Stages Of Myeloid Differentiation Research Articles

Identification of biomarkers for acute leukemia via machine learning-based stemness index.

Traditional methods to understand leukemia stem cell (LSC)'s biological characteristics include constructing LSC-like cells and mouse models by transgenic or knock-in methods. However, there are some potential pitfalls in using this method, such as retroviral insertion mutagenesis, non-physiological level gene expression, non-physiological expansion, and difficulty to construct. The mRNAsi index for each sample of the Cancer Genome Atlas (TCGA) could avoid these potential pitfalls by machine learning. In this work, we aimed to construct a network of LSC genes utilizing the mRNAsi. First, mRNAsi value was analyzed with expressions distributions, survival analysis, age, and gender in acute myeloid leukemia (AML) samples. Then, we used the weighted gene co-expression network analysis (WGCNA) to construct modules of stemness genes. The correlation of the LSC genes transcription and interplay among LSC proteins was analyzed. We performed functional and pathway enrichment analysis to annotate stemness genes. Survival analysis further identified prognostic biomarkers by clinical data of TCGA and the Gene Expression Omnibus (GEO) database. We found that the result of mRNAsi overall survival is not significant, which may be due to the heterogeneity of AML in the stage of myeloid differentiation, French-American-British (FAB) classification systems. Enrichment analysis indicated that the stemness genes were biologically clustered as a group and mainly associated with cell cycle and mitosis. Moreover, 10 key genes (SNRNP40, RFC4, RFC5, CDC6, HSPE1, PA2G4, SNAP23P, DARS2, MIS18A, and HPRT1) were screened by survival analysis with the data from TCGA and GEO. Among them, RFC4 and RFC5 were the distinguished biomarkers for their double-validated prognostic value in both databases. Additionally, the expression of RFC4 and RFC5 had the same trend as mRNAsi score in FAB subtypes. In conclusion, our result demonstrated that mRNAsi based LSC-related genes were found to have strong interactions as a cluster. These genes, especially RFC4 and RFC5, could be the therapeutic targets for inhibiting the stemness characteristics of AML. This work is also a comprehensive pipeline for future cancer stem cell studies.

Single-Cell Drug Profiling Reveals Maturation Stage-Dependent Drug Responses in AML

IntroductionAcute myeloid leukemia (AML) is a disease of myeloid lineage precursors with poor overall survival and limited treatment options in relapsed patients. Ex vivo drug testing is an approach to identify potential responders for novel targeted agents. However, AML bone marrow is comprised of a mixture of malignant and non-malignant cell types, and may contain myeloid cells at distinct differentiation stages. Here, we evaluated the ex vivo sensitivity of primary AML samples at a detailed cell-population level using a multicolor high-throughput flow cytometer (FACS).MethodsBone marrow (BM) mononuclear cells of 23 AML patients were plated on 96-well plates in mononuclear cell media (PromoCell). Seven FDA approved drugs were used, including signal transduction inhibitors that had shown AML specific responses in our earlier studies (Pemovska et al. Cancer Discovery 2013 ): trametinib, ruxolitinib, everolimus, sunitinib, venetoclax, plus standard of care drugs idarubicin and cytarabine. After 3-day incubation, cells were stained with six cell surface markers (CD45, CD34, CD33, CD14, CD123, CD38) to identify major cell populations present in many AML samples: blasts, promonocytes/monocytes, lymphocytes and granulopoietic cells. Flow cytometric analysis was performed using the iQue Screener PLUS instrument (Intellicyt). Apoptotic and dead cells were removed from the analysis (Annexin-V/7-AAD) and live cell counts of different cell populations after drug treatment were measured. For comparison, the viability of the samples was also assessed using the CellTiter-Glo (CTG) assay that uses the amount of ATP as an indicator of metabolically active cells.ResultsImmunophenotyping of the AML samples showed large inter-sample variation in cell composition. The BM blast cell population out of the CD45+ positive cells ranged between 17-94% and the non-malignant lymphocyte population ranged from 0.4 to 46%. High numbers of promonocytes/monocytes were observed in samples from FAB M4/5 patients as expected, while FAB M0/1/2 patient samples mainly consisted of blasts and lymphocytes. FACS analysis of cell population specific drug responses revealed that blast cells of different patients had highly different drug sensitivities whereas lymphocytes and granulocytes did not show differences in drug sensitivities between the AML samples (Figure 1A). Interestingly, promonocytes/monocytes that are mainly present in FAB M4/5 samples but also in small quantities in some FAB M0/1/2 samples were systematically more sensitive to MEK inhibition, but more resistant to BCL-2 inhibition when compared to blasts (Figure 1A-B). In line with this observation, the average overall BM cell sensitivity of myelomonocytic/monocytic (FAB M4/5) samples as measured by CTG showed decreased sensitivity to venetoclax (P<0.01) and increased sensitivity to trametinib (P<0.05) when compared to less mature (FAB M0/1/2) AMLs. Initial differential gene expression analysis revealed enrichment of genes in the RAS/MAPK pathway in samples diagnosed with FAB M4/5. Amongst the tested compounds, venetoclax showed the highest toxic effect against blast cells even though 40% of the samples were at least partly resistant. Intriguingly, we observed that by combining venetoclax (50nM) with trametinib (25nM) or ruxolitinib (300nM), chemo- and venetoclax-resistant blast cells were effectively killed ex vivo (>80%) in all tested samples.ConclusionFACS-based drug testing ex vivo enables accurate drug sensitivity profiling of AML blast cells even in samples with high numbers of more mature leukemic cells and healthy cells. Our data indicate heterogeneity in drug responses of AML cells at different stages of myeloid differentiation, with high sensitivity to BCL-2 inhibition and resistance to MEK inhibition of the primitive blasts compared to more mature monocytic cells. MEK inhibitor sensitivity in FAB M4/5 AMLs might be linked to the inherent characteristics of monocyte lineage cells. Furthermore, we show that venetoclax-resistant blast cells can be effectively targeted by combining venetoclax with either MEK inhibitor trametinib or JAK inhibitor ruxolitinib. [Display omitted] DisclosuresMustjoki:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Celgene: Honoraria; Ariad: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Heckman:Novartis: Research Funding; IMI2 project HARMONY: Research Funding; Pfizer: Research Funding; Orion Pharma: Research Funding; Celgene: Research Funding.

Langerhans cell histiocytosis: Version 2021.

Children with Langerhnans cell histiocytosis (LCH) develop granulomatous lesions with characteristic clonal CD207+ dendritic cells that can arise as single lesions or life-threatening disseminated disease. Despite the wide range of clinical presentations, LCH lesions are histologically indistinguishable based on severity of disease, and uncertain classification as an immune versus neoplastic disorder has historically challenged the development of optimal clinical strategies for patients with LCH. Recently, activating somatic mutations in MAPK pathway genes, most notably BRAFV600E, have been discovered in almost all cases of LCH. Further, the stage of myeloid differentiation in which the mutation arises defines the extent of disease and risk of developing LCH-associated neurodegeneration. MAPK activation in LCH precursor cells drives myeloid differentiation, inhibits migration, and inhibits apoptosis, resulting in accumulation of resilient pathologic dendritic cells that recruit and activate T cells. Recurrent somatic mutations in MAPK pathway genes have also been identified in related histiocytic disorders: juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease. New insights into pathogenesis support reclassification of these conditions as a myeloid neoplastic disorders. Continued research will uncover opportunities to identify novel targets and inform personalized therapeutic strategies based on cell of origin, somatic mutation, inherited risk factors, and residual disease.

Lysophosphatidic acid receptors 2 and 3 regulate erythropoiesis at different hematopoietic stages

Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA2 is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA3 displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA2 agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA2 predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA3 increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA3 signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA2 and LPA3 at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.

Granulocytic Myeloid-Derived Suppressor Cells as Negative Regulators of Anticancer Immunity.

The immune system plays a critical role in cancer progression and response to therapy. However, the immune system can be compromised during the neoplastic process. Notably, the myeloid lineage, which gives rise to granulocytic cells, including neutrophils, is a well-recognized target of tumor-mediated immune suppression. Ordinarily, granulocytic cells are integral for host defense, but in neoplasia the normal process of granulocyte differentiation (i.e., granulopoiesis) can be impaired leading instead to the formation of granulocytic (or PMN)-myeloid-derived suppressor cells (MDSCs). Such cells comprise various stages of myeloid differentiation and are defined functionally by their highly pro-tumorigenic and immune suppressive activities. Thus, considerable interest has been devoted to impeding the negative contributions of PMN-MDSCs to the antitumor response. Understanding their biology has the potential to unveil novel therapeutic opportunities to hamper PMN-MDSC production in the bone marrow, their mobilization, or their effector functions within the tumor microenvironment and, therefore, bolster anticancer therapies that require a competent myeloid compartment. In this review, we will highlight mechanisms by which the neoplastic process skews granulopoiesis to produce PMN-MDSCs, summarize mechanisms by which they execute their pro-tumorigenic activities and, lastly, underscore strategies to obstruct their role as negative regulators of antitumor immunity.

Multidimensional study of the heterogeneity of leukemia cells in t(8;21) acute myelogenous leukemia identifies the subtype with poor outcome

t(8;21)(q22;q22) acute myelogenous leukemia (AML) is morphologically characterized by a continuum of heterogeneous leukemia cells from myeloblasts to differentiated myeloid elements. Thus, t(8;21) AML is an excellent model for studying heterogeneous cell populations and cellular evolution during disease progression. Using integrative analyses of immunophenotype, RNA-sequencing (RNA-seq), and single-cell RNA-sequencing (scRNA-seq), we identified three distinct intrapatient leukemic cell populations that were arrested at different stages of myeloid differentiation: CD34+CD117dim blasts, CD34+CD117bri blasts, and abnormal myeloid cells with partial maturation (AM). CD117 is also known as c-KIT protein. CD34+CD117dim cells were blocked in the G0/G1 phase at disease onset, presenting with the regular morphology of myeloblasts showing features of granulocyte-monocyte progenitors (GMP), and were drug-resistant to chemotherapy. Genes associated with cell migration and adhesion (LGALS1, EMP3, and ANXA2) were highly expressed in the CD34+CD117dim population. CD34+CD117bri blasts were blocked a bit later than the CD34+CD117dim population in the hematopoietic differentiation stage and displayed high proliferation ability. AM cells, which bear abnormal myelocyte morphology, especially overexpressed granule genes AZU1, ELANE, and PRTN3 and were sensitive to chemotherapy. scRNA-seq at different time points identified CD34+CD117dim blasts as an important leukemic cluster that expanded at postrelapse refractory stage after several cycles of chemotherapy. Patients with t(8;21) AML with a higher proportion of CD34+CD117dim cells had significantly worse clinical outcomes than those with a lower CD34+CD117dim proportion. Univariate and multivariate analyses identified CD34+CD117dim proportion as an independent factor for poor disease outcome. Our study provides evidence for the multidimensional heterogeneity of t(8;21)AML and may offer new tools for future disease stratification.

NPAS2 regulates proliferation of acute myeloid leukemia cells via CDC25A-mediated cell cycle progression and apoptosis.

Promoted proliferation and associated suppression of apoptosis at various stages of myeloid differentiation are well-known features of acute myeloid leukemia (AML), but understanding of the molecular processes involved remains limited. As a crucial circadian agent, neuronal PAS domain protein 2 (NPAS2) is widely recognized as a promising predictor of clinical outcome in various malignancies. Nevertheless, the understanding of its influence on AML is insufficient. Using KD cells and expression assays, we carried out detailed investigation of the role of NPAS2 in AML in vivo and in vitro. Firstly, we found that NPAS2 expression was elevated in AML cells both in vivo and in vitro. NPAS2 knockdown via lentiviral infection clearly suppressed proliferation of MV4-11 and MOLM-14 cells. Additionally, NPAS2 knockdown caused G1/S cell cycle arrest (CCA), which inhibited CDC25A expression. Moreover, NPAS2 knockdown promoted cell death, as evidenced by increased caspase-3 cleavage, and change in Bcl2/Bax production. Excessive CDC25A expression eliminated G1/S CCA triggered by NPAS2 knockdown and death of NPAS2 knocked down MOLM and MV4-11 cells. The expression of CDC25A was stabilized by NPAS2, which induced cell cycle progression and participated in suppression of cell death by modulating caspase-3 cleavage, and expression of Bcl2/Bax. We therefore indicated NPAS2 to be a crucial modulator of survival as well as proliferation. Our research sheds light on the etiology of the proliferation of promyelocytes modulated via NPAS2 with regard to AML.

Hexokinase Proteins Impart Distinct Functions in Myeloid Development and Cell Death

Many key oncogenic pathways converge to adapt tumor cell metabolism to requirements of cancer cells. Aberrant proliferation that is frequently associated with cancer cells is also linked to an adjustment of metabolism in order to fuel cell growth and division. Cancer cells prefer utilizing glycolysis for energy production and providing essential building blocks for a variety of macromolecules. Hexokinases (HKs) are rate-limiting enzymes that catalyze the first and irreversible step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. Four HK isoforms are expressed in mammalian cells, HK1, HK2, HK3 and HK4 (also known as glucokinase). HKs promote and sustain a concentration gradient that facilitates glucose entry, which ensures the initiation of glucose dependent pathways. In general, HKs have a cytoprotective role that was highlighted by enhanced sensitivity of cancer cells to drugs when HKs were inhibited. Previously we have reported that HK3 was transcriptionally regulated by PU.1 (SPI-1) in myeloid cells. Further, HK3 expression was significantly reduced in patient acute myeloid leukemia (AML) cells, particularly in acute promyelocytic leukemia (APL) cells expressing the PML-RARA oncofusion protein.We now report on the expression and regulatory function of HKs, particularly HK3, during myeloid differentiation and granulocyte associated cell death. First, we analyzed mRNA HK levels in human CD34+ hematopoietic progenitors cells differentiated towards granulocytes or macrophages by qPCR. Interestingly, while HK1 and HK2 levels remain stable during all stages of myeloid differentiation, HK3 mRNA levels significantly increased. The same pattern of HK mRNA expression was seen in NB4 APL and HL60 AML cell lines differentiated towards granulocytes and monocytes using all-trans retinoic acid (ATRA) and vitamin D3, respectively.To determine a specific role for HK1-3 function in myeloid cells, HK1-3 knockdowns (KD) and knockouts (KO) in NB4 and HL60 cell lines, using shRNA or gRNAs (Cas9/CRISPR technology), respectively, were generated. NB4 HK KD AML cells were tested for their differentiation upon ATRA treatment. Knockdown of HKs generally resulted in a decreased differentiation response of about 20% as assessed by the differentiation marker CD11b. We next determined energy metabolism of HK altered KD and KO cells, relative to parental cells, using a seahorse analyzer. While lowering HK1 or HK3 levels in NB4 and HL60 AML cells did not affect glycolytic capacity at steady state, HK2 inhibition significantly reduced steady state glycolytic capacity. In contrast, knocking down or knocking out HK3 resulted in a higher sensitivity to ATRA-induced cell death during differentiation, which was coupled with higher glycolytic capacity.Together, our findings suggest that HK2 has an important role in steady state metabolism of AML cells while HK3 appears to be a metabolic switch for cell survival during myeloid differentiation. DisclosuresNo relevant conflicts of interest to declare.

Acquired Hematologic Disorders of Ras-MAPK Activation

Histiocytic disorders represent a collection of conditions characterized by aberrant function, differentiation and/or proliferation of cells of the mononuclear phagocyte system (MPS). “Histiocyte” is an archaic term (meaning “tissue cell”) used to describe phagocytic cells with mononuclear morphology. Clinical approaches to histiocytic disorders have historically been challenged by incomplete understanding of mechanisms of pathogenesis, with debate over classification as cancer versus immune dysregulation. Langerhans cell histiocytosis (LCH), the most common histiocytic disorder, presents with granulomatous lesions with clonal CD207+ dendritic cells (DCs) that can arise as single lesions or life-threatening disseminated disease. Despite the wide range of clinical presentations, LCH lesions are histologically indistinguishable regardless of disease severity. Erdheim-Chester disease (ECD) arises in adults and is characterized by distribution of inflammatory lesions with CD163+ foamy histiocytes involving bone, retroperitoneum/kidney, skin and/or brain. Juvenile xanthogranuloma (JXG), histologically similar to ECD, is typically skin-limited in children, but may also manifest as life-threatening systemic disease. Classical Rosai-Dorfman disease (RDD) presents in children as lymphadenopathy with CD163+ histiocytes and emperipolesis identified in lymph node biopsy, though RDD likely represents a spectrum of conditions in children and adults with a common histiological endpoint. Classification of histiocytic disorders has been based on phenotype of the pathologic cell: LCH (DC-like), non-LCH (macrophage-like), or malignant histiocytosis. BRAFV600E was the first recurrent somatic mutation identified in LCH and ECD. While occurring in 7% of all human cancers, BRAFV600E mutations are also frequently found in benign conditions such as melanocytic nevi and colon polyps. Whole exome sequencing of LCH, ECD, RDD and JXG biopsies has revealed mutually exclusive MAPK pathway activating mutations within otherwise “quiet” genomic landscapes. In LCH, the stage of myeloid differentiation in which the mutation arises defines the extent of disease, and MAPK activation in precursor cells drives myeloid differentiation, blocks migration, and inhibits apoptosis, resulting in accumulation of resilient pathologic DCs that recruit and activate T cells. These new insights support reclassification of these histiocytic disorders as myeloproliferative neoplasms (MPN). Early phase trials in adults with LCH and ECD and emerging pediatric case studies demonstrate promising responses to MAPK pathway inhibitors, though potential for cure and long-term safety are not known. MAPK pathway activation clearly drives pathogenesis in histiocytic MPNs. However, it remains unclear how pathway activation, in many cases with the same mutation in the same CD34+ hematopoietic stem cell, can produce distinct cellular phenotypes with characteristic tissue distribution. While we now are beginning to understand the framework for mechanisms of pathogenesis of histiocytic disorders, continued research will uncover opportunities to identify additional targets and inform personalized therapeutic strategies based on cell of origin, somatic mutation and inherited risk factors. DisclosuresNo relevant conflicts of interest to declare.

Increased basal intracellular signaling patterns do not correlate with JAK2 genotype in human myeloproliferative neoplasms

AbstractMyeloproliferative neoplasms (MPNs) are associated with recurrent activating mutations of signaling proteins such as Janus kinase 2 (JAK2). However, the actual downstream signaling events and how these alter myeloid homeostasis are poorly understood. We developed an assay to measure basal levels of phosphorylated signaling intermediates by flow cytometry during myeloid differentiation in MPN patients. Our study provides the first systematic demonstration of specific signaling events and their comparison with disease phenotype and JAK2 mutation status. We demonstrate increased basal signaling in MPN patients, which occurs in both early and later stages of myeloid differentiation. In addition, the pattern of signaling is not correlated with JAK2 mutation status and signaling intensity is poorly correlated with mutant JAK2 allele burden. In contrast, signaling differences are detected between different MPN disease phenotypes. Finally, we demonstrate that signaling can be inhibited by a JAK2-selective small molecule, but that this inhibition is not JAK2 V617F specific, because MPN patients with mutant JAK2, wild-type JAK2, and control patients were inhibited to a similar degree. Our data suggest that, in addition to JAK2 mutations, other factors contribute significantly to the MPN phenotype, results that are relevant to both the pathogenesis and therapy of MPN.

Global and HOX Gene DNA Methylation In Normal Karyotype Acute Myeloid Leukemia: Clinical Implications and Molecular Correlations

AbstractAbstract 231 Background:DNA methylation induces gene silencing in a nonrandom fashion in many types of malignancies. In AML there is no consensus regarding the clinical implications of DNA methylation on a global level. Normal karyotype AML (NK AML) can be prognostically stratified by molecular mutations in genes such as FLT3 and NPM1. We have previously reported that increased gene promoter methylation levels may have positive prognostic implications in AML. Now we have focused and expanded our analysis in a homogenous group of NK AML in an effort to illuminate these issues. Methods and patients:We analyzed genome wide DNA methylation signatures from the diagnostic bone marrow samples of 58 de novo NK AML with the IlluminaHuman27 Methylation array, covering 27000 CpG sites, mainly located in the promoter regions of 15000 individual genes. Global methylation was defined as the average methylation values of all measured CpG sites in the specific sample. All patients were between 18 and 67 years of age and received standard induction chemotherapy. All were eligible for intensive consolidation therapy including allogeneic transplantation. FACS sorted normal bone marrow separated into four stages of myeloid differentiation were analyzed as normal controls. Methylation data were correlated to clinical outcomes and molecular mutational status of NPM1 and FLT3. Functional annotation analyses were performed on differentially methylated genes to find epigenetically perturbed pathways. Further molecular analysis of CEBPA, IDH1 and IDH2 is currently performed. Results and discussion:Global methylation levels varied substantially between AML samples but remained mainly unchanged during normal myeloid differentiation. Methylation levels were significantly higher in AML cases than in the normal myeloid progenitors (p<0.001). There were no correlations between methylation levels, age, white blood cell (WBC) count or bone marrow blast percentage at diagnosis. Increased global methylation correlated significantly to increased in vivo chemosensitivity and to patient survival. CR rates after one course of induction chemotherapy were 64% in the quartile of patients with the highest level of global DNA methylation compared to 32% in others (HR 3.9, p=0.04). Median overall survival of patients of the most methylated quartile was not reached and significantly longer than the 16 months median survival of the least methylated quartile (p=0.001) (see figure below). In a Cox Regression model stratified for FLT3-mutational status, methylation level was an independent prognostic factor for survival (p=0.01) together with WBC count (p=0.01). [Display omitted] A functional annotation analysis revealed that NPM1 wild type samples had an enrichment of Homeobox (HOX) gene methylation as compared to NPM1 mutated cases (p=1.7×10−11), providing a mechanism for the previously described difference in HOX gene expression in NPM1 wt vs. NPM1 mutated AML. The average methylation levels of all HOX genes were higher in wild type NPM1 cases compared to NPM1 mutated cases (p=0.02). Additional gene expression array data in our cohort of patients also showed methylation levels of individual HOX genes to be correlated inversely with expression. HOX genes were also enriched in non-FLT3ITD cases compared to FLT3ITD-cases, however, not to a statistically significant degree.In conclusion we show that global DNA methylation levels are predictive of response to chemotherapy and an independent prognostic factor for survival in normal karyotype AML. Furthermore, our results suggest that HOX gene methylation may be the mechanism underlying the previously known difference in HOX-gene expression between NPM1 mutated and unmutated AML cases. Disclosures:Nahi:Jansen-Cilag: Honoraria; Celgene: Honoraria.

Abstract 3491: Differential antileukemic activity of plant polyphenol combinations in acute myeloid leukemia (AML) cells

Abstract AML is the most common acute leukemia in adults with high mortality rate. This malignancy results from the block of terminal differentiation of the multipotent hematopoietic progenitors at early stages of myelopoiesis. The standard approach for AML treatment is cytotoxic chemotherapy with cytarabine and anthracyclines which has low efficiency and is accompanied by severe side effects. Therefore, AML represents a largely unsolved challenge for chemotherapy of malignant disease. Plant polyphenols, which are abundant in diet rich in vegetables and fruits, have been shown to act as chemopreventive and anticancer agents. In this study we demonstrate the in-vitro antileukemic activity of curcumin (CUR), carnosic acid (CA) and silibinin (SIL), alone and in combination, in several human AML cell lines representing different stages of myeloid differentiation: KG-1a (FAB: M1; WHO: AML with minimal differentiation), HL60 (FAB: M2; WHO: AML with maturation), and U937 (FAB: M4; WHO: acute myelomonocytic leukemia). All polyphenols tested inhibited cell proliferation in a concentration-dependent manner. Remarkably, when administered together even at non-cytotoxic concentrations, CUR (2.5-5 µM) and CA (5-10 µM) induced robust cell death in all cell lines tested. This effect was due to the induction of apoptosis concomitant with activation of caspase −8, −9 and −3, and was associated with cell cycle arrest in the S-phase. Interestingly, the induction of cell death was not accompanied either by generation of the intracellular reactive oxygen species or by a decrease in the glutathione content. In addition, the combined treatment induced caspase-8-dependent cleavage of the BH3-only protein Bid, but had no effect on the other Bcl-2 family pro- and anti- apoptotic proteins tested, including Bcl-2, Bcl-xL, Mcl-1, Bax and Bak. In contrast to the CUR/CA combination, cell treatment with SIL (30-60 µM) combined with either CUR or CA resulted in an enhanced inhibition of proliferation with a minor decrease in cell viability. This antiproliferative effect was accompanied by a marked G0/G1 cell cycle arrest with a concomitant decrease in the protein levels of cyclin E and cyclin-dependent kinase-4, and elevation of the cyclin-dependent kinase inhibitor p27Kip1 levels. Importantly, the polyphenol combinations tested here did not affect the viability of human normal skin fibroblasts as well as of both proliferating and non-proliferating peripheral blood mononuclear cells. Our findings indicate that distinct combinations of plant polyphenols exert strong but differential in vitro antileukemic effects by selectively inducing synergistic growth arrest and/or apoptotic cell death. These results suggest that such combinations may have potential for the treatment of AML. Supported by RO1-CA117942-02 grant from the NIH-NCI (to both G.P.S. and M.D.) and by Israel Science Foundation grant 778/07 (to M.D.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3491.

Myeloid blasts are the mouse bone marrow cells prone to differentiate into osteoclasts

Cells of the myeloid lineage at various stages of maturity can differentiate into multinucleated osteoclasts. Yet, it is unclear which developmental stages of this lineage are more prone to become osteoclasts than others. We investigated the osteoclastogenic potential of three successive stages of myeloid development isolated from mouse bone marrow. Early blasts (CD31hi/Ly-6C-), myeloid blasts (CD31+/Ly-6C+), and monocytes (CD31-/Ly-6Chi), as well as unfractionated marrow cells, were cultured in the presence of M-CSF and receptor activator of NF-B ligand (RANKL), and the differentiation toward multinucleated cells and their capacity to resorb bone was assessed. Myeloid blasts developed rapidly into multinucleated cells; in only 4 days, maximal numbers were reached, whereas the other fractions required 8 days to reach maximal numbers. Bone resorption was observed after 6 (myeloid blasts and monocyte-derived osteoclasts) and 8 (early blast-derived osteoclasts) days. This difference in kinetics in osteoclast-forming capacity was confirmed by the analysis of osteoclast-related genes. In addition, the myeloid blast fraction proved to be most sensitive to M-CSF and RANKL, as assessed with a colony-forming assay. Our results show that osteoclasts can develop from all stages of myeloid differentiation, but myeloid blasts are equipped to do so within a short period of time.

Elevated Leukocyte Alkaline Phosphatase Scores Induced by Jak2 V617F Mutation

Leukocyte alkaline phosphatase (LAP) enzymatic activity is a marker of the last stages of myeloid differentiation. The level of LAP is quantitated as the LAP score. Estimation of the LAP score has been useful for distinguishing chronic myelogenous leukemia (CML) from BCR-ABL–negative chronic myeloproliferative disorders (MPDs) and neutrophilic reactions in severe infections. CML patients usually have a low LAP score, whereas elevated LAP scores are seen in patients with polycythemia vera (PV), primary myelofibrosis (PMF), and leukocytosis caused by infections. An acquired Jak2 V617F mutation is seen in approximately 95% of patients with PV and in about 50% of patients with essential thrombocythemia or PMF. It has been shown that Jak2 V617F mutation induced constitutive activation of the JAK-STAT signaling pathway. We speculated that an elevated LAP score might be caused due to activation of JAK-STAT signaling through a Jak2 V617F mutation, and conducted this study to address this question. We analyzed the LAP scores in Jak2 V617F-positive and -negative MPD patients. Jak2 V617F-positive MPD patients had higher LAP scores than Jak2 V617F-negative patients. Moreover, patients carrying homozygous mutations had higher LAP scores than patients with heterozygous mutations. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of Jak2 V617F-positive patients. We lentivirally transfected the acute promyelocytic leukemia cell line NB4 with the Jak2 V617F mutation and wild-type Jak2 V617F. The expression level of Jak2 was not significantly different between the Jak2 V617F mutation and wild-type Jak2 V617F. We then examined the LAP scores of transfected NB4 cells after these cells were differentiated by all-trans retinoic acid and granulocyte colony stimulating factor. It was observed that the Jak2 V617F mutation and not the wild-type Jak2 induced elevated LAP scores. Furthermore, we showed that Jak2 followed the MAP kinase pathway and not the PI3 kinase pathway, as a downstream signaling pathway to elevate the LAP scores using MEK 1/2 (U0126) and PI3 kinase (LY294002) inhibitors. In conclusion, we obtained direct evidence that Jak2 V617F mutation induces elevated LAP scores via the MAP kinase pathway.

Pegylated granulocyte colony-stimulating factor mobilizes CD34+ cells with different stem and progenitor subsets and distinct functional properties in comparison with unconjugated granulocyte colony-stimulating factor

Pegylated granulocyte colony-stimulating factor (G-CSF) has recently been introduced as a new compound for mobilization of CD34(+) hematopoietic stem and progenitor cells. In this study, we compared the molecular and functional characteristics of CD34(+) cells mobilized by pegylated G-CSF with those mobilized by unconjugated G-CSF. Gene expression of immunomagnetically enriched CD34(+) cells from leukapheresis products of patients who were given pegylated-G-CSF or unconjugated G-CSF was analyzed using Affymetrix HG Focus microarrays and quantitative reverse transcriptase polymerase chain reaction. Flow cytometry and fluorescence activated cell sorting was conducted to assess the CD34(+) subset composition and to obtain Lin(-), CD34(+), CD38(-) hematopoietic stem cells. Cell cycle assays and clonogenic assays were performed for functional corroboration. Pegylated G-CSF and unconjugated G-CSF mobilized CD34(+) and hematopoietic stem cells with different molecular phenotypes and functional properties. The CD34(+) cells mobilized by pegylated G-CSF had higher expression levels of genes indicative of early hematopoiesis, including HOXA9, MEIS1 and GATA3. We found lower expression of genes characteristic of erythroid and later stages of myeloid differentiation and a lower functional burst-forming unit erythroid/colony-forming unit-granulocyte-macrophage ratio. Consistently, greater numbers of hematopoietic stem cells and common myeloid progenitors and fewer megakaryocyte-erthrocyte progenitors were found in the pegylated-G-CSF-mobilized CD34(+) cells. Additionally, sorted pegylated-G-CSF-mobilized hematopoietic stem cells displayed higher expression of HOXA9 in comparison to G-CSF-mobilized hematopoietic stem cells. In line with the gene expression data, CD34(+) cells mobilized by pegylated G-CSF, as well as sorted hematopoietic stem cells, showed a significantly greater cell cycle activity. Stimulation with pegylated-G-CSF or G-CSF results in different expression of key regulatory genes and different functional properties of mobilized hematopoietic stem cells as well as their progeny, a finding that might be relevant for the application of these cells in blood stem cell transplantation.

Expression patterns of CD33 and CD15 predict outcome in patients with acute myeloid leukemia

Expression patterns of CD33 and CD15 in normal/reactive bone marrow (n = 13) and in leukemic blasts from patients with acute myeloid leukemia (n = 129) were determined using multiparameter flow cytometry and a standard panel of triple antibody combinations. Five patterns, corresponding to the consecutive stages of myeloid differentiation, were identified [I: CD33–/CD15– (n = 18), II: CD33+/CD15– (n = 43), III: CD33+/CD15 heterogeneous (n = 10), IV: CD33+/CD15+ (n = 50), V: CD33–/CD15+ (n = 8)]. Patients with pattern II had the highest relapse rate and shortest median overall survival (OS, 8 months), but they were also the oldest (median age 72 years) and had the highest frequency of unfavorable cytogenetic aberrations. Pattern V patients had a short OS (median 14 months) even though they were the youngest (median age 50 years), had high remission rate and did not have unfavorable cytogenetics. In multivariate analysis, age, cytogenetics, CD15 expression and the presented immunophenotypic classification were significant for OS (age p = 0.004, cytogenetics p = 0.011, immunophenotype pattern p = 0.024, CD15 p = 0.031). Age (p = 0.001) and immunophenotypic classifications (p = 0.015) were significant for disease-free survival in patients who achieved complete remission.

Pegylated G-CSF Mobilizes CD34+ Cells with Different Stem and Progenitor Cell Subsets and Distinct Functional Properties in Comparison with Unconjugated G-CSF.

Current regimens for peripheral blood stem cell (PBSC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of G-CSF starting shortly after cytotoxic therapy. Recently a polyethylenglycole (PEG)-conjugated G-CSF has been introduced which has a substantially longer half-life than the original formula and therefore provides the basis for long-lasting G-CSF serum-levels after a single injection. In this study, we compared gene expression patterns, subset composition and functional properties of CD34+ cells and highly purified HSC mobilized with cyclophosphamide and either Peg-G-CSF or G-CSF. Cells were derived from peripheral blood of patients with multiple myeloma. After the end of chemotherapy, 7 patients got a single injection of Peg-G-CSF whereas 9 patients received daily G-CSF resulting in an equal cumulative dose. Gene expression analysis was performed using Affymetrix HG Focus GeneChips. Key functional genes were verified by RT-PCR. Subset analysis and fluorescence based cell sorting has been conducted to assess the effects of stimulation with either pegylated or unconjugated G-CSF on CD34+ subset composition and to obtain HSCs. Cell cycle and apoptosis assays as well as clonogenic assays were for functional corroboration. The same patients with multiple myeloma who had donated CD34+ cells for the molecular and biological studies were transplantated with Peg-G-CSF- or G-CSF-mobilized PBSC.Peg-G-CSF-mobilized cells showed lower expression of genes characteristic for erythroid and later stages of myeloid differentiation as well as a lower BFU-E/CFU-GM ratio compared to G-CSF-mobilized cells. In turn, we found higher expression levels of genes indicative of early hematopoiesis including HOXA9, MEIS1, MLL and GATA3. Subset analyses revealed a greater number of HSC and CMP (common myeloid progenitors) and a lower number of MEP (megakaryocyte-erthrocyte progenitors) in Peg-G-CSF-mobilized CD34+ cells.Cell cycle-promoting genes including cyclins and kinases were higher expressed in Peg-G-CSF-mobilized cells. On the other hand human HTm4, which causes cell cycle arrest in hematopoietic cells, was lower expressed compared to G-CSF-mobilized cells. This is emphasized by a significant higher proportion of actively cycling CD34+ cells after pegfilgrastim-mobilization. Higher gene expression levels of HOXA9, MEIS1 and GATA3 were also found in sorted Peg-G-CSF-mobilized HSC in comparison to G-CSF-mobilized HSC. Moreover, Peg-G-CSF-mobilized HSC showed a lower apoptosis rate and a greater proportion of cells in S- and G2/M phase of cell cycle. After transplantation of Peg-G-CSF-mobilized stem- and progenitor cells we observed earlier leukocyte recovery compared to G-CSF-mobilized transplants.Our data demonstrate that Peg-G-CSF and G-CSF stimulation differentially affects the expression of key regulatory genes and functional properties of mobilized HSC as well as their progeny, which might be important for their application in stem cell transplantation.

Identification of PU.1 Target Genes That Are Dependent on Specific Functional Domains of the Transcription Factor PU.1.

The expression of PU.1, a member of the ets transcription factor family, is limited to the hematopoietic lineage. Using knockout and reduced PU.1 expression and gain of function mice as a model system, it has been demonstrated that PU.1 plays a key role in early myeloid and lymphoid fate decision and at later stages of myeloid differentiation and function. In PU.1 (Sfpi1) null mice, dendritic cells and monocytes could not be identified, but early myeloid progenitors and immature neutrophils were present. In mice with reduced PU.1 expression monocyte development was hindered, immature neutrophil development ensued, and the mice developed acute myeloid leukemia. The different domains of the PU.1 transcription factors have been attributed to distinct functions in early myeloid fate decision. The PU.1 transcription factor is composed of 3 major domains: transactivation, PEST, and DNA binding. We have used an immature myeloid cell line (503) derived from the Sfpi1-null mouse to analyze the effect of lentiviral gene delivery of PU.1 mutants lacking specific PU.1 domains, on differentiation of 503 cells, and to identify PU.1 target genes that are dependent on these specific domains for their expression. We found that a PU.1 mutant lacking the acidic domain leads to differentiation of the 503 cells towards neutrophil lineage, measured by specific lineage specific surface markers. Expression of a PU.1 mutant lacking the glutamine rich region of the transactivation domain did not promote differentiation to neutrophils. Moreover, the glutamine rich and acidic motifs are not necessary for the expression of the CD11b. Despite the independence of CD11b expression from both transactivation domain motifs, its expression was dependent on PU.1 DNA binding. Using cDNA microarray analysis, we identified PU.1 target genes that are regulated by specific domains of the PU.1 transcription factor. In our Affymetrix cDNA microarray analysis we identified over 1300 gene changes that are exclusively dependent on either the acidic domain or the glutamine rich region of the PU.1 transactivation domain for regulation. In addition, we found 33 PU.1- genes that are repressed by the PU.1 mutant lacking the acidic domain, but activated by the glutamine rich region PU.1 mutant, and 38 genes that are regulated in the opposite direction, thus allowing us to begin to identify the hierarchy of the PU.1-regulated gene cascade assocated with cellular function. Currently, we are investigating the biological significance of some of these differentially expressed target genes. For example, one of the identified genes, Neutrophil Elastase (ELA2), is highly upregulated in the 503 cells expressing full length PU.1, or the PU.1 mutant lacking the acidic domain, but is profoundly downregulated in the 503 cell line expressing the glutamine rich region deletion mutant. It has been shown that ELA2 deficient mice are protected form acute promyelocytic leukemia. We are currently investigating the functional role of the PU.1-dependent regulation of ELA2 in our model system. In addition, we are identifying target genes that are regulated exclusively by other PU.1 domains, i.e. the PEST domain. Taken together, we identified and are analyzing the functional importance of specific target genes in PU.1-dependent myeloid differentiation that are regulated by different functional domains of the PU.1 transcription factor.

Brg1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, is required for myeloid differentiation to granulocytes

Many mammalian SWI/SNF complexes use Brahma-related gene 1 (Brg1) as a catalytic subunit to remodel nucleosomes for transcription regulation. In several mesenchymal cells and tissues, expression of a defective Brg1 protein negates the normal activity of the SWI/SNF complex and delays or blocks differentiation. To investigate the role of SWI/SNF complexes during myelopoiesis, we stably expressed a dominant negative (dn) Brg1 mutant in the myeloid lineage. Forced expression of dnBrg1 in IL-3-dependent murine 32Dcl3 myeloid progenitor cells results in a profound delay in the granulocyte-colony stimulating factor (G-CSF) induced granulocytic maturation. These cells also exhibit a significant decrease in the expression of both CD11b and Gr-1 surface receptors, which are normally upregulated during granulopoiesis, and show sustained expression of myeloperoxidase, which is synthesized primarily during the promyelocytic (blast) stage of myeloid development. Thus, dnBrg1 expression causes a developmental block at the promyelocytic/metamyelocytic stage of myeloid differentiation. Our findings indicate that the normal chromatin remodeling function of Brg1 is necessary for the G-CSF dependent differentiation of myeloid cells towards the granulocytic lineage. This dependency on Brg1 may reflect a stringent requirement for chromatin remodeling at a critical stage of hematopoietic cell maturation.

Mutations of the SHP-2 Proteine Tyrosine Phosphatase in Adult Patients with Acute Myeloid Leukemia.

Acute myeloid leukaemia (AML) represents a heterogeneous group of early stem cell malignancies characterized by an uncontrolled expansion of malignant cells blocked at certain stages of myeloid differentiation. Several genetic abnormalities have been described, including translocations involving genes of the CBF family, as well as activating mutations of signal transduction proteins, e.g. N-RAS, K-RAS or the Flt3- receptor tyrosine kinase (RTK). More recently, mutations in the SHP-2 protein, a tyrosine phosphatase involved in RTK signal transduction, have been described in patients with juvenile myelomonocytic leukaemia (JMML) as well as paediatric patients with AM. Only limited data on this aberration are available in older patients. In order to investigate the prevalence and the prognostic relevance of this gene in adults, we analyzed the mutational status of SHP-2 in a cohort of patients with AML.Patients and Methods: DNA samples prepared from bone marrow or peripheral blood samples taken at diagnosis from 977 patients treated in the AML96 protocol of the SHG Dresden were analyzed. Screening for mutations in the mutational hot spot in exon 3 of the PTPN11 gene coding for SHP-2 was performed on a dHPLC system (Transgenomic). Samples with aberrant dHPLC chromatogram were sequenced.Results: In the 977 patients investigated so far, 29 PTPN11 exon 3 mutations were found (3%). All mutations consisted of single base pair exchanges in codons 60–76, mostly affecting codons 69 (n=6), 72 (n=7) and 76 (n=6). Patients with PTPN11 mutations were more likely to have FAB M0 (p= .038) or M5b (p=.045) morphology than negative cases and had a significantly lower prevalence of FLT3-ITD mutations (p= .017). PTPN11 mutations were associated with a normal karyotype in 15/28 evaluable patients, whereas 13 had cytogenetic abnormalities, all of which were either high risk (−7/7q-, n=4; inv3, n=2, complex, n=2, −5, n=1) or intermediate risk (n=4) aberrations. No significant differences were found in the complete remission rate as well as the overall and event free survival, but a trend for a shorter disease free survival was observed in patients with PTPN11 mutations (median 7.8 vs 14.4; p= 0.06) associated with a significantly increased rate of relapse (p= .02). Although not significant yet, a similar trend was seen for patients with normal karyotype.Conclusion: PTPN11 mutations can be found in a small but considerable number of adult patients with AML. Consistent with the data in children, this mutation seems to more prevalent in patients with monosomy 7 and leukemias with monocytic differentiation. Although very preliminary, the data point to a worse prognosis of patients with SHP-2 mutations. We are currently further extending the number of patients and analyze additional exons to delineate the potential prognostic role of this aberration.