF-) I. Since IL-4 and IGF-I promote growth of the protozoan Leishmania major, we here addressed their interaction in downregulating the expression of Igf-I mRNA using small interfering RNA (siRNA) in Leishmania major-infected macrophages. Parasitism was decreased in the siRNA-treated cells compared with the nontreated cells, reversed by the addition of recombinant IGF-I (rIGF-I). In IL-4-stimulated macrophages, parasitism and the Igf-I mRNA amount were increased, and the effects were nullified upon siRNA transfection. IGF-I downregulation inhibited both parasite and macrophage arginase activation even in IL-4-stimulated cells. Searching for intracellular signaling components shared by IL-4 and IGF-I, upon siRNA transfection, phosphorylated p44, p38, and Akt proteins were decreased, affecting the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. In L. major-infected C57BL6-resistant mice, the preincubation of the parasite with rIGF-I changed the infection profile to be similar to that of susceptible mice. We conclude that IGF-I constitutes an effector element of IL-4 involving the PI3K/Akt pathway during L. major infection.

Preservation Method and Phosphate Buffered Saline Washing Affect the Acute Myeloid Leukemia Proteome

Acute myeloid leukemia (AML) primary cells can be isolated from peripheral blood, suspended with media containing bovine serum and cryoprotectant, and stored in liquid nitrogen before being processed for proteomic analysis by mass spectrometry (MS). The presence of bovine serum and human blood proteins in AML samples can hamper the identifications of proteins, and thereby reduce the proteome coverage of the study. Herein, we have established the effect of phosphate buffered saline (PBS) washing on AML patient samples stored in media. Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss. We also compared different methods to preserve the AML proteome from THP-1 and Molm-13 cell lines before MS analysis: (1) stored in media containing bovine serum and dimethyl sulfoxide (DMSO); (2) stored as dried cell pellets; and (3) stored as cell lysates in 4% sodium dodecyl sulfate (SDS). MS analysis of differently preserved AML cell samples shows that preservation with DMSO produce a high number of fragile cells that will burst during freezing and thawing. Our studies encourage the use of alternative preservation methods for future MS analysis of the AML proteome.