Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations.

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased responsiveness) and possibly also bromodomain inhibition (increased responsiveness). FAB-M4/M5 patients are heterogeneous with regard to genetic abnormalities, even though monocytic differentiation is common for patients with Nucleophosmin 1 (NPM1) insertions/mutations; to further study the heterogeneity of FAB-M4/M5 patients we did a proteomic and phosphoproteomic comparison of FAB-M4/M5 patients with (n = 13) and without (n = 12) NPM1 mutations. The proteomic profile of NPM1-mutated FAB-M4/M5 patients was characterized by increased levels of proteins involved in the regulation of endocytosis/vesicle trafficking/organellar communication. In contrast, AML cells without NPM1 mutations were characterized by increased levels of several proteins involved in the regulation of cytoplasmic translation, including a large number of ribosomal proteins. The phosphoproteomic differences between the two groups were less extensive but reflected similar differences. To conclude, even though FAB classification/monocytic differentiation are associated with differences in responsiveness to new targeted therapies (e.g., Bcl-2 inhibition), our results shows that FAB-M4/M5 patients are heterogeneous with regard to important biological characteristics of the leukemic cells.

Vacuolar ATPase Is a Possible Therapeutic Target in Acute Myeloid Leukemia: Focus on Patient Heterogeneity and Treatment Toxicity.

Vacuolar ATPase (V-ATPase) is regarded as a possible target in cancer treatment. It is expressed in primary acute myeloid leukemia cells (AML), but the expression varies between patients and is highest for patients with a favorable prognosis after intensive chemotherapy. We therefore investigated the functional effects of two V-ATPase inhibitors (bafilomycin A1, concanamycin A) for primary AML cells derived from 80 consecutive patients. The V-ATPase inhibitors showed dose-dependent antiproliferative and proapoptotic effects that varied considerably between patients. A proteomic comparison of primary AML cells showing weak versus strong antiproliferative effects of V-ATPase inhibition showed a differential expression of proteins involved in intracellular transport/cytoskeleton functions, and an equivalent phosphoproteomic comparison showed a differential expression of proteins that regulate RNA processing/function together with increased activity of casein kinase 2. Patients with secondary AML, i.e., a heterogeneous subset with generally adverse prognosis and previous cytotoxic therapy, myeloproliferative neoplasia or myelodysplastic syndrome, were characterized by a strong antiproliferative effect of V-ATPase inhibition and also by a specific mRNA expression profile of V-ATPase interactome proteins. Furthermore, the V-ATPase inhibition altered the constitutive extracellular release of several soluble mediators (e.g., chemokines, interleukins, proteases, protease inhibitors), and increased mediator levels in the presence of AML-supporting bone marrow mesenchymal stem cells was then observed, especially for patients with secondary AML. Finally, animal studies suggested that the V-ATPase inhibitor bafilomycin had limited toxicity, even when combined with cytarabine. To conclude, V-ATPase inhibition has antileukemic effects in AML, but this effect varies between patients.

High Mitochondrial Protein Expression as a Potential Predictor of Relapse Risk in Acute Myeloid Leukemia Patients with the Monocytic FAB Subtypes M4 and M5.

AML is a highly aggressive and heterogeneous form of hematological cancer. Proteomics-based stratification of patients into more refined subgroups may contribute to a more precise characterization of the patient-derived AML cells. Here, we reanalyzed liquid chromatography-tandem mass spectrometry (LC-MS/MS) generated proteomic and phosphoproteomic data from 26 FAB-M4/M5 patients. The patients achieved complete hematological remission after induction therapy. Twelve of them later developed chemoresistant relapse (RELAPSE), and 14 patients were relapse-free (REL_FREE) long-term survivors. We considered not only the RELAPSE and REL_FREE characteristics but also integrated the French-American-British (FAB) classification, along with considering the presence of nucleophosmin 1 (NPM1) mutation and cytogenetically normal AML. We found a significant number of differentially enriched proteins (911) and phosphoproteins (257) between the various FAB subtypes in RELAPSE patients. Patients with the myeloblastic M1/M2 subtype showed higher levels of RNA processing-related routes and lower levels of signaling related to terms like translation and degranulation when compared with the M4/M5 subtype. Moreover, we found that a high abundance of proteins associated with mitochondrial translation and oxidative phosphorylation, particularly observed in the RELAPSE M4/M5 NPM1 mutated subgroup, distinguishes relapsing from non-relapsing AML patient cells with the FAB subtype M4/M5. Thus, the discovery of subtype-specific biomarkers through proteomic profiling may complement the existing classification system for AML and potentially aid in selecting personalized treatment strategies for individual patients.

Patient Heterogeneity in Acute Myeloid Leukemia: Leukemic Cell Communication by Release of Soluble Mediators and Its Effects on Mesenchymal Stem Cells.

Acute myeloid leukemia (AML) is an aggressive bone marrow malignancy, and non-leukemic stromal cells (including mesenchymal stem cells, MSCs) are involved in leukemogenesis and show AML-supporting effects. We investigated how constitutive extracellular mediator release by primary human AML cells alters proteomic profiles of normal bone marrow MSCs. An average of 6814 proteins (range 6493-6918 proteins) were quantified for 41 MSC cultures supplemented with AML-cell conditioned medium, whereas an average of 6715 proteins (range 6703-6722) were quantified for untreated control MSCs. The AML effect on global MSC proteomic profiles varied between patients. Hierarchical clustering analysis identified 10 patients (5/10 secondary AML) showing more extensive AML-effects on the MSC proteome, whereas the other 31 patients clustered together with the untreated control MSCs and showed less extensive AML-induced effects. These two patient subsets differed especially with regard to MSC levels of extracellular matrix and mitochondrial/metabolic regulatory proteins. Less than 10% of MSC proteins were significantly altered by the exposure to AML-conditioned media; 301 proteins could only be quantified after exposure to conditioned medium and 201 additional proteins were significantly altered compared with the levels in control samples (153 increased, 48 decreased). The AML-modulated MSC proteins formed several interacting networks mainly reflecting intracellular organellar structure/trafficking but also extracellular matrix/cytokine signaling, and a single small network reflecting altered DNA replication. Our results suggest that targeting of intracellular trafficking and/or intercellular communication is a possible therapeutic strategy in AML.

Effects of the Autophagy-Inhibiting Agent Chloroquine on Acute Myeloid Leukemia Cells; Characterization of Patient Heterogeneity.

Autophagy is a highly conserved cellular degradation process that prevents cell damage and promotes cell survival, and clinical efforts have exploited autophagy inhibition as a therapeutic strategy in cancer. Chloroquine is a well-known antimalarial agent that inhibits late-stage autophagy. We evaluated the effects of chloroquine on cell viability and proliferation of acute myeloid leukemia acute myeloid leukemia (AML) cells derived from 81 AML patients. Our results show that chloroquine decreased AML cell viability and proliferation for the majority of patients. Furthermore, a subgroup of AML patients showed a greater susceptibility to chloroquine, and using hierarchical cluster analysis, we identified 99 genes upregulated in this patient subgroup, including several genes related to leukemogenesis. The combination of chloroquine with low-dose cytarabine had an additive inhibitory effect on AML cell proliferation. Finally, a minority of patients showed increased extracellular constitutive mediator release in the presence of chloroquine, which was associated with strong antiproliferative effects of chloroquine as well as cytarabine. We conclude that chloroquine has antileukemic activity and should be further explored as a therapeutic drug against AML in combination with other cytotoxic or metabolic drugs; however, due to the patient heterogeneity, chloroquine therapy will probably be effective only for selected patients.

Proteomic Comparison of Bone Marrow Derived Osteoblasts and Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, and therapeutic targeting of these cells is considered both for malignant and non-malignant diseases. We analyzed global proteomic profiles for osteoblasts derived from ten and MSCs from six healthy individuals, and we quantified 5465 proteins for the osteoblasts and 5420 proteins for the MSCs. There was a large overlap in the profiles for the two cell types; 156 proteins were quantified only in osteoblasts and 111 proteins only for the MSCs. The osteoblast-specific proteins included several extracellular matrix proteins and a network including 27 proteins that influence intracellular signaling (Wnt/Notch/Bone morphogenic protein pathways) and bone mineralization. The osteoblasts and MSCs showed only minor age- and sex-dependent proteomic differences. Finally, the osteoblast and MSC proteomic profiles were altered by ex vivo culture in serum-free media. We conclude that although the proteomic profiles of osteoblasts and MSCs show many similarities, we identified several osteoblast-specific extracellular matrix proteins and an osteoblast-specific intracellular signaling network. Therapeutic targeting of these proteins will possibly have minor effects on MSCs. Furthermore, the use of ex vivo cultured osteoblasts/MSCs in clinical medicine will require careful standardization of the ex vivo handling of the cells.

Proteomic Studies of Primary Acute Myeloid Leukemia Cells Derived from Patients Before and during Disease-Stabilizing Treatment Based on All-Trans Retinoic Acid and Valproic Acid.

All-trans retinoic acid (ATRA) and valproic acid (VP) have been tried in the treatment of non-promyelocytic variants of acute myeloid leukemia (AML). Non-randomized studies suggest that the two drugs can stabilize AML and improve normal peripheral blood cell counts. In this context, we used a proteomic/phosphoproteomic strategy to investigate the in vivo effects of ATRA/VP on human AML cells. Before starting the combined treatment, AML responders showed increased levels of several proteins, especially those involved in neutrophil degranulation/differentiation, M phase regulation and the interconversion of nucleotide di- and triphosphates (i.e., DNA synthesis and binding). Several among the differentially regulated phosphorylation sites reflected differences in the regulation of RNA metabolism and apoptotic events at the same time point. These effects were mainly caused by increased cyclin dependent kinase 1 and 2 (CDK1/2), LIM domain kinase 1 and 2 (LIMK1/2), mitogen-activated protein kinase 7 (MAPK7) and protein kinase C delta (PRKCD) activity in responder cells. An extensive effect of in vivo treatment with ATRA/VP was the altered level and phosphorylation of proteins involved in the regulation of transcription/translation/RNA metabolism, especially in non-responders, but the regulation of cell metabolism, immune system and cytoskeletal functions were also affected. Our analysis of serial samples during the first week of treatment suggest that proteomic and phosphoproteomic profiling can be used for the early identification of responders to ATRA/VP-based treatment.

The Constitutive Extracellular Protein Release by Acute Myeloid Leukemia Cells-A Proteomic Study of Patient Heterogeneity and Its Modulation by Mesenchymal Stromal Cells.

Extracellular protein release is important both for the formation of extracellular matrix and for communication between cells. We investigated the extracellular protein release by in vitro cultured normal mesenchymal stem cells (MSCs) and by primary human acute myeloid leukemia (AML) cells derived from 40 consecutive patients. We observed quantifiable levels of 3082 proteins in our study; for the MSCs, we detected 1446 proteins, whereas the number of released proteins for the AML cells showed wide variation between patients (average number 1699, range 557-2380). The proteins were derived from various cellular compartments (e.g., cell membrane, nucleus, and cytoplasms), several organelles (e.g., cytoskeleton, endoplasmatic reticulum, Golgi apparatus, and mitochondria) and had various functions (e.g., extracellular matrix and exosomal proteins, cytokines, soluble adhesion molecules, protein synthesis, post-transcriptional modulation, RNA binding, and ribonuclear proteins). Thus, AML patients were very heterogeneous both regarding the number of proteins and the nature of their extracellularly released proteins. The protein release profiles of MSCs and primary AML cells show a considerable overlap, but a minority of the proteins are released only or mainly by the MSC, including several extracellular matrix molecules. Taken together, our observations suggest that the protein profile of the extracellular bone marrow microenvironment differs between AML patients, these differences are mainly caused by the protein release by the leukemic cells but this leukemia-associated heterogeneity of the overall extracellular protein profile is modulated by the constitutive protein release by normal MSCs.

The Extracellular Bone Marrow Microenvironment-A Proteomic Comparison of Constitutive Protein Release by In Vitro Cultured Osteoblasts and Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) and osteoblasts are bone marrow stromal cells that contribute to the formation of stem cell niches and support normal hematopoiesis, leukemogenesis and development of metastases from distant cancers. This support is mediated through cell-cell contact, release of soluble mediators and formation of extracellular matrix. By using a proteomic approach, we characterized the protein release by in vitro cultured human MSCs (10 donors) and osteoblasts (nine donors). We identified 1379 molecules released by these cells, including 340 proteins belonging to the GO-term Extracellular matrix. Both cell types released a wide range of functionally heterogeneous proteins including extracellular matrix molecules (especially collagens), several enzymes and especially proteases, cytokines and soluble adhesion molecules, but also several intracellular molecules including chaperones, cytoplasmic mediators, histones and non-histone nuclear molecules. The levels of most proteins did not differ between MSCs and osteoblasts, but 82 proteins were more abundant for MSC (especially extracellular matrix proteins and proteases) and 36 proteins more abundant for osteoblasts. Finally, a large number of exosomal proteins were identified. To conclude, MSCs and osteoblasts show extracellular release of a wide range of functionally diverse proteins, including several extracellular matrix molecules known to support cancer progression (e.g., metastases from distant tumors, increased relapse risk for hematological malignancies), and the large number of identified exosomal proteins suggests that exocytosis is an important mechanism of protein release.

Biological characteristics of aging in human acute myeloid leukemia cells: the possible importance of aldehyde dehydrogenase, the cytoskeleton and altered transcriptional regulation.

Patients with acute myeloid leukemia (AML) have a median age of 65-70 years at diagnosis. Elderly patients have more chemoresistant disease, and this is partly due to decreased frequencies of favorable and increased frequencies of adverse genetic abnormalities. However, aging-dependent differences may also contribute. We therefore compared AML cell proteomic and phosphoproteomic profiles for (i) elderly low-risk and younger low-risk patients with favorable genetic abnormalities; and (ii) high-risk patients with adverse genetic abnormalities and a higher median age against all low-risk patients with lower median age. Elderly low-risk and younger low-risk patients showed mainly phosphoproteomic differences especially involving transcriptional regulators and cytoskeleton. When comparing high-risk and low-risk patients both proteomic and phosphoproteomic studies showed differences involving cytoskeleton and immunoregulation but also transcriptional regulation and cell division. The age-associated prognostic impact of cyclin-dependent kinases was dependent on the cellular context. The protein level of the adverse prognostic biomarker mitochondrial aldehyde dehydrogenase (ALDH2) showed a similar significant upregulation both in elderly low-risk and elderly high-risk patients. Our results suggest that molecular mechanisms associated with cellular aging influence chemoresistance of AML cells, and especially the cytoskeleton function may then influence cellular hallmarks of aging, e.g. mitosis, polarity, intracellular transport and adhesion.

The Progression of Acute Myeloid Leukemia from First Diagnosis to Chemoresistant Relapse: A Comparison of Proteomic and Phosphoproteomic Profiles.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy. Nearly 50% of the patients who receive the most intensive treatment develop chemoresistant leukemia relapse. Although the leukemogenic events leading to relapse seem to differ between patients (i.e., regrowth from a clone detected at first diagnosis, progression from the original leukemic or preleukemic stem cells), a common characteristic of relapsed AML is increased chemoresistance. The aim of the present study was to investigate at the proteomic level whether leukemic cells from relapsed patients present overlapping molecular mechanisms that contribute to this chemoresistance. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare the proteomic and phosphoproteomic profiles of AML cells derived from seven patients at the time of first diagnosis and at first relapse. At the time of first relapse, AML cells were characterized by increased levels of proteins important for various mitochondrial functions, such as mitochondrial ribosomal subunit proteins (MRPL21, MRPS37) and proteins for RNA processing (DHX37, RNA helicase; RPP40, ribonuclease P component), DNA repair (ERCC3, DNA repair factor IIH helicase; GTF2F1, general transcription factor), and cyclin-dependent kinase (CDK) activity. The levels of several cytoskeletal proteins (MYH14/MYL6/MYL12A, myosin chains; VCL, vinculin) as well as of proteins involved in vesicular trafficking/secretion and cell adhesion (ITGAX, integrin alpha-X; CD36, platelet glycoprotein 4; SLC2A3, solute carrier family 2) were decreased in relapsed cells. Our study introduces new targetable proteins that might direct therapeutic strategies to decrease chemoresistance in relapsed AML.

Targeting Cellular Metabolism in Acute Myeloid Leukemia and The Role of Patient Heterogeneity.

Acute myeloid leukemia (AML) is an aggressive blood cancer resulting in accumulation of immature, dysfunctional blood cells in the bone marrow. Changes in cell metabolism are features of many cancers, including AML and this may be exploited as a therapeutic target. In this study we investigated the in vitro antileukemic effects of seven metabolic inhibitors that target different metabolic pathways. The metabolic inhibitors were tested on AML cells derived from 81 patients using proliferation and viability assays; we also compared global gene expression and proteomic profiles for various patient subsets. Metformin, 2DG, 6AN, BPTES and ST1326 had strong antiproliferative and proapoptotic effects for most patients, whereas lonidamine and AZD3965 had an effect only for a minority. Antiproliferative effects on AML cells were additive when combined with the chemotherapeutic agent AraC. Using unsupervised hierarchical clustering, we identified a strong antiproliferative effect on AML cells after treatment with metabolic inhibitors for a subset of 29 patients. Gene expression and proteomic studies suggested that this subset was characterized by altered metabolic and transcriptional regulation. In addition, the Bcl-2 inhibitor venetoclax, in combination with 2DG or 6AN, increased the antiproliferative effects of these metabolic inhibitors on AML cells. Therapeutic targeting of cellular metabolism may have potential in AML, but the optimal strategy will likely differ between patients.

Proteome and Phosphoproteome Changes Associated with Prognosis in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly. Although complete remission (CR) is achieved for the majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has become of major clinical interest in AML. We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a five-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells. This suggests that therapy against the upregulated kinases could also target the factors inducing their upregulation rather than their activity. This study, therefore, presents markers that could help predict AML relapse and direct therapeutic strategies.

Mice depleted for Exchange Proteins Directly Activated by cAMP (Epac) exhibit irregular liver regeneration in response to partial hepatectomy.

The exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2) are expressed in a cell specific manner in the liver, but their biological functions in this tissue are poorly understood. The current study was undertaken to begin to determine the potential roles of Epac1 and Epac2 in liver physiology and disease. Male C57BL/6J mice in which expression of Epac1 and/or Epac2 are deleted, were subjected to partial hepatectomy and the regenerating liver was analyzed with regard to lipid accumulation, cell replication and protein expression. In response to partial hepatectomy, deletion of Epac1 and/or Epac2 led to increased hepatocyte proliferation 36 h post surgery, and the transient steatosis observed in wild type mice was virtually absent in mice lacking both Epac1 and Epac2. The expression of the protein cytochrome P4504a14, which is implicated in hepatic steatosis and fibrosis, was substantially reduced upon deletion of Epac1/2, while a number of factors involved in lipid metabolism were significantly decreased. Moreover, the number of Küpffer cells was affected, and Epac2 expression was increased in the liver of wild type mice in response to partial hepatectomy, further supporting a role for these proteins in liver function. This study establishes hepatic phenotypic abnormalities in mice deleted for Epac1/2 for the first time, and introduces Epac1/2 as regulators of hepatocyte proliferation and lipid accumulation in the regenerative process.

High Constitutive Cytokine Release by Primary Human Acute Myeloid Leukemia Cells Is Associated with a Specific Intercellular Communication Phenotype.

Acute myeloid leukemia (AML) is a heterogeneous disease, and this heterogeneity includes the capacity of constitutive release of extracellular soluble mediators by AML cells. We investigated whether this capacity is associated with molecular genetic abnormalities, and we compared the proteomic profiles of AML cells with high and low release. AML cells were derived from 71 consecutive patients that showed an expected frequency of cytogenetic and molecular genetic abnormalities. The constitutive extracellular release of 34 soluble mediators (CCL and CXCL chemokines, interleukins, proteases, and protease regulators) was investigated for an unselected subset of 62 patients, and they could be classified into high/intermediate/low release subsets based on their general capacity of constitutive secretion. FLT3-ITD was more frequent among patients with high constitutive mediator release, but our present study showed no additional associations between the capacity of constitutive release and 53 other molecular genetic abnormalities. We compared the proteomic profiles of two contrasting patient subsets showing either generally high or low constitutive release. A network analysis among cells with high release levels demonstrated high expression of intracellular proteins interacting with integrins, RAC1, and SYK signaling. In contrast, cells with low release showed high expression of several transcriptional regulators. We conclude that AML cell capacity of constitutive mediator release is characterized by different expression of potential intracellular therapeutic targets.

Effects of insulin and pathway inhibitors on the PI3K-Akt-mTOR phosphorylation profile in acute myeloid leukemia cells.

The phosphatidylinositol 3-kinase (PI3K)-Akt-mechanistic target of rapamycin (mTOR) pathway is constitutively activated in human acute myeloid leukemia (AML) cells and is regarded as a possible therapeutic target. Insulin is an agonist of this pathway and a growth factor for AML cells. We characterized the effect of insulin on the phosphorylation of 10 mediators in the main track of the PI3K-Akt-mTOR pathway in AML cells from 76 consecutive patients. The overall results showed that insulin significantly increased the phosphorylation of all investigated mediators. However, insulin effects on the pathway activation profile varied among patients, and increased phosphorylation in all mediators was observed only in a minority of patients; in other patients, insulin had divergent effects. Global gene expression profiling and proteomic/phosphoproteomic comparisons suggested that AML cells from these two patient subsets differed with regard to AML cell differentiation, transcriptional regulation, RNA metabolism, and cellular metabolism. Strong insulin-induced phosphorylation was associated with weakened antiproliferative effects of metabolic inhibitors. PI3K, Akt, and mTOR inhibitors also caused divergent effects on the overall pathway phosphorylation profile in the presence of insulin, although PI3K and Akt inhibition caused a general reduction in Akt pT308 and 4EBP1 pT36/pT45 phosphorylation. For Akt inhibition, the phosphorylation of upstream mediators was generally increased or unaltered. In contrast, mTOR inhibition reduced mTOR pS2448 and S6 pS244 phosphorylation but increased Akt pT308 phosphorylation. In conclusion, the effects of both insulin and PI3K-Akt-mTOR inhibitors differ between AML patient subsets, and differences in insulin responsiveness are associated with differential susceptibility to metabolic targeting.

An Overview on G Protein-coupled Receptor-induced Signal Transduction in Acute Myeloid Leukemia.

BACKGROUND: Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by uncontrolled proliferation of precursor myeloid-lineage cells in the bone marrow. AML is also characterized by patients with poor long-term survival outcomes due to relapse. Many efforts have been made to understand the biological heterogeneity of AML and the challenges to develop new therapies are therefore enormous. G Protein-coupled Receptors (GPCRs) are a large attractive drug-targeted family of transmembrane proteins, and aberrant GPCR expression and GPCR-mediated signaling have been implicated in leukemogenesis of AML. This review aims to identify the molecular players of GPCR signaling, focusing on the hematopoietic system, which are involved in AML to help developing novel drug targets and therapeutic strategies. METHODS: We undertook an exhaustive and structured search of bibliographic databases for research focusing on GPCR, GPCR signaling and expression in AML. RESULTS AND CONCLUSION: Many scientific reports were found with compelling evidence for the involvement of aberrant GPCR expression and perturbed GPCR-mediated signaling in the development of AML. The comprehensive analysis of GPCR in AML provides potential clinical biomarkers for prognostication, disease monitoring and therapeutic guidance. It will also help to provide marker panels for monitoring in AML. We conclude that GPCR-mediated signaling is contributing to leukemogenesis of AML, and postulate that mass spectrometrybased protein profiling of primary AML cells will accelerate the discovery of potential GPCR related biomarkers for AML.

The Capacity of Long-Term in Vitro Proliferation of Acute Myeloid Leukemia Cells Supported Only by Exogenous Cytokines Is Associated with a Patient Subset with Adverse Outcome.

Acute myeloid leukemia (AML) is an aggressive malignancy, which is highly heterogeneous with regard to chemosensitivity and biological features. The AML cell population is organized in a hierarchy that is reflected in the in vitro growth characteristics, with only a minority of cells being able to proliferate for more than two weeks. In this study, we investigated the ability of AML stem cells to survive and proliferate in suspension cultures in the presence of exogenous mediators but without supporting non-leukemic cells. We saw that a high number of maintained stem cells (i.e., a large number of clonogenic cells after five weeks of culture) was associated with decreased overall survival for patients receiving intensive chemotherapy; this prognostic impact was also detected in the multivariate/adjusted analysis. Furthermore, the patients with many clonogenic cells presented more frequently with mutations in transcription-related genes, and also showed a higher abundance of proteins involved in transcription at the time of diagnosis. In conclusion, the growth characteristics of the long-term proliferating leukemic stem cells seem to have an independent prognostic impact in human AML, and these characteristics appear to be reflected by the mutational landscape and the proteome of the patients at the time of diagnosis.

Proteomic Profiling of Primary Human Acute Myeloid Leukemia Cells Does Not Reflect Their Constitutive Release of Soluble Mediators.

Acute myeloid leukemia (AML) is a heterogeneous disease, and communication between leukemic cells and their neighboring leukemia-supporting normal cells is involved in leukemogenesis. The bone marrow cytokine network is therefore important, and the mediator release profile seems more important than single mediators. It is not known whether the characterization of primary AML cell proteomes reflects the heterogeneity of the broad and dynamic constitutive mediator release profile by these cells. To address this, we compared the intracellular levels of 41 proteins in 19 AML patients with the constitutive extracellular release during in vitro culture, including chemokines, growth factors, proteases, and protease regulators. The constitutive release of most mediators showed a wide variation (up to 2000-fold differences) between patients. Detectable intracellular levels were seen for 10 of 41 mediators, but for most of these 10 mediators we could not detect significant correlations between the constitutive release during in vitro culture and their intracellular levels. Intracellular protein levels in primary human AML cells do not reflect the dynamics, capacity, and variation between patients in constitutive mediator release profiles. Measurements of these profiles thus add complementary information to proteomic detection/quantification regarding the heterogeneity of the AML cell contributions to the bone marrow cytokine network.

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.

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.